WO2025032069A1 - Mono and multispecific anti-trem2 antibodies, methods and uses thereof - Google Patents

Abstract

Herein is reported an anti-TREM2/Abeta protein antibody comprising a first binding site binding to human TREM2 comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 305, a HVR-H2 of SEQ ID NO: 306 and a HVR-H3 of SEQ ID NO: 307, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 309, a HVR-L2 of SEQ ID NO: 310 and a HVR-L3 of SEQ ID NO: 311 (TREM2 3295), or a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 329, a HVR-H2 of SEQ ID NO: 330 and a HVR-H3 of SEQ ID NO: 331, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 333, a HVR-L2 of SEQ ID NO: 334 and a HVR-L3 of SEQ ID NO: 335 (TREM2 3306), and a second binding site binding to human Abeta protein comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 09, a HVR-H2 of SEQ ID NO: 10 and a HVR-H3 of SEQ ID NO: 11, and in the light chain variable domain a HVR- L1 of SEQ ID NO: 13, a HVR-L2 of SEQ ID NO: 14 and a HVR-L3 of SEQ ID NO: 15 (Abeta 8675).

Description

MONO AND MULTISPECIFIC ANTI-TREM2 ANTIBODIES, METHODS

AND USES THEREOF

The current invention is in the field of mono- and multispecific antibodies. In more detail herein are reported novel mono-, bi- and trispecific anti-TREM2 antibodies with improved properties.

Background of the Invention

Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed on the cell surface of myeloid lineage-derived cells such as monocytes/macrophages, microglia, osteoclasts, neutrophils, and dendritic cells. It is a transmembrane receptor of the innate immune system.

Without being bound to a particular theory, it is believed that upon certain ligand binding, TREM2 forms a signaling complex with a transmembrane adapter protein, DNAX-activating protein 12 (DAP12), which in turn is tyrosine phosphorylated by the protein kinase SRC. It is believed that the activated TREM2/DAP12 signaling complex mediates intracellular signaling by recruiting and phosphorylating kinases such as Syk kinase. TREM2/DAP12 signaling modulates cellular activities such as phagocytosis, cell growth and survival, cytokine secretion, and the migration of cells such as microglia and macrophages. TREM2 may also activate phosphatidylinositol 3 -kinase (PI3K) in a TREM2/DAP12/DAP10 heterodimer complex, which in turn can be inhibited by Src homology 2 (SH2) domain-containing inositol phosphatase- 1 (SHIP1). TREM2 undergoes regulated proteolysis, in which the membrane- associated full-length TREM2 is cleaved by the alpha-secretases disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) and ADAMIO at the H157-S158 peptide bond into a sTREM2 portion that can be shed from the cell and a membrane-retained C-terminal fragment that is further degraded by a gamma- secretase. Altered levels of sTREM2 have been reported in cerebrospinal fluid of patients in a prodromal stage of Alzheimer’s disease (AD) and after diagnosis of AD, in patients with frontotemporal dementia, or having a certain mutation in TREM2. Some mutations are associated with a higher genetic risk to develop Alzheimer’s disease. Additionally, certain mutations in TREM2 are associated with altered functions such as impaired phagocytosis and reduced microglial function (see, e.g., WO 2020/172450).

TREM2 has been found to be localize to microglia around plaques and neurons in the brains of TgCRND8 mice (see, e.g., US 2015/0065567) and to be enriched at those microglia cell surface regions, which contact amyloid plaques or neuronal debris (see, e.g., WO 2020/079580). Knockdown of TREM2 or DAP12 in microglia resulted in reduced phagocytosis of apoptotic neurons, whereas TREM2 overexpression lead to increased phagocytosis of dying neurons by microglia, and similarly increases phagocytosis by other myeloid lineage cells (see, e.g., WO 2014/074942; WO 2016/023019; WO 2019/118513; Kleinberger et al., Sci. Transl. Med. 6 (2014) 243ra86, pp. 1-12).

Cells expressing high levels of TREM2 are thought to participate in immune surveillance, cell-cell interactions, tissue debris clearance, and the resolution of latent inflammatory reactions (see, e.g., WO 2019/118513)

However, the mechanism by which TREM2 contributes to neurodegeneration remains obscure. Furthermore, studies investigating the impact of TREM2 signaling on the inflammatory response have produced contradictory results, demonstrating either an anti-inflammatory or a pro-inflammatory role for TREM2 (Jay, et al., J. Exp. Med. 212:287-295 (2015), Jay, et al., J. Neurosci. 37:637-647 (2017), Sieber, et al., PLoS One 8:e52982 (2013), Turnbull, et al., J. Immunol 177:3520-3524 (2006)). Other studies have identified a role for TREM2 in microglial survival (Wang, et al., Cell 160: 1061-1071 (2015)), as well as in regulating energy metabolism (Ulland, et al., Cell 170:649-663 (2017)). Several studies have pointed to a role for TREM2 in phagocytosis (Hsieh, et al., J. Neurochem. 109: 1144-1156 (2009), Kawabori, et al., J. Neurosci. 35:3384-3396 (2015), Kleinberger, et al., Sci. Transl. Med. 6:243ra86 (2014), Takahashi, et al., J. Exp. Med. 201 :647-657 (2005), Xiang, et al., EMBO Mol. Med. 8:992-1004 (2016)), while others have observed no effect (e.g., Wang, et al., Cell 160: 1061-1071 (2015)) (see, e.g., WO 2020/121195).

WO 2017/062672 reported anti-TREM2 antibodies and methods of use thereof. WO 2007/068429 reported antibodies against amyloid beta 4 with glycosylated in the variable region.

WO 2022/032293 reported treatment of diseases related to colony-stimulating factor 1 receptor dysfunction using TREM2 agonists.

WO 2019/055841 reported anti-TREM2 antibodies and methods of use thereof.

Fassler, M., et al., reported that engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer"s disease models (J. Neuroinfl. 18 (2021) 19).

Van Lengerich, B., et al., reported that a TREM2-activating antibody with a bloodbrain barrier transport vehicle enhances microglial metabolism in Alzheimer's disease models (Nat. Neurosci. 26 (2023) 416-429).

Summary of the Invention

Herein are reported mono- and multispecific antibodies that bind to human TREM2. The antibodies according to the current invention have improved properties. Such improved properties relate, amongst other things, to improved therapeutic properties. For example, the antibodies according to the current invention induce phagocytosis as an effector function silent antibody, i.e. induce antibody-mediated uptake or phagocytosis in the absence of the canonical Fc-effector function-mediated ADCP. Thereby, it is possible to use effector function silent TREM2-specific antibodies according to the current invention advantageously for the treatment of brain diseases with a putative lower risk of Fc-region mediated negative side effects after systemic application.

The antibodies according to the current invention bind to a different epitope in the extracellular domain of human TREM2 as antibodies known from the art.

The antibodies according to the current invention are bispecific antibodies.

Herein are reported agonistic anti-TREM2/Abeta protein bispecific antibodies without Fc-effector function which modulate neuroprotective activity of microglia in the vicinity of plaques or brain vasculature covered by amyloid in Alzheimer’s disease as well as pharmaceutical composition comprising said antibodies for the treatment of early AD or preclinical AD patients who are amyloid positive or other indications with amyloid plaques such as Dementia with Lewy bodies or Parkinson’ s dementia or similar neurodegenerative disorder with amyloid plaque co-morbidity.

The antibodies according to the current invention have in vitro the ability to induce cellular migration and phagocytosis by the same molecule. That is the antibodies according to the current invention do not induce pSyk in the absence of Abeta, but increase LPC- and C5a stimulated cellular migration. In more detail, the antibodies according to the current invention induce in vitro the migration of THP-1 and iPSC- derived MO cells in the absence of any Abeta but as an amplifier of an existing inflammatory or damage signal (C5a or lyso-phosphatidylcholine, respectively) that induces an elevated baseline migration. This indicates that the TREM2/ Abeta protein mAbs can also modulate TREM2 on microglia under certain disease settings devoid of Abeta plaques.

The antibodies according to the current invention show after peripheral administration in vivo enrichment at plaques in APPswePS2 tg mice for targeted and sustained microglial modulation there over other brain regions.

The antibodies according to the current invention will show TREM2 oligomerization and longer brain retention via plaque targeting.

The antibodies according to the current invention have a non-inflammatory neuroprotective profile due to lack of FcgRII and FcgRIII-engagement while supporting uptake of amyloid plaques and potentially also cellular debris. Thus, the antibodies according to the current invention have the potential to prove for a therapy with lower risk to develop ARIA in contrast to anti-Abeta antibody therapy or the Fc-region effector function competent and Fc-region-modified anti-TREM2 antibodies from the art or in clinical trials.

One aspect according of the current invention is a multispecific antibody that is binding to human TREM2 and human amyloid beta protein, comprises an effector-function silent Fc-region, and induces phagocytosis in a bead-based phagocytosis assay as reported herein.

In one preferred embodiment, the antibody is a bispecific antibody that is binding to human TREM2 and human amyloid beta protein.

In another preferred embodiment, the antibody is a trispecific antibody that is binding to human TREM2, human amyloid beta protein and human transferrin receptor.

In one embodiment the antibody is binding to the extracellular domain of TREM2 or/and the human transferrin receptor.

In certain embodiments, the antibody according to the current invention induces phagocytosis in a bead-based phagocytosis assay and has reduced effector function, or is without effector function, or does not bind to FcgRII and FcgRIII.

In certain embodiments, the antibody according to the current invention comprises a human IgGl Fc-region comprising the L234A, L235A, and P329G substitutions (LALAPG substitutions) (numbering according to Kabat).

In certain embodiments, the antibody according to the current invention is conjugated covalently or non-covalently to at least one other molecule. In some cases, the antibody is conjugated covalently or non-covalently to at least one other molecule, wherein the at least one other molecule comprises a detection label and/or a drug.

One aspect according to the current invention is an anti-TREM2/Abeta protein antibody specifically binding to human TREM2 and human Abeta protein.

In one embodiment, the anti-TREM2/ Abeta protein antibody has one or more of the following properties: a) the antibody induces phagocytosis of amyloid plaques by macrophages, and/or b) the antibody induces microglial amyloid uptake in the absence of FcgRII as well as FcgRIII receptor engagement, and/or c) the antibody induces amyloid uptake in the absence of engagement of FcgR, and/or d) the antibody in Fc-effector function competent form induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, and/or e) the antibody without effector-function induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, preferably in the absence of Abeta and FcgR crosslinking, and/or f) the antibody induces migration of macrophages, preferably at a concentration of 0.14 to 34 nM in a migration assay as described herein, and/or g) the antibody increases LPC- and C5a stimulated cellular migration of THP-1 and iPSC-derived MO cells in the absence of any Abeta protein, and/or h) the antibody does not induce pSyk in the absence or presence of human Abeta protein, preferably the antibody is an antagonist of the Syk pathway, and/or i) the antibody does induce pSyk in the presence of human Abeta protein, and/or j) the antibody does not show a dose dependent Syk and S6 phosphorylation in DAP12 and TREM2 expressing HEK cells, and/or k) the antibody does not induce pSyk in TREM2/DAP12 overexpressing HEK or iPSC macrophages in the absence of cross-linking, and/or l) the antibody shows after peripheral administration in vivo enrichment at plaques in APPswePS2 transgenic mice, and/or m) the antibody modulates neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid, and/or n) the antibody has non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and/or o) the antibody provides for plaque retention and plaque-targeted brain exposures, preferably with higher local concentrations as an anti- TREM2 monospecific antibody, and/or p) the antibody does not induce TNF alpha, MIP-1 alpha or IL-8 release from iPSC-derived macrophages, and/or q) the antibody blocks shedding of sTREM2, and/or r) the antibody stabilizes sTREM2 in biological fluids, and/or s) the antibody binds to the ECD of TREM2 and thereby results i the accumulation of sTREM2 in biological fluids, and/or t) the antibody induces the shift from homeostatic or less engaged towards activated microglia, and/or u) the antibody induces ARIA at a lower level than a monospecific antibody, preferably does not induce ARIA.

In one embodiment the anti-TREM2/Abeta protein antibody comprises a) a first binding site binding to human TREM2 comprising i) a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 305, a HVR-H2 of SEQ ID NO: 306 and a HVR-H3 of SEQ ID NO: 307, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 309, a HVR-L2 of SEQ ID NO: 310 and a HVR-L3 of SEQ ID NO: 311 (TREM2 3295), or ii) a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 329, a HVR-H2 of SEQ ID NO: 330 and a HVR-H3 of SEQ ID NO: 331, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 333, a HVR-L2 of SEQ ID NO: 334 and a HVR-L3 of SEQ ID NO: 335 (TREM2 3306), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 09, a HVR-H2 of SEQ ID NO: 10 and a HVR-H3 of SEQ ID NO: 11, and in the light chain variable domain a HVR-L1 of SEQ ID NO: 13, a HVR-L2 of SEQ ID NO: 14 and a HVR-L3 of SEQ ID NO: 15 (Abeta 8675).

In one embodiment the anti-TREM2/ Abeta protein antibody comprises a) a first binding site binding to human TREM2 comprising i) a heavy chain variable domain of SEQ ID NO: 308 and a light chain variable domain of SEQ ID NO: 312 (TREM2 3295), or ii) a heavy chain variable domain of SEQ ID NO: 332 and a light chain variable domain of SEQ ID NO: 336 (TREM2 3306), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain of SEQ ID NO: 12 and in the light chain variable domain of SEQ ID NO: 16 (Abeta 8675).

In one embodiment, the anti-TREM2/ Abeta protein antibody binds to human TREM2 with an affinity of less than 1 nM.

In one embodiment, the anti-TREM2/ Abeta protein antibody induce migration in vitro at a concentration of 5-15 nM.

In one embodiment, the anti-TREM2/Abeta protein antibody comprises a fist light chain of SEQ ID NO: 462, a second light chain of SEQ ID NO: 463, a first heavy chain of SEQ ID NO: 464 and a second heavy chain of SEQ ID NO: 465.

One aspect of the current invention is an isolated nucleic acid or set of two or more nucleic acids encoding the antibody according to the current invention.

One aspect of the current invention is an isolated vector comprising one or more nucleic acids encoding the heavy chain and the light chain of the antibody according to the current invention.

One aspect of the current invention is a mammalian cell comprising the nucleic acid or vector according to the current invention.

One aspect of the current invention is a method of producing an antibody according to the current invention comprising the steps of culturing the mammalian according to the invention cell under conditions suitable for the expression of the antibody according to the current invention and recovering the antibody according to the current invention from the mammalian cell or the cultivation medium. One aspect is an antibody produced by the method.

One aspect of the current invention is a pharmaceutical composition comprising an antibody according to the current invention and a pharmaceutically acceptable carner. One aspect of the current invention is an antibody according to the current invention for use as a medicament.

One aspect of the current invention is an antibody according to the current invention for use in the treatment of a disease.

One aspect according to the current invention is a method of treating a condition associated with TREM2 loss of function in a subject in need thereof, comprising administering an antibody according to the current invention or a pharmaceutical composition according to the current invention to the subject.

One aspect according to the current invention is a method of treating a condition associated with toxic gain of TREM2 function in a subject in need thereof, comprising administering an antibody according to the current invention or a pharmaceutical composition according to the current invention to the subject.

One aspect according to the current invention is a method of treating a condition associated hyperactivity of microglia in a subject in need thereof, comprising administering an antibody according to the current invention or a pharmaceutical composition according to the current invention to the subject.

One aspect according to the current invention is a method of reducing levels of amyloid plaques in a subject in need thereof, comprising administering an antibody according to the current invention or a pharmaceutical composition according to the current invention to the subject.

One aspect according to the current invention is an antibody or a pharmaceutical composition according to the current invention for use in treating a condition associated with TREM2 loss of function in a subject in need thereof.

One aspect according to the current invention is an antibody or a pharmaceutical composition according to the current invention for use in reducing levels of amyloid plaques in a subject in need thereof. One aspect according to the current invention is the use of an antibody or a pharmaceutical composition according to the current invention in the preparation of a medicament for treating a condition in a subject in need thereof.

One aspect according to the current invention is the use of an antibody or a pharmaceutical composition according to the current invention in the preparation of a medicament for reducing levels of amyloid plaques in a subject in need thereof.

In certain embodiments of all aspects and embodiments of the invention, the disease or the condition is, or the subject suffers from, a neuroinfl ammatory or neurodegenerative disease. In certain embodiments, the neuroinflammatory or neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hakola disease, Guillain-Barre Syndrome (GBS), lysosomal storage disease, sphingomyelinlipidosis (Neimann-Pick C), mucopolysaccharidosis II/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuroBehcet’s disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In one preferred embodiment, the disease is Alzheimer’s disease. In another preferred embodiment, the disease is MS. In yet another preferred embodiment, the disease is Parkinson’s disease.

Thus, the current invention encompasses at least the following embodiments:

1. An anti-TREM2 antibody with one or more of the following properties: a) the antibody as bispecific anti-TREM2/Abeta protein antibody induces phagocytosis of amyloid plaques by macrophages, and/or b) the antibody as bispecific anti-TREM2/Abeta protein antibody induces microglial amyloid uptake in the absence of FcgRII as well as FcgRIII receptor engagement, and/or c) the antibody as bispecific anti-TREM2/Abeta protein antibody induces amyloid uptake in the absence of engagement of FcgR, and/or d) the antibody in Fc-effector function competent form induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, and/or e) the antibody as bispecific anti-TREM2/Abeta protein antibody without effector-function induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, preferably in the absence of Abeta and FcgR crosslinking, and/or f) the antibody induces migration of macrophages, preferably at a concentration of 0.14 to 34 nM in a migration assay as described herein, and/or g) the antibody increases LPC- and C5a stimulated cellular migration of THP-1 and iPSC-derived MO cells in the absence of any Abeta protein, and/or h) the antibody does not induce pSyk in the absence or presence of human Abeta protein, preferably the antibody is an antagonist of the Syk pathway, and/or i) the antibody as bispecific anti-TREM2/ Abeta protein antibody does induce pSyk in the presence of human Abeta protein, and/or j) the antibody does not show a dose dependent Syk and S6 phosphorylation in DAP12 and TREM2 expressing HEK cells, and/or k) the antibody does not induce pSyk in TREM2/DAP12 overexpressing HEK or iPSC macrophages in the absence of cross-linking, and/or l) the antibody as bispecific anti-TREM2/Abeta protein antibody shows after peripheral administration in vivo enrichment at plaques in APPswePS2 transgenic mice, and/or m) the antibody as bispecific anti-TREM2/Abeta protein antibody modulates neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid, and/or n) the antibody has non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and/or o) the antibody as bispecific anti-TREM2/Abeta protein antibody provides for plaque retention and plaque-targeted brain exposures, preferably with higher local concentrations as an anti-TREM2 monospecific antibody, and/or p) the antibody does not induce TNF alpha, MIP-1 alpha or IL-8 release from iPSC-derived macrophages, and/or q) the antibody blocks shedding of sTREM2, and/or r) the antibody stabilizes sTREM2 in biological fluids, and/or s) the antibody binds to the ECD of TREM2 and thereby results in the accumulation of sTREM2 in biological fluids, and/or t) the antibody as bispecific anti-TREM2/Abeta protein antibody induces the shift from homeostatic or less engaged towards activated microglia, and/or u) the antibody as bispecific anti-TREM2/Abeta protein antibody induces ARIA at a lower level than a monospecific antibody, preferably does not induce ARIA. An anti-TREM2 antibody binding to the same epitope as anti-TREM2 antibody TREM2 3295 or TREM2 3306. An anti-TREM2 antibody specifically binding to human TREM2 comprising a) in the heavy chain variable domain a HVR-H1 of SEQ ID NO: 305,

(DYAMS) a HVR-H2 of SEQ ID NO: 306 (IIGDSGDNTYYADSVKG) and a HVR-H3 of SEQ ID NO: 307 (YDIDV), and in the light chain variable domain a HVR-L1 of SEQ ID NO: 309 (RASQSISSYLN), a HVR-L2 of SEQ ID NO: 310 (AASDLQS) and a HVR-L3 of SEQ ID NO: 311 (QQANSFPPT) (TREM2 3295), or b) in the heavy chain variable domain a HVR-H1 of SEQ ID NO: 329 (SYAMN), a HVR-H2 of SEQ ID NO: 330 (TMSGSGGDTFYADSVKG) and a HVR-H3 of SEQ ID NO: 331 (EGGTVFDN), and in the light chain variable domain a HVR-L1 of SEQ ID NO: 333 (RASQDISNDLG), a HVR-L2 of SEQ ID NO: 334 (AASFLQS) and a HVR-L3 of SEQ ID NO: 335 (LQDYNLPFT) (TREM2 3306). The anti-TREM2 antibody according to any one of embodiments 2 to 3, wherein the antibody has one or more of the following properties: a) the antibody as bispecific anti-TREM2/Abeta protein antibody induces phagocytosis of amyloid plaques by macrophages, and/or b) the antibody as bispecific anti-TREM2/Abeta protein antibody induces microglial amyloid uptake in the absence of FcgRII as well as FcgRIII receptor engagement, and/or c) the antibody as bispecific anti-TREM2/Abeta protein antibody induces amyloid uptake in the absence of engagement of FcgR, and/or d) the antibody in Fc-effector function competent form induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, and/or e) the antibody as bispecific anti-TREM2/Abeta protein antibody without effector-function induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, preferably in the absence of Abeta and FcgR crosslinking, and/or f) the antibody induces migration of macrophages, preferably at a concentration of 0.14 to 34 nM in a migration assay as described herein, and/or g) the antibody increases LPC- and C5a stimulated cellular migration of THP-1 and iPSC-derived MO cells in the absence of any Abeta protein, and/or h) the antibody does not induce pSyk in the absence or presence of human Abeta protein, preferably the antibody is an antagonist of the Syk pathway, and/or i) the antibody as bispecific anti-TREM2/ Abeta protein antibody does induce pSyk in the presence of human Abeta protein, and/or j) the antibody does not show a dose dependent Syk and S6 phosphorylation in DAP12 and TREM2 expressing HEK cells, and/or k) the antibody does not induce pSyk in TREM2/DAP12 overexpressing HEK or iPSC macrophages in the absence of cross-linking, and/or l) the antibody as bispecific anti-TREM2/Abeta protein antibody shows after peripheral administration in vivo enrichment at plaques in APPswePS2 transgenic mice, and/or m) the antibody as bispecific anti-TREM2/Abeta protein antibody modulates neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid, and/or n) the antibody has non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and/or o) the antibody as bispecific anti-TREM2/Abeta protein antibody provides for plaque retention and plaque-targeted brain exposures, preferably with higher local concentrations as an anti-TREM2 monospecific antibody, and/or p) the antibody does not induce TNF alpha, MIP-1 alpha or IL-8 release from iPSC-derived macrophages, and/or q) the antibody blocks shedding of sTREM2, and/or r) the antibody stabilizes sTREM2 in biological fluids, and/or s) the antibody binds to the ECD of TREM2 and thereby results i the accumulation of sTREM2 in biological fluids, and/or t) the antibody as bispecific anti-TREM2/Abeta protein antibody induces the shift from homeostatic or less engaged towards activated microglia, and/or u) the antibody as bispecific anti-TREM2/Abeta protein antibody induces ARIA at a lower level than a monospecific antibody, preferably does not induce ARIA. The anti-TREM2 antibody according to any one of embodiments 2 to 4, wherein the antibody comprises a) a heavy chain variable domain of SEQ ID NO: 308 (VQLVESGGGLVQPGRSLRLSCAASGFTFGDYAMSWFRQAP GKGLEWVSIIGDSGDNTYYADSVKGRFAISRDNSKNTLYLQ MNSLRAEDTAVYYCMNYDIDVWGQGTTVTVSS) and a light chain variable domain of SEQ ID NO: 312 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKRLIYAASDLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATY YCQQANSFPPTFGGGTKVEIK), or b) a heavy chain variable domain of SEQ ID NO: 332 (VQLLESGGGLVQPGGSLRLSCVASGFIFNSYAMNWVRQAPG KGLEWVSTMSGSGGDTFYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAIYYCAKEGGTVFDNWGQGTLVTVSS) and a light chain variable domain of SEQ ID NO: 336 (AIQMTQSPSSLSTSVGDRVTITCRASQDISNDLGWYQQKPGK APKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCLQDYNLPFTFGPGTKVDFK). The anti-TREM2 antibody according to any one of embodiments 2 to 5, wherein the antibody binds to human TREM2 with an affinity of less than 1 nM. The anti-TREM2 antibody according to any one of embodiments 2 to 6, wherein the antibodies induce migration in vitro at a concentration of 5-15 nM. The anti-TREM2 antibody according to any one of embodiments 2 to 7, wherein the antibody is a bivalent, monospecific antibody. The anti-TREM2 antibody according to any one of embodiments 2 to 7, wherein the antibody is a multispecific antibody. The anti-TREM2 antibody according to embodiment 9, wherein the antibody is a bivalent, bispecific antibody, or a trivalent, bispecific antibody, or a trivalent, trispecific antibody, or a tetravalent, bispecific antibody. The anti-TREM2 antibody according to any one of embodiments 9 to 10, wherein the antibody comprises a first binding site binding to human TREM2 and at least one further binding site binding to a different epitope on human TREM2 than the first binding site, or binding to human A-beta protein, or binding to human transferrin receptor 1, or binding to human tau protein, or binding to human alpha-synuclein, or binding to human TDP-43, or binding to human huntingtin protein, or binding to human apolipoprotein E, or binding to delipidated human apolipoprotein E, or binding to human myelin basic protein. The anti-TREM2 antibody according to any one of embodiments 9 to 11, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising i) a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 305, a HVR-H2 of SEQ ID NO: 306 and a HVR-H3 of SEQ ID NO: 307, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 309, a HVR-L2 of SEQ ID NO: 310 and a HVR-L3 of SEQ ID NO: 311 (TREM2 3295), or ii) a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 329, a HVR-H2 of SEQ ID NO: 330 and a HVR-H3 of SEQ ID NO: 331, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 333, a HVR-L2 of SEQ ID NO: 334 and a HVR-L3 of SEQ ID NO: 335 (TREM2 3306), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 09, a HVR-H2 of SEQ ID NO: 10 and a HVR-H3 of SEQ ID NO: 11, and in the light chain variable domain a HVR-L1 of SEQ ID NO: 13, a HVR-L2 of SEQ ID NO: 14 and a HVR-L3 of SEQ ID NO: 15 (Abeta 8675). The anti-TREM2 antibody according to any one of embodiments 9 to 11, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising i) a heavy chain variable domain of SEQ ID NO: 308 and a light chain variable domain of SEQ ID NO: 312 (TREM2 3295), or ii) a heavy chain variable domain of SEQ ID NO: 332 and a light chain variable domain of SEQ ID NO: 336 (TREM2 3306), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain of SEQ ID NO: 12 (VELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVSAINATGTRTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYC ARGKGS SGYVRYFD VWGQGTL VTVS S) and in the light chain variable domain of SEQ ID NO: 16 (DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPG QAPRLLIYGAS SRATGVPARF SGSGSGTDFTLTIS SLEPEDF AT YYCLQIYNMPITFGQGTKVEIK) (Abeta 8675). The anti-TREM2 antibody according to any one of embodiments 9 to 11, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 305, a HVR-H2 of SEQ ID NO: 306 and a HVR-H3 of SEQ ID NO: 307, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 309, a HVR-L2 of SEQ ID NO: 310 and a HVR-L3 of SEQ ID NO: 311 (TREM2 3295), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 329, a HVR-H2 of SEQ ID NO: 330 and a HVR-H3 of SEQ ID NO: 331, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 333, a HVR-L2 of SEQ ID NO: 334 and a HVR-L3 of SEQ ID NO: 335 (TREM2 3306). 15. The anti-TREM2 antibody according to any one of embodiments 9 to 11, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising a heavy chain variable domain of SEQ ID NO: 308 and a light chain variable domain of SEQ ID NO: 312 (TREM2 3295), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain of SEQ ID NO: 332 and a light chain variable domain of SEQ ID NO: 336 (TREM2 3306).

16. The anti-TREM2 antibody according to any one of embodiments 9 to 11, wherein the antibody comprises a fist light chain of SEQ ID NO: 462 (AIQMTQSPSSLSTSVGDRVTITCRASQDISNDLGWYQQKPGKAPKLLI YAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNLPFT FGPGTKVDFKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC), a second light chain of SEQ ID NO: 463 (QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSAINATGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARGKGSSGYVRYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), a first heavy chain of SEQ ID NO: 464

(DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL LIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPI TFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG) and a second heavy chain of SEQ ID NO: 465 (EVQLLESGGGLVQPGGSLRLSCVASGFIFNSYAMNWVRQAPGKGLE WVSTMSGSGGDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAI YYCAKEGGTVFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGOL VEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG). The anti-TREM2 antibody according to any one of embodiments 2 to 16, wherein the antibody is mouse cross-reactive. The anti-TREM2 antibody according to any one of embodiments 2 to 16, wherein the antibody is not mouse cross-reactive. The anti-TREM2 antibody according to any one of embodiments 2 to 18, wherein the antibody is a) a full-length antibody of the human subclass IgGl, b) a full-length antibody of the human subclass IgG4, c) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A and P329G, d) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, e) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, f) a full-length antibody of the human subclass IgG4 with the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, g) a full-length antibody of the human subclass IgG4 with the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, h) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and H435A in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, i) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and H435A in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, j) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, k) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, or l) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A in both heavy chains and the mutations i) T366W, and ii) S354C or Y349C, in one heavy chain and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other heavy chain, or m) one of a) to 1) without the C-terminal lysine residue. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 19 and a pharmaceutically acceptable carrier. The anti-TREM2 antibody according to any one of embodiments 2 to 19 for use as a medicament. The anti-TREM2 antibody according to any one of embodiments 2 to 19 or the medicament according to embodiment 21 for use in the treatment of a disease. A method of treating a condition associated with TREM2 loss of function in a subject in need thereof, comprising administering an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 to the subject. A method of treating a condition associated with toxic gain of TREM2 function in a subject in need thereof, comprising administering an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 to the subject. A method of treating a condition associated with hyperactivated microglia in a subject in need thereof, comprising administering an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according to embodiment 20 to the subject. 26. A method of reducing levels of amyloid plaques in a subject in need thereof, comprising administering an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 to the subject.

27. A method of reducing slowing cognitive and functional decline in a subject in need thereof by targeted promotion and enrichment of beneficial and neuroprotective microglial functions at amyloid plaques or the brain vasculature covered by amyloid, comprising administering an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 to the subject.

28. A method of modulating neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid in a subject in need thereof, comprising administering an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 to the subject.

29. An antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 for use in treating a condition associated with TREM2 loss of function in a subject in need thereof.

30. An antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 for use in treating a condition associated with toxic gain of TREM2 function in a subject in need thereof.

31. An antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 for use in treating a condition associated with hyperactivated microglia in a subject in need thereof.

32. An antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 for use in reducing levels of amyloid plaques in a subject in need thereof. 33. An antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 in the preparation of a medicament for treating a condition in a subject in need thereof.

34. The use of an antibody according to any one of embodiments 2 to 19 or a pharmaceutical composition according embodiment 20 in the preparation of a medicament for reducing levels of amyloid plaques in a subject in need thereof.

35. The anti-TREM2 antibody or method or use according to any one of embodiments 21 to 34, wherein the disease or the condition is, or the subject suffers from, a neuroinflammatory or neurodegenerative disease.

36. The anti-TREM2 antibody or method or use according to embodiment 35, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hakola disease, Guillain-Barre Syndrome (GBS), lysosomal storage disease, sphingomyelinlipidosis (Neimann-Pick C), mucopolysaccharidosis II/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet’s disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury.

37. The anti-TREM2 antibody or method or use according to embodiment 36, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer’s disease.

38. The anti-TREM2 antibody or method or use according to any one of embodiments 36 to 37, wherein the neuroinflammatory or neurodegenerative disease is early Alzheimer’s disease.

39. The anti-TREM2 antibody or method or use according to any one of embodiments 36 to 38, wherein the subject is amyloid positive. 40. The anti-TREM2 antibody or method or use according to embodiment 35, wherein the neuroinflammatory or neurodegenerative disease is MS.

41. The anti-TREM2 antibody or method or use according to any one of embodiments 21 to 35, wherein the disease or the condition is, or the subject suffers from, Dementia with Lewy bodies.

42. The anti-TREM2 antibody or method or use according to any one of embodiments 21 to 35, wherein the disease or the condition is, or the subject suffers from, Parkinson’s disease with Dementia.

Additional objects and advantages will be set forth in part in the description that follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. For example, in addition to the various embodiments depicted and claimed herein, the subject matter of the current invention is also directed to other embodiments having other combinations of the features reported and claimed herein. As such, the particular features presented herein, especially presented as aspects or embodiments, can be combined with each other in other manners within the scope of the subject matter of the current invention such that the invention includes any suitable combination of the features reported herein. The description of specific embodiments of the current invention is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the current invention to those embodiments reported verbatim.

Detailed Description of the Invention

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention induce microglial uptake of Abeta coated beads in the absence of FcgR receptor engagement, e.g. in THP-1 cells, whereas no uptake is induced in THP-1 TREM2-knockout cells; the antibodies are binding to TREM2 on the THP-1 cells and Abeta coated on beads and induce subsequent potent uptake by the THP-1 cells; the antibodies induce migration in vitro at surprisingly low concentrations (5-15 nM) compared to molecules known from the art (34 nM).

The antibodies according to the current invention have an enhanced neuroprotective phenotype in microglia at plaques or brain vasculature covered by amyloid by modulating microglial activity via TREM2 and induce microglial amyloid uptake in the absence of FcgRII as well as FcgRIII receptor engagement.

The antibodies according to the current invention have sub-nM TREM2 binding, with sufficient affinity for Abeta to induce potent amyloid uptake in vitro and vivo.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention trigger a local neuroprotective phenotype in microglia at plaques or at brain vasculature covered by amyloid by activating TREM2 signaling in the absence of FcgR signaling which is different than it is proposed for antibodies from the prior art (i.e., antibodies from prior art act more globally on all microglia that express TREM2 independent of the presence of Abeta plaques or Abeta covering brain vasculature).

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention provide for plaque retention and thereby for locally higher and plaque- targeted brain exposures due to the presence of an Abeta binding site.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention have reduced effects on peripheral TREM2 positive cells due to absence of Abeta- and FcgR-mediated cross-linking.

Although less potent in vitro and with inferior plaque retention, the effector function incompetent 1+1 format showed in vivo potency (amyloid uptake by microglia) comparable to that of the 2+2 format, driven by a surprising superiority in brain uptake and lower peripheral clearance than the 2+2 format; only minor differences observed between TREM2 8008 (PGLALA reference version of antibody AL2p58 from the art) and TREM2 3306 regarding their target mediated drug disposition behavior in vitro. With respect to DMPK, TREM2 4524 (1+1 surrogate antibody) plaque decoration was higher than that of TREM2 0116 (2+2 format) (2 weeks); no obvious off-target binding in Retrogenix assay was identified.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention differ from anti-TREM2 antibodies known from the art by being a TREM2/Abeta protein bispecific antibody, by absence of FcgRII and FcgRIII binding, by microglia modulation and by being localized at the amyloid plaques.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention differ from anti-TREM2 antibodies known from the art as the antibodies according to the current invention do not induce pSyk in TREM2/DAP12 overexpressing HEK or iPSC MO cells in the absence of cross-linking (i.e. without Abeta beads); in TREM2/DAP12 overexpressing HEK cells a single high concentration of bispecific anti-TREM2/ Abeta protein antibodies (e.g. TREM23306 and TREM2 3306-based bispecific antibodies in 1+1 and 2+2 format) upregulate pSyk levels in the presence of Abeta coated beads or Abeta coated on the plate while this is absent without Abeta coated on beads or Abeta on the plate; pSyk upregulation is also absent without antibodies or in the presence of DP47/Abeta antibody or Abeta antibody; in contrast, anti-TREM2 antibodies known from the art upregulate pSyk with as well as without Abeta beads; without being bound by this theory it is assumed that the finding with the bispecific mAbs indicates a potential clustering effect of surface TREM2 due to opsonization of Abeta coated on beads or the plate by the bispecific anti-TREM2/Abeta protein antibodies.

The antibodies according to the current invention differ from anti-Abeta antibodies known from the art by induction of amyloid uptake in the absence of engagement of FcgR; potentially also uptake of cellular debris; neuroprotective effect.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention are the first amyloid plaque-targeted TREM2 agonist inducing a protective microglial phenotype; these antibodies have the potential to be used as replacement of or add-on to anti-amyloid antibodies in early-to-moderate AD and could also be used in combo-therapy with other amyloid lowering molecules or non-amyloid targeting therapies in Alzheimer’s disease.

It has been found that a 2+1 format was inferior in potency (i.e., uptake of Abeta coated beads) to 1+1 and 2+2 formats.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention can be used in slowing cognitive and functional decline in AD patients by targeted promotion and enrichment of beneficial and neuroprotective microglial functions at amyloid plaques or at brain vasculature covered by amyloid.

The anti-TREM2 antibodies according to the current invention can be used in combo with an anti -Ab eta antibody.

Thus, the current invention is based, at least in part on the findings that

- only anti-Abeta/TREM2 bispecific antibodies induce significant Abeta-beads uptake in an THP-1 Abeta-bead engagement and uptake assay (Example 3, Figures 8 and 9), whereas none of the anti-TREM2 monospecific antibodies does; this is independent of Fc-region isotype (wt or LALAPG);

- all monospecific anti-TREM2 antibodies according to the current invention of epitope region 2 and 3 (TREM2 3306, TREM2 3308, TREM2 3292, TREM2 3297, TREM2 3293, TREM2 3295, TREM2 0819), their Fc-effector function silent LALAPG variants and biparatopic formats containing TREM2 3306 and 3295, or TREM2 0885/2907 from the prior art, but not anti-Abeta monospecific mAbs induce migration in a THP-1 LPC-induced migration assay;

- anti-TREM2/Abeta protein bispecific antibodies according to the current invention with effector-function silent Fc-region (LALAPG) with TREM2 epitope region 2 (TREM2 3295 paratope) and 3 (TREM2 3306 paratope) and biparatopic formats containing TREM2 3306 increase migration similar or better than TREM2 0885/2907 from the prior art with effector-function silent Fc-region (LALAPG) in an human iPSC M0 C5a-induced migration assay; - in contrast to anti-TREM2 antibodies from the art (Alector, Denali, Cognyxx, WashU) neither the anti-TREM2 monoclonal antibody according to the current invention with effector-function competent Fc-region nor the anti- TREM2/Abeta protein bispecific antibodies with effector-function silent Fc- region (LALAPG) according to the current invention induced TNFalpha/MIP- lalpha/IL-8 release from iPSC-derived macrophages;

- none of the anti-TREM2/Abeta protein bi specific antibodies according to the current invention, nor the anti-TREM2 antibodies according to the current invention, nor Alector TREM2 0885 showed a dose dependent Syk and S6 phosphorylation in HEK/DAP12/TREM2 cells, whereas anti-TREM2 antibodies known from the art (Alector, Denali, Cognyxx, WashU, Amgen) induce pSyk or pS6 in the absence of Abeta beads or Abeta coating of the plate;

- the anti-TREM2/ Abeta protein bispecific antibodies according to the current invention do induce pSyk or pS6 in the presence of Abeta beads or Abeta coating of the plate, whereas the anti-TREM2 monoclonal antibodies according to the current invention didn’t induce pSyk or pS6 in the presence of Abeta beads or Abeta coating of the plate;

- the anti-TREM2 antibodies according to the current invention (TREM23306, TREM2 3295) bind epitopes in the ligand binding Ig-like domain of the ECD of TREM2; anti-TREM2 antibodies TREM2 0819, TREM2 3292 and TREM2 3295 according to the current invention have epitopes in the same region as TREM2 0885 from the prior art, whereas anti-TREM2 antibodies TREM2 3297 and TREM2 3306 have no overlap with any antibody known from the art; antibodies from the art (i.e., those from Denali and Alector) bind near the ADAM cleavage site (antibodies disclosed by Denali and Alector apparently bind the C-terminal sTREM2 part);

- the anti-TREM2 antibody TREM2 3306 is not mouse TREM2 cross-reactive (same for prior art antibodies from Denali and Alector), but mouse cross- reactive Fc-effector function competent anti-TREM2 antibody TREM2 3295 as well as TREM2 0885 from the prior art as well as the TREM2/ Abeta protein bispecific antibodies with effector-function silent Fc-region (LALAPG) according to the current invention induce acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 tg mice.

GENERAL DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used in connection with the current invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987). The content of which is incorporated herein by reference,

The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

The term “about” denotes a range of +/- 20 % of the following numerical value. In certain embodiments, the term about denotes a range of +/- 10 % of the thereafter following numerical value. In certain embodiments, the term about denotes a range of +/- 5 % of the thereafter following numerical value.

The term “activity” as used herein with respect to activity of a protein refers to any activity of a protein including, but not limited to, enzymatic activity, ligand binding, drug transport, ion transport, protein localization, receptor binding, and/or structural activity.

The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term “comprising” also encompasses the term “consisting of’. Herein are also contemplated other embodiments “comprising”, “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

The term “effective amount” herein refers to an amount that is sufficient to result in a desired outcome, such as treatment, inhibition, or reduction as described above.

As used herein, the term “exogenous” indicates that a nucleotide sequence or nucleic acid does not originate from a specific cell and is introduced into said cell by DNA delivery methods, e.g., by transfection, electroporation, or transformation methods. Thus, an exogenous nucleic acid is an artificial element wherein the artificiality can originate, e.g., from the combination of parts of nucleic acids of different origin (e.g. a combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence of green fluorescent protein is an artificial nucleic acid) or from the deletion of parts of a nucleic acid (e.g. a sequence coding only the extracellular domain of a membrane-bound receptor or a cDNA) or the mutation of nucleobases. The term “endogenous” refers to a nucleotide sequence originating from a cell. An “exogenous” nucleotide sequence can have an “endogenous” counterpart that is identical in base compositions, but where the “exogenous” sequence is introduced into the cell, e.g., via recombinant DNA technology.

The terms “expression” and “expresses” are used herein to refer to transcription and translation occurring within a cell. The level of expression of a nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the nucleic acid that is produced by the cell. For example, mRNA transcribed from a nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

As used herein, the term “heterologous” indicates that a polypeptide does not originate from a specific cell and the respective encoding nucleic acid has been introduced into said cell by DNA delivery methods, e.g., by transfection, electroporation, or transformation methods. Thus, a heterologous polypeptide is a polypeptide that is artificial to the cell expressing it, whereby this is independent whether the polypeptide is a naturally occurring polypeptide originating from a different cell/organism or is a synthetic polypeptide.

The term “integration site” denotes a location within a cell’s genome into which an exogenous nucleic acid is/can be/has been inserted. In certain embodiments, an integration site is inserted between two adjacent nucleotides in the cell’s genome. In certain embodiments, an integration site includes a stretch of nucleotides. In certain embodiments, the integration site is located within a specific locus of the genome of a mammalian cell. In certain embodiments, the integration site is within an endogenous gene of a mammalian cell.

An “isolated” nucleic acid refers to a nucleic acid that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid contained in cells that ordinarily contain the nucleic acid, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

An “isolated nucleic acid encoding an antibody” refers to one or more nucleic acids encoding the heavy and light chains (or fragments thereof) of the antibody according to the invention. Such nucleic acid(s) include those in a single vector or separate vectors, and such nucleic acid(s) present at one or more locations in a host cell.

The terms “(mammalian) cell” and “(mammalian) cell line” are used interchangeably herein refer to cells into which an exogenous nucleic acid(s) has been introduced, including the progeny of such cells.

A “mammalian cell comprising an exogenous nucleotide sequence” and a “recombinant mammalian cell” are both "transformed cells". This term includes the primary transformed cell as well as progeny derived therefrom without regard to the number of passages. Progeny may, e.g., not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are encompassed.

The term “nucleic acid” or “polynucleotide” includes any molecule and/or compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, a nucleic acid is described by its sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid. The sequence of bases is typically represented in 5’- to 3 ’-direction, i.e. from the 5 ’-end to the 3 ’-end. Herein, the term nucleic acid encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA as well as synthetic forms of DNA. The nucleic acid may be linear or circular. In addition, the term nucleic acid includes both sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA molecules which are suitable as a vector for direct expression of an antibody according to the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) vectors can be unmodified or modified.

As used herein, the term “operably linked” refers to a juxtaposition of two or more components, wherein the components are in a relationship permitting them to function in their intended manner. For example, a promoter and/or an enhancer is operably linked to a coding sequence if the promoter and/or enhancer acts to modulate the transcription of the coding sequence. In certain embodiments, nucleic acid sequences that are “operably linked” are contiguous and adjacent on a single chromosome. In certain embodiments, e.g., when it is necessary to join two protein encoding regions, such as a secretory leader and a polypeptide, the sequences are contiguous, adjacent, and in the same reading frame. In certain embodiments, an operably linked promoter is located upstream of the coding sequence and can be adjacent to it. In certain embodiments, e.g., with respect to enhancer sequences modulating the expression of a coding sequence, the two components can be operably linked although not adjacent. An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. Operably linked enhancers can be located upstream, within, or downstream of coding sequences and can be located at a considerable distance from the promoter of the coding sequence. Operable linkage can be accomplished by recombinant methods known in the art, e.g., using PCR methodology and/or by ligation at convenient restriction sites. If convenient restriction sites do not exist, then synthetic oligonucleotide adaptors or linkers can be used in accord with conventional practice. An internal ribosomal entry site (IRES) is operably linked to an open reading frame (ORF) if it allows initiation of translation of the ORF at an internal location in a 5’- end-independent manner.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference 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 for the purposes of the alignment. 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, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction. The term "recombinant mammalian cell” as used herein denotes a mammalian cell comprising an exogenous nucleotide sequence capable of expressing a polypeptide. Such recombinant mammalian cells are cells into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such cells. Thus, the term “a mammalian cell comprising a nucleic acid encoding an antibody” denotes cells comprising an exogenous nucleic acid integrated in the genome of the mammalian cell and capable of expressing the antibody. In certain embodiments, the mammalian cell comprising an exogenous nucleic acid is a cell comprising an exogenous nucleic acid integrated at a single site within a locus of the genome of the mammalian cell, wherein the exogenous nucleic acid comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and all the recombination recognition sequences are different. The integration has been effected in this case by a recombinase mediated cassette exchange (RMCE).

As used herein, the term “selection marker” denotes a nucleic acid that allows cells carrying the nucleic acid to be specifically selected for or against, in the presence of a corresponding selection agent. For example, but not by way of limitation, a selection marker can allow the mammalian cell transformed with the selection marker nucleic acid to be positively selected for in the presence of the respective selection agent (selective cultivation conditions); a non-transformed mammalian cell would not be capable of growing or surviving under the selective cultivation conditions. Selection markers can be positive, negative or bi-functional. Positive selection markers can allow selection for cells carrying the marker, whereas negative selection markers can allow cells carrying the marker to be selectively eliminated. A selection marker can confer resistance to a drug or compensate for a metabolic or catabolic defect in the host cell. Resistance genes useful as selection markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. Further marker genes are described in WO 92/08796 and WO 94/28143. Beyond facilitating a selection in the presence of a corresponding selection agent, a selection marker can alternatively encode a molecule normally not present in the cell, e.g., green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells expressing such a molecule can be distinguished from cells not harboring this nucleic acid, e.g., by the detection or absence, respectively, of the fluorescence emitted by the encoded polypeptide.

The term “shedding” as used herein refers to the process of creating soluble TREM2 from membrane-bound TREM2 by proteolytic cleavage of the stalk region of the protein. In vivo, “shedding” may occur at the cell surface, for example, due to cleavage of TREM2 on the cell membrane by a metalloproteinase or other enzyme. In some cases, cleavage occurs between Hl 57 and SI 58 (consecutive numbering; SEQ ID NO: 424) of the protein, forming sTREM2 comprising the ectodomain residues 19-157 of SEQ ID NO: 424.

The term “signal sequence” or “leader sequence” refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Non-limiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, an antibody lacks a leader sequence. In some embodiments, an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.

The terms “subject” and “patient” are used interchangeably herein to refer to a human. In some embodiments, methods of treating other mammals, including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided.

The term “treatment,” as used herein, covers any administration or application of a therapeutic for disease in a human, or other mammal, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting or slowing its development, inhibiting, reducing, or slowing development of at least one symptom of the disease, slowing the time to onset of the disease, preventing onset of at least one disease symptom, slowing the time to onset of at least one disease symptom, partially or fully relieving the disease, or curing the disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. The terms “inhibition” or “inhibit” refer to a decrease or cessation of any symptom or phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that symptom or characteristic.

The term “triggering receptor expressed on myeloid cells-2” and its abbreviation “TREM2”, as used herein, refers to a human TREM2 protein (“hTREM2”), unless expressly noted otherwise (i.e., murine TREM2, or cynomolgus TREM2, or the like). An exemplary hTREM2 amino acid sequence, including the signal sequence of amino acids 1-18, is shown in SEQ ID NO: 424, while an exemplary sequence without the signal sequence is shown in SEQ ID NO: 425. TREM2 including the signal sequence may also be referred to as the “precursor” or “preprotein” form of the protein, while TREM2 without the signal sequence may be referred to as the “mature” form of the protein. The membrane-bound form of the protein includes a V-type immunoglobulin (Ig) domain (at amino acids 19-128) followed by a TREM2 “stalk” domain (at amino acids 129-174; SEQ ID NO: 424), which collectively form the “extracellular domain” of the protein, followed by a transmembrane domain (residues 175-197), and a cytosolic domain (residues 198-230). A soluble form of the protein, “soluble TREM2” or “sTREM2” herein, may be generated in the body either by cleavage in the stalk domain or by alternative splicing. For example, proteases may cleave the protein between amino acid residues H157 and S158 (consecutive numbering; SEQ ID NO: 424), resulting in soluble TREM2 comprising the N-terminal portion of the protein up to the H157, e.g., residues 19-157. The portion of the mature TREM2 protein that may be cleaved off to form sTREM2 is also known as the “ectodomain”, and comprises residues 19-157 of SEQ ID NO: 424. In general herein, the term “TREM2” as used herein refers to the mature form of the protein. In addition, hTREM2 comprises several isoforms or alleles whose native sequences or splicing may differ from that shown in SEQ ID NO: 424. The term TREM2 encompasses all of these native forms of TREM2 unless a particular isoform or sequence is referred to.

The term “vector”, as used herein, refers to a nucleic acid capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a mammalian cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

RECOMBINANT PRODUCTION METHODS

Antibodies may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods, one or more isolated nucleic acid(s) encoding an antibody are provided.

In one aspect of the current invention, a method of producing a multispecific antibody is provided, wherein the method comprises culturing a recombinant mammalian cell comprising one or more nucleic acid(s) encoding an antibody according to the current invention, under conditions suitable for expression of the antibody, recovering the antibody from the cell (or cell culture medium), and optionally purifying the multispecific antibody with one or more chromatography steps, thereby producing the multispecific antibody..

Expression vectors

For recombinant production of an antibody, nucleic acids encoding the antibody are generated or isolated and inserted into one or more vectors for further cloning and/or expression in a mammalian cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to nucleic acids encoding the heavy and light chains of the antibody) or produced by recombinant methods or obtained by chemical synthesis.

Generally, for the recombinant large-scale production of, e.g., a therapeutic antibody, a mammalian cell stably expressing and secreting said antibody is required. This cell is termed “recombinant cell” or “recombinant production cell” and the process used for generating such a cell is termed “cell line development”.

In the first step of the cell line development process, a suitable mammalian cell, such as, e.g., a CHO cell, is transfected with a nucleic acid suitable for expression of said antibody. In a second step, a cell stably expressing the antibody is selected based on the co-expression of a selection marker, which had been co-transfected with the nucleic acid encoding the antibody.

A nucleic acid encoding the amino acid sequence of a chain of an antibody, i.e. the coding nucleic acid, is pure information and additional regulatory elements are required for expression thereof. Therefore, normally a coding nucleic acid is integrated in a so-called expression cassette. The minimal regulatory elements needed for an expression cassette to be functional in a mammalian cell are a promoter functional in said mammalian cell, which is located upstream, i.e., 5’, to the coding nucleic acid, and a polyadenylation signal sequence functional in said mammalian cell, which is located downstream, i.e., 3’, to the coding nucleic acid. The promoter, the coding nucleic acid and the polyadenylation signal sequence are arranged in an operably linked form.

In case the polypeptide of interest is a heteromultimeric protein that is composed of different (monomeric) polypeptides, such as an antibody, not only a single expression cassette is required but a multitude of expression cassettes differing from each other at least in the contained coding nucleic acid are required, i.e., at least one expression cassette for each of the different (monomeric) polypeptides of the heteromultimeric protein. For example, a full-length monospecific antibody is a heteromultimeric protein comprising two copies of a light chain as well as two copies of a heavy chain. Thus, a full-length antibody is a tetramultimer composed of two different polypeptides. Therefore, at least two expression cassettes are required for the expression of a full- length antibody, one expression cassette for the light chain and one expression cassette for the heavy chain.

If, for example, the full-length antibody is a bispecific antibody, i.e., the antibody comprises two different binding sites specifically binding to two different antigens, the two light chains as well as the two heavy chains are also different from each other. Thus, such a bispecific, full-length antibody is composed of four different polypeptides and, therefore, four expression cassettes are required.

The expression cassettes for the antibody are in turn integrated into one or more so called “expression vector(s)”. An “expression vector" is a nucleic acid providing all required elements for the amplification of said vector in prokaryotic cells as well as those required for the expression of the comprised coding nucleic acids(s) in a mammalian cell. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, e.g. for E.coli, comprising an origin of replication, and a prokaryotic selection marker, as well as a eukaryotic selection marker, and further the expression cassettes required for the expression of the coding nucleic acid(s) of the antibody. An “expression vector” is a transport vehicle for the introduction of expression cassettes into a mammalian cell to produce a recombinant mammalian cell.

As outlined in the previous paragraphs, the more complex the antibody is, the higher is also the number of required different expression cassettes. Inherently with the number of expression cassettes also the total size, i.e., the number of base pairs (bps), of the nucleic acid to be integrated into the genome of the host cell increases. Concomitantly also the size of the expression vector increases. However, there is a practical upper limit to the size of a vector in the range of about 15 kbps above which handling and processing efficiency profoundly drops. This issue can be addressed by using two or more expression vectors. Thereby the expression cassettes are split between different expression vectors each comprising only some of the expression cassettes resulting in a reduction of the size (number of bps) of the individual vectors.

Cell Line Development

Cell line development (CLD) for the generation of recombinant mammalian cells expressing an antibody employs either random integration (RI) or targeted integration (TI) of the nucleic acid(s) comprising the respective expression cassettes required for the expression and production of the antibody.

Using RI, in general, several vectors or fragments thereof integrate into the cell’s genome at the same or different loci.

Using TI, in general, a single copy of the transgene comprising the different expression cassettes is integrated at a predetermined “hot-spot” in the host cell’s genome.

Targeted Integration

Targeted integration (TI) allows exogenous nucleic acids to be integrated into a predetermined site of a mammalian cell’s genome.

In certain embodiments, a TI mammalian cell is used in the methods according to the current invention for the introduction of an exogenous nucleic acid encoding an antibody. Employing such a recombinant TI mammalian cell will provide for robust, stable cell culture performance and lower risk of sequence variants in the resulting recombinantly produced antibody. TI cells and strategies for the use of the same are described in detail in, e.g., US 2021/0002669, the contents of which are incorporated by reference in their entirety.

In certain embodiments employing targeted integration, the exogenous nucleic acid is integrated at a site within a specific locus of the genome of a TI cell.

In targeted integration, site-specific recombination is employed for the introduction of an exogenous nucleic acid into a specific locus in the genome of a mammalian TI cell. This is an enzymatic process wherein a sequence at the site of integration in the genome is exchanged for the exogenous nucleic acid. One system used to effect such nucleic acid exchanges is the Cre-lox system. The enzyme catalyzing the exchange is the Cre recombinase. The sequence to be exchanged is defined by the position of two lox(P)-sites in the genome as well as in the exogenous nucleic acid. These lox(P)-sites are recognized by the Cre recombinase. Nothing more is required, i.e., no ATP etc. Originally, the Cre-lox system has been found in bacteriophage Pl.

In certain embodiments of all aspects and embodiments of the current invention, the cell used in the methods according to the current invention has been subjected to targeted integration of nucleic acid(s) encoding an antibody.

In certain embodiments of all aspects and embodiments of the current invention, the targeted integration is mediated by a recombinase that recognizes one or more recombination recognition sequences (RRSs), which are present in the genome of the mammalian cell and in the exogenous nucleotide sequence to be integrated into the genome of the mammalian cell.

In certain embodiments of all aspects and embodiments of the current invention, the targeted integration is mediated by homologous recombination.

A “recombination recognition sequence” (RRS) is a nucleotide sequence recognized by a recombinase and is necessary and sufficient for recombinase-mediated recombination events. A RRS can be used to define the position where a recombination event will occur in a nucleotide sequence.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is recognized by a Cre recombinase.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is a LoxP site and a Cre recombinase mediates the targeted integration by recombination.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is recognized by a FLP recombinase. In certain embodiments of all aspects and embodiments of the current invention, the RRS is a FRT site and a FLP recombinase mediates the targeted integration by recombination.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is recognized by a Bxbl integrase.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is a Bxbl attP or a Bxbl attB site and a Bxbl integrase mediates the targeted integration by recombination.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is recognized by a cpC31 integrase.

In certain embodiments of all aspects and embodiments of the current invention, the RRS is a (pC31 attP or a cpC31 attB site and the cpC31 integrase mediates the targeted integration by recombination.

The recombinases can be introduced into a cell using an expression vector comprising coding sequences of the enzymes or as protein or an mRNA.

With respect to TI, any known or future mammalian cell comprising a landing site as described herein integrated at a single site within a locus of the genome can be used in the methods according to the current invention. Such a cell is denoted as a mammalian TI cell.

In certain embodiments of all aspects and embodiments of the current invention, the mammalian TI host cell is a hamster cell, a human cell, a rat cell, or a mouse cell comprising a landing site as described herein. In one preferred embodiment, the mammalian TI cell is a CHO cell. In certain embodiments, the mammalian TI CHO cell is a CHO KI cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell, or a CHO KIM cell comprising a landing site as described herein integrated at a single site within a locus of the genome.

In certain embodiments of all aspects and embodiments of the current invention, a mammalian TI cell comprises an integrated landing site, wherein the landing site comprises one or more recombination recognition sequence (RRS). The RRS can be recognized by a recombinase, for example, a Cre recombinase, an FLP recombinase, a Bxbl integrase, or a cpC31 integrase. The one or more RRS can be selected independently of each other from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, aLox71 sequence, aLox66 sequence, aFRT sequence, a Bxbl attP sequence, a Bxbl attB sequence, a q>C31 attP sequence, and a q>C31 attB sequence. If multiple RRSs have to be present, the selection of each of the sequences is dependent on the other insofar as non-identical RRSs are chosen.

In certain embodiments of all aspects and embodiments of the current invention, the landing site comprises one or more recombination recognition sequence (RRS), wherein the RRS can be recognized by a recombinase. In certain embodiments, the integrated landing site comprises at least two RRSs. In certain embodiments, an integrated landing site comprises three RRSs, wherein the third RRS is located between the first and the second RRS. In certain preferred embodiments, all three RRSs are different. In certain embodiments, the landing site comprises a first, a second and a third RRS, and at least one selection marker located between the first and the second RRS, and the third RRS is different from the first and/or the second RRS. In certain embodiments, the landing site further comprises a second selection marker, and the first and the second selection markers are different. In certain embodiments, the landing site further comprises a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker. The third selection marker can be different from the first or the second selection marker.

An exemplary mammalian TI cell that is suitable for use in a method according to the current invention is a CHO cell harboring a landing site integrated at a single site within a locus of its genome wherein the landing site comprises three heterospecific loxP sites for Cre recombinase mediated DNA recombination. In certain embodiments, the heterospecific loxP sites are L3 (SEQ ID NO: 426), LoxFas (SEQ ID NO: 427) and 2L (SEQ ID NO: 428) (see e.g., Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) el47), whereby L3 and 2L flank the landing site at the 5 ’-end and 3 ’-end, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further contains a bicistronic unit linking the expression of a selection marker via an IRES to the expression of the fluorescent GFP protein allowing to stabilize the landing site by positive selection as well as to select for the absence of the site after transfection and Cre-recombination (negative selection). Green fluorescence protein (GFP) serves for monitoring the RMCE reaction.

Such a configuration of the landing site as outlined in the previous paragraph allows for the simultaneous integration of two vectors, e.g., of a so called front vector harboring an L3 and a LoxFas site and a back vector harboring a LoxFas and an 2L site. The functional elements of a selection marker gene different from that present in the landing site can be distributed between both vectors: promoter and start codon can be located on the front vector whereas coding region and poly A signal are located on the back vector. Only correct recombinase-mediated integration of said nucleic acids from both vectors induces resistance against the respective selection agent.

Generally, a mammalian TI cell is a mammalian cell comprising a landing site integrated at a single site within a locus of the genome of the mammalian cell, wherein the landing site comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and all the recombination recognition sequences are different.

The selection marker(s) can be selected from the group consisting of an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. The selection marker(s) can also be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced GFP (eGFP), a synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

An exogenous nucleic acid is a nucleic acid that does not originate from a specific cell but can be introduced into said cell by DNA delivery methods, such as, e.g., by transfection, electroporation, or transformation methods. In certain embodiments, a mammalian TI cell comprises at least one landing site integrated at one or more integration sites in the mammalian cell’s genome. In certain embodiments, the landing site is integrated at one or more integration sites within a specific locus of the genome of the mammalian cell.

In certain embodiments of all aspects and embodiments according to the current invention, the integrated landing site comprises at least one selection marker. In certain embodiments, the integrated landing site comprises a first, a second and a third RRS, and at least one selection marker. In certain embodiments, a selection marker is located between the first and the second RRS. In certain embodiments, two RRSs flank at least one selection marker, i.e., a first RRS is located 5’ (upstream) and a second RRS is located 3’ (downstream) of the selection marker. In certain embodiments, a first RRS is adjacent to the 5’-end of the selection marker and a second RRS is adjacent to the 3’-end of the selection marker. In certain embodiments, the landing site comprises a first, second, and third RRS, and at least one selection marker located between the first and the third RRS.

In certain embodiments of all aspects and embodiments according to the current invention, in the integrated landing site a selection marker is located between a first and a second RRS and the two flanking RRSs are different. In certain preferred embodiments, the first flanking RRS is a LoxP L3 sequence and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, a LoxP L3 sequence is located 5’ of the selection marker and a LoxP 2L sequence is located 3’ of the selection marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is a Bxbl attP sequence and the second flanking RRS is a Bxbl attB sequence.

In certain embodiments, the first flanking RRS is a cpC31 attP sequence and the second flanking RRS is a cpC31 attB sequence. In certain embodiments, the two RRSs are positioned in the same orientation. In certain embodiments, the two RRSs are both in the forward or reverse orientation. In certain embodiments, the two RRSs are positioned in opposite orientations.

In certain embodiments of all aspects and embodiments according to the current invention, the integrated landing site comprises a first and a second selection marker, which are flanked by two RRSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, the two selection markers are both independently of each other selected from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated landing site comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection maker is selected from the group consisting of an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid, and the second selection maker is selected from the group consisting of a GFP, an eGFP, a synthetic GFP, a YFP, an eYFP, a CFP, an mPlum, an mCherry, a tdTomato, an mStrawberry, a J-red, a DsRed-monomer, an mOrange, an mKO, an mCitrine, a Venus, a YPet, an Emerald, a CyPet, an mCFPm, a Cerulean, and a T-Sapphire fluorescent protein. In certain embodiments, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein. In certain embodiments, the two RRSs flanking both selection markers are different.

In certain embodiments, the selection marker is operably linked to a promoter sequence. In certain embodiments, the selection marker is operably linked to an SV40 promoter. In certain embodiments, the selection marker is operably linked to a human cytomegalovirus (CMV) promoter.

In certain embodiments of all aspects and embodiments according to the current invention, the exogenous nucleic acid(s) encoding an antibody has been integrated into the mammalian TI cell by single or double recombinase mediated cassette exchange (RMCE). Thereby a recombinant mammalian cell, such as a recombinant CHO cell, is obtained, in which a defined and specific expression cassette sequence has been integrated into the genome at a single locus, which in turn results in the efficient expression and production of the antibody.

The Cre-LoxP site-specific recombination system has been widely used in many biological experimental systems. Cre recombinase is a 38-kDa site-specific DNA recombinase that recognizes 34 bp LoxP sequences. Cre recombinase is derived from bacteriophage Pl and belongs to the tyrosine family site-specific recombinase. Cre recombinase can mediate both intra- and intermolecular recombination between LoxP sequences. The LoxP sequence is composed of an 8 bp non-palindromic core region flanked by two 13 bp inverted repeats. Cre recombinase binds to the 13 bp repeat thereby mediating recombination within the 8 bp core region. Cre-LoxP- mediated recombination occurs at a high efficiency and does not require any other host factors. If two LoxP sequences are placed in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise DNA sequences located between the two LoxP sequences as a covalently closed circle. If two LoxP sequences are placed in an inverted position on the same nucleotide sequence, Cre recombinase-mediated recombination will invert the orientation of the DNA sequences located between the two sequences. If two LoxP sequences are on two different DNA molecules and if one DNA molecule is circular, Cre recombinase- mediated recombination will result in integration of the circular DNA sequence. The term “matching RRSs” indicates that a recombination occurs between two RRSs. In certain embodiments, the two matching RRSs are the same. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wildtype FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxbl attP sequence and the second matching RRS is a Bxbl attB sequence. In certain embodiments, the first matching RRS is a cpC31 attB sequence and the second matching RRS is a cpC31 attB sequence.

A “two-plasmid RMCE” strategy or “double RMCE” is employed in the method according to the current invention when using a two-vector combination. For example, but not by way of limitation, an integrated landing site could comprise three RRSs, e.g., an arrangement where the third RRS (“RRS3”) is present between the first RRS (“RRS1”) and the second RRS (“RRS2”), while a first vector comprises two RRSs matching the first and the third RRS on the integrated exogenous nucleotide sequence, and a second vector comprises two RRSs matching the third and the second RRS on the integrated exogenous nucleotide sequence.

The two-plasmid RMCE strategy involves using three RRS sites to carry out two independent RMCEs. Therefore, a landing site in the mammalian TI cell using the two-plasmid RMCE strategy includes a third RRS site (RRS3) that has no cross activity with either the first RRS site (RRS1) or the second RRS site (RRS2). The two plasmids to be targeted require the same flanking RRS sites for efficient targeting, one plasmid (front) flanked by RRS1 and RRS3 and the other (back) by RRS3 and RRS2. In addition, two selection markers are needed in the two-plasmid RMCE. One selection marker expression cassette was split into two parts. The front plasmid would contain the promoter followed by a start codon and the RRS3 sequence. The back plasmid would have the RRS3 sequence fused to the N-terminus of the selection marker coding region, minus the start-codon (ATG). Additional nucleotides may need to be inserted between the RRS3 site and the selection marker sequence to ensure in-frame translation for the fusion protein, i.e., operable linkage. Only when both plasmids are correctly inserted, the full expression cassette of the selection marker will be assembled and, thus, rendering cells resistant to the respective selection agent.

Two-plasmid RMCE involves double recombination crossover events, catalyzed by a recombinase, between the two heterospecific RRSs within the target genomic locus and the donor DNA molecule. Two-plasmid RMCE is designed to introduce a copy of the DNA sequences from the front- and back-vector in combination into the predetermined locus of a mammalian TI cell’s genome. RMCE can be implemented such that prokaryotic vector sequences are not introduced into the mammalian TI cell’s genome, thus, reducing and/or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

In certain embodiments of all aspects and embodiments according to the current invention, targeted integration is achieved by two RMCEs, wherein two different DNA sequences, each comprising at least one expression cassette encoding a part of a multispecific antibody according to the current invention and/or at least one selection marker or part thereof flanked by two heterospecific RRSs, are both integrated into a predetermined site of the genome of a RRSs matching mammalian TI cell. In certain embodiments, targeted integration is achieved by multiple RMCEs, wherein DNA sequences from multiple vectors, each comprising at least one expression cassette encoding a part of a heteromultimeric polypeptide and/or at least one selection marker or part thereof flanked by two heterospecific RRSs, are all integrated into a predetermined site of the genome of a mammalian TI cell. In certain embodiments the selection marker can be partially encoded on the first the vector and partially encoded on the second vector such that only the correct integration of both by double RMCE allows for the expression of the selection marker.

Cell Lines

Suitable mammalian cells for the expression of an antibody are generally derived from multicellular organisms such as, e.g., vertebrates. Examples of mammalian cells that can be used in the methods of the current invention are human amniocyte cells (e.g., CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) P133); monkey kidney cells (CV1); monkey kidney CV1 cells transformed by SV40 (COS-7); human embryonic kidney cells (HEK293 or HEK293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59- 74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells.

Especially useful mammalian cells to be used in the methods according to the current invention include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220) and CHO KI cells; as well as human embryonic kidney cells (HEK293 cells). For a review of certain mammalian cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

In certain embodiments of all aspects and embodiments of the current invention, the mammalian cell used in a method or use according to the current invention is a Chinese Hamster Ovary (CHO) cell (e.g. CHO KI, CHO DG44, etc.) or a Human Embryonic Kidney (HEK) cell.

ANTIBODIES ACCORDING TO THE CURRENT INVENTION AND THEIR PROPERTIES

The anti-TREM2 antibodies according to the current invention

It has been found that the anti-TREM2 antibody according to the current invention provides for advantageous properties, which are, amongst others, the induction of microglia migration The anti-TREM2 antibodies according to the current invention have sub-nM (double digit pM) to low nM affinity to human TREM2.

The anti-Abeta antibody according to the current invention

It has been found that the anti-Abeta antibody according to the current invention provides for advantageous properties, which are, amongst others, a more homogeneous and fully glycosylation in the FAB, improved cynomolgus pharmacokinetic properties and improved stability.

The bispecific anti-TREM2/Abeta protein antibodies according to the current invention

It has been found that the format of the antibody according to the current invention provides for advantageous properties, which are, amongst others, the induction of microglia migration, efficient in vitro and in vivo amyloid plaque decoration, in vivo target engagement and amyloid plaque uptake as shown on basis of Abeta beads phagocytosis potency and in vivo in an amyloid uptake paradigm as analyzed ex vivo by flow cytometry.

The TREM2 paratopes have 2-digit pM to low nM affinity to human TREM2.

The Abeta binding is comparable to gantenerumab in the 2+2 format.

The antibodies show overall good stability under shelf-life conditions.

Especially, the antibodies have good concentratability/viscosity results of 131-183 mg/ml at 20 °C to reach 20 cP.

In two independent studies in aged APPswePS2 transgenic mice it has been found that the antibodies in 1+1 format and 2+2 format have similar potency to increase the number of MX04 loaded microglia. The absence of such an increase in the DP47/Abeta group indicates a TREM2-mediated engagement of microglia on amyloid plaques consequently leading to uptake of amyloid into microglia.

It has been found that decoration of plaques by antibodies in 1+1 format vs. the 2+2 format correlates with brain exposure of the molecules. When normalized to plaque load decoration the 1+1 format shows 2-3 -times more decoration than the 2+2 format over 336 hours. However, the 2+2 format shows continuous but low overall decoration over 504 hours.

The invention is based, at least in part, on the finding that the predominant effect of TREM2/Abeta protein bispecific antibodies according to the current invention is a shift from homeostatic or “less engaged” towards “activated” microglia (i.e., more plaque engagement based on increase in MX04 positive microglia), whereas both populations show very similar gene expression profiles in both antibody and vehicle treated animals. Thus, without being bound by this theory, it is assumed that the treatment does not induce any detrimental effects (e.g. cellular stress).

Thus, it has been found that the antibodies according to the current invention do not induce release of detrimental soluble factors in iPSC-derived MO macrophages. The antibodies

Antibodies against human TREM2 (SEQ ID NO: 424) have been generated by immunizing wild-type rabbits, human IgG locus transgenic rabbits (tg rabbit), human IgG locus transgenic rats (tg rat) and by phage display. The resulting antibodies have been characterized and tested in different formats with different paratope combinations. The respective monospecific, biparatopic and bispecific antibodies are listed in the following table. A sketch of the respective formats is shown in Figure 1.

The antibodies generated by immunization have been characterized with respect to their activity in two cellular migration assays, a phagocytosis assay, cytokine release assay, pSyk activation assay, epitope binding, human and murine TREM2 monomer binding affinity, human and cynomolgus TREM2 dimer binding affinity and IHC staining of murine TREM2 in murine brain samples.

Epitope

The epitopes of different anti-TREM2 antibodies are shown in the sequence of human TREM2 (SEQ ID NO: 424) in the following alignment. s: signal peptide/sequence 1 : TREM2 0885 2: TREM2 0886

3: TREM2 3306 (conformational epitope) 4: TREM2 3297 (conformational epitope) 5: TREM2 2903, 3247-3259, 3164, 3263, 3264 6: TREM2 2900

7: TREM2 2904 8: cleavage site soluble TREM2 9: TREM2 0705 (conformational epitope) A: TREM2 0702 B: TREM2 8010 C: TREM2 3295

MEPLRLLILLFVTELSGAHNT TVFQGVAGQS LQVS CP YDSMKHWGRRKAWCRQLG ssssssssssssssssss 11111111

2222222 99999999 ccccc

EKGPCQRWSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQC

22 33 3333333

444 4 4

999 999 QSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHS ISRSLLE 5555555555

66666666666

77777777777

8 AAAAAAAAAA BBBBBBB

GEIPFPPTS ILLLLACI FLIKILAASALWAAAWHGQKPGTHPPSELDCGHDPGYQ

LQTLPGLRDT

As can be seen the epitopes of the antibodies according to the current invention are different from the epitopes of anti-TREM2 antibodies known from the art. This can further be seen by the properties of the anti-TREM2 antibodies according to the current invention, which are different from those known from the art. Binding to the same epitope would result in the same properties.

Non-specific binding

No non-specific binding, i.e. cell membrane interaction, was observed (ARC score at pH 7.4; the lower the better).

All antibodies according to the current invention showed an FcRn-recy cling efficiency in the range between AAA (I253A/H310A/H435A mutation; non-binder IgG) and wild-type IgG. FcgRIA is the only FcgR that shows high enough affinity to contribute to internalization of monomeric, non-complexed IgGs, which is not relevant for LALAPG variants.

The results are shown in the following Tables.

It can be seen that TREM2 2272 performs best in the ARC assay.

Pharmacokinetic

Pharmacokinetic (PK) parameters were determined in human FcRn transgenic wild- type mice and human FcRn transgenic SCID mice.

The results are shown in the following Tables and Figure 2.

The PK parameters in SCID mice are similar to those in immunocompetent mice for TREM2 0116 and TREM2 4483.

TREM2 1094 has high clearance similar to TREM2 0116.

Abeta 8655 has a higher clearance than TREM2 4483 and TREM2 3306. TREM2 4483 or its YTE variant TREM2 2272 are the best from solely a PK perspective.

Plaque decoration study in APPswePS2 tg mice

A single dose study APPswePS2 mice with termination on day 8, day 15 and day 22 (168 h, 336 h, 504 h) after application, respectively, and serial blood sampling, bioanalytics of serum and staining of antibodies in brain tissue was performed. The blood tracer DP47 with cynomolgus Fc-region (DP47 5213) was administered 15 minutes prior to sacrifice of the animals to assess potential blood contamination in the brain. See WO 2021/136772 for further details.

Serum PK was measured at 0.5, 7, 24, 72 and 168 hours post dosing. CSF levels and brain exposure was measured at termination on days 8, 15 and 22.

The study outline is shown in the following Table.

In APPswePS2 mice TREM2 4524 has the best PK.

Brain exposure was the highest in animals treated with TREM2 4524 and declined over time.

DMPK analysis shows that 1+1 formats have a 2.7-fold higher brain uptake rate than 2+2 formats.

The results are shown in Figures 3 and 4.

Developability

All antibodies could be produced with good yield.

Thermal stability was analyzed by storage at 40 °C for 2 weeks (pH 6, His/NaCl buffer, 1 mg/mL; denaturing conditions) and 37 °C for 4 weeks (pH 7.4, PBS, 10 mg/mL; native conditions mimicking in vivo situation).

All candidates show LMW formation after PBS stress for 4 weeks.

Nevertheless, none of the tested formats was unstable under the employed conditions indicating a good shelf-life stability. Overall, a better stability for the 1+1 format could be seen.

In more detail, under native conditions TREM2 2269 and TREM2 2270 showed more than 20 % LMW formation in vivo mimicking PBS conditions, whereas under denaturizing conditions TREM2 2267, 2269, and 2270 had 80 % purity (CE-SDS) and TREM2 4483 and 2272 had 88 % purity (CE-SDS), Abeta 42 aa peptide binding is reduced after PBS pH 7.4 stress in the 2+2 formats only (TREM2 2267 -12%, TREM2 2269 -16%, TREM2 2270 -18%) whereas binding to TREM2 is not affected.

Occupancy during this relative active concentration (RAC; determined by SPR) assay was ~1.7 to 1.8 TREM2 per molecule for 2+2 constructs and -0.8 for 1+1 constructs. For TREM2 binding the antibody was captured via an anti-Fc antibody and soluble TREM2 was injected in the mobile phase; for Abeta binding, Abeta was immobilized to the chip and the antibody was applied in the mobile phase.

Viscosity was determined in 20 mM histidine buffer at pH 6. The results are shown in the following Tables.

Target-mediated drug disposition (TMDD)

Mouse

Anti-TREM2 antibodies are binding to mouse macrophage’s cell surface. Both, Ml and M2 macrophages show TREM2 expression on the cell surface. M2 macrophages show a -2 -fold higher TREM2 expression compared to Ml macrophages.

The TREM2 binders show a characteristic TMDD-like behavior with respect to internalization with Ml and M2 macrophages. Human

Same as with murine macrophages can be seen with human M2 macrophages.

Bivalent TREM2 binders show a saturating kinetics, whereas monovalent TREM2 binder shows mild TMDD-like behavior. The Abeta antibodies show linear uptake. In more detail, the bivalent TREM2 binders TREM2 1094 and TREM2 3306 show significantly higher Vmax and lower K_m compared to monovalent binder TREM2 4483. See the following Table and Figure 5.

In human M2 macrophages the non-specific uptake rate is higher (~3-5 fold) but of similar magnitude compared to mouse hFcRn Tg32 +/+ M2 macrophages (4 - 15 mAbs/cell/min/nM). The bivalent, monospecific anti-Abeta antibody has the lowest linear uptake rate, followed by the bispecific anti-TREM2/Abeta protein antibodies in the 1+1 format. Bivalent anti-TREM2 antibodies in 1+1 format, the reference antibody motavizumab and the bispecific anti-Abeta/TREM2 antibodies in 2+2 format have a higher non-specific uptake rate (see Figure 6). Thus, a lower TMDD for monovalent compared to bivalent formats can be expected.

Thus, from an in vitro TMDD perspective, TREM2 4483 is the preferred choice for reducing TMDD.

Migration assay with THP-1 or iPSC-derived macrophages (MO cells)

5 THP-1 and iPSC-derived MO cells were cultured in a transwell-assay, exposed to a gradient of a chemotactic agent (lyso-PC or complement C5a, respectively) and coincubated with various concentrations of different antibodies. Cells migrating through the transwell were counted by flow cytometry (THP-1) or in an Incucyte imaging system (iPSC-derived macrophages). A sketch of the assay is shown in 10 Figure 7.

In a first set of experiments Fc-competent versions of TREM2 for epitope regions 2 and 3 were tested side-by-side with reference antibody TREM2 0885 (mAb21) at 10 pg/ml (-EC50 in the THP-1 migration assay). The antibodies listed in the following Table achieved maximal migration before 10 pg/ml. Most antibodies showed strong 15 induction of migration compared to vehicle control with 2.0-3.6 fold increase (indicated as % change compared to vehicle). The reference antibody reached a plateau at 0.015 pg/ml (calculated based on dose response curve) whereas the antibodies according to the current invention reached a maximal response at 0.02- 0.62 pg/ml. Maximal response for the reference antibody was set at 100%. The 20 antibodies according to the current invention showed a range of 49-117% of that response. There was no obvious difference in potency between the different epitope regions.

Negative data indicate a potential reduction of migration

Likewise, antibodies were tested for migration in iPSC derived macrophages with similar effects as in THP-1 cells as shown in the following Table.

The paratopes of TREM2 3295 and TREM2 3306 were used to engineer further antibody formats, such as shown in Figure 1 in different 1+1, 2+1 and 2+2 formats, and different specificities, such as monospecific, monoparatopic or monospecific, biparatopic or bispecific. Additionally, different linker lengths and Fc-region variations were tested. However, all antibody formats comprised the LALAPG mutation (L234A/L235A/P329G mutation) in order to reduce engagement of the Fc- region with FcgR and complement Clq. Without being bound by this theory, it is assumed that thereby the risk of inducing inflammation mediated by the Fc-region of the antibodies is reduced or even abolished. The antibodies of the different formats and specificities were tested in the THP- 1 and the iPSC-derived MO migration assay and compared to the LALAPG version of the reference antibody mAb21 (TREM22907). It has to be pointed out that the reference antibody with the LALAPG mutations (TREM2 2907) showed a migration index that was usually only about 66% of the reference antibody without LALAPG mutations (TREM2 0885), indicating that the reference antibody most likely exerts some of its efficacy via FcgR binding. As negative control an anti-Abeta antibody (Abeta 8653) was also included, which never showed any induction of migration. The respective results are shown in the following Table.

¹⁾ Max % of TREM2 2907: TREM2 2907 reached its maximum signal at 34 nM. This column indicates what % of TREM2 2907 maximum was achieved at 34 nM for the respective antibody. Values above 100% indicate that the antibodies were more potent to induce a maximum already a lower concentrations than TREM2 2907

²⁾ 50% TREM2 2907: indicates the concentration at which 50% of the TREM2 2907 signal was achieved; knowing that TREM2 2907 maximum is at 34 nM, values below 17 nM (50% of 34 nM)

5 indicate stronger potency for that particular mAb compared with TREM2 2907 n/d = not done as only one or none biological replicate was available

This data shows amongst other things that the bispecific TREM2/Abeta protein bispecific antibodies according to the current invention with LALAPG mutation in the Fc-region and paratopes derived from TREM2 3295 or TREM2 3306 induce 10 migration in THP-1 cells similar or better than the modified reference antibody

TREM2 2907. Surprisingly, it has been found that this is independent of the format of the antibody, i.e., 1+1 and 2+2. Without being bound by this theory, it is assumed that no cross-linking of TREM2 may be required in order to induce this functional phenotype, which is in contrast to literature reports about mechanisms to activate 15 TREM2.

Also surprisingly, antibodies known from the art sometimes only minimally induced migration above the baseline in the same dose range of 0.14-34 nM and some reduced baseline migration at concentrations >1 nM indicating a functionally inhibitory effect. None of the antibodies according to the current invention listed reduced 20 baseline migration at any concentration tested.

Phagocytosis Assay

It has further been tested if the format of the anti-TREM2/Abeta bi specific antibody may affect interaction of the antibodies with Abeta and how this compares to antibodies known from the art that lack Abeta binding.

5 To evaluate this an assay employing Abeta coated beads, labeled with pHRodo was used. In case of uptake of these coated beads and transport to the lysosome, the pH sensitive dye would become detectable and uptake can be quantified.

The data were generated with Abeta 42 coated pHRodo labeled beads, co-incubated for 16 hours with phagocytic THP-1 cells. The baseline uptake into these cells was 10 about 10 % and was subtracted from the mean values shown in Figure 8 and the following Table. The ‘no mAb adjusted % of total cells with uptake of amyloid coated beads’ shows that TREM2/ Abeta protein bispecific antibodies according to the current invention with LALAPG mutation in the Fc-region induce at a concentration of 2 nM or at concentration of 4 nM in about 20-25 % of cells uptake 15 of the labeled Abeta beads which is comparable to effect observed with 4 nM Fc- region effector function competent Abeta antibody, i.e. without the LALAPG mutation (black bar). Antibodies known from the art stay mostly below 5 % of uptake or are undetectable. Monospecific anti-TREM2 antibodies according to the current invention without the LALAPG mutation did not show any uptake of Abeta beads 20 either (data not shown).

The lack of uptake of Abeta coated beads by Fc-region effector function silent LALAPG constructs of the monospecific anti-Abeta antibody evidence that the effect of bispecific anti-TREM2/ Abeta protein antibodies with effector function silent Fc-region in LALAPG format is due to the co-binding of Abeta on beads with 25 TREM2 on cells. The results of the bispecific anti-TREM2/ Abeta protein antibodies in 1+1 and 2+2 formats are similar. Without being bound by this theory, it can be considered that as bispecific 1+1 formats have only one binder for each antigen (TREM2 and Abeta) when corrected for valency, the 1+1 constructs are 2x more potent at the same nM concentration as their 2+2 construct counterparts. Unexpectedly, the YTE variants of the same 1+1 construct seem to have reduced potency compared with its parent variant (TREM2 2272 vs TREM2 4483, respectively).

The respective results are shown in Figure 8 and the following Table.

The same effect can be seen in case an anti-TREM2 antibody from the art is combined with an anti-Abeta antibody, as exemplarily shown for TREM2 2807 in Figure 9. Likewise for monospecific, biparatopic anti-TREM2 antibodies according to the current invention this effect could be shown. However, and in contrast to the bispecific formats, the monospecific, biparatopic with regular IgG format (TREM2 3737) was performing best. Most unexpectedly, beads uptake for TREM2 3737 happened even in the absence of an Abeta binder and as LALAPG molecule.

Biparatopic anti-TREM2 antibodies in different formats are TREM2 3737, TREM2 3738, TREM2 3846, TREM2 3857, TREM2 3847, TREM2 3858, TREM2 3739, TREM2 2727, and TREM2 3859.

Additionally, SPR and FACS data show that monospecific, biparatopic anti-TREM2 antibodies show enhanced binding compared to their original binders (e.g., TREM2 3737, TREM2 3738, TREM2 3739). TREM2 3737 even shows activation of phagocytosis in the absence of FcgR or Abeta binding. The data is shown in the following Table.

Based on EC50 and AUC values, all antibodies are strong phagocytosis inducers. Antibodies derived from TREM2 4491 are more efficient than those in 1+1 format. The introduction of the YTE mutation does not seem to have an effect. No effect can be seen in TREM2 knockout THP-1 cells.

In vivo amyloid uptake in the Methoxy-X04 paradigm with APPswePS2 transgenic mice

The Methoxy-X04 (MX04) paradigm to label amyloid plaques in vivo in APP transgenic mice followed by ex-vivo analysis of isolated microglia by flow cytometry was adapted from literature (Bolmont et al J Neurosci, 2008; Heneka et al., Nature 2013; Kummer et al., EMBO Journal 2021). Briefly, APPswePS2 transgenic mice were aged in order to develop amyloid plaques and injected i.v. with antibodies according to the current invention or inert IgG control. One week after antibody injection, mice were injected with MX04 and brains collected for isolation of microglia and flow cytometry analysis the next day. In a first set of experiments (Experiments 1 and 2), Fc-region effect function competent anti-TREM2 antibodies according to the current invention that previously showed positive response in the migration and Abeta coated beads uptake assay were injected. Reference antibodies TREM2 0885 (mAb 21) and TREM2 0886 (mAb52) from the art were included for comparison. Except for TREM2 0886, these antibodies showed moderate to strong increase of amyloid into microglia, which reached statistical significance for some of them. The results are shown in Figure 10.

The experiments show that Fc-region effector function competent anti-TREM2 antibodies can induce increased uptake of amyloid into microglia. Without being bound by this theory, it can be assumed that bispecific anti-TREM2/ Abeta protein antibodies may improve engagement of microglia with amyloid plaques and thus provide a way to optimize uptake of amyloid. Binding of bispecific anti- TREM2/ Abeta protein antibodies to amyloid plaques may also result in better crosslinking of TREM2 receptors and thus optimized signal induction. As an alternative to the bispecific anti-TREM2/Abeta protein antibodies according to the invention monospecific, biparatopic anti-TREM2 antibodies can be used. Without being bound by this theory, it can be assumed that this approach optimizes TREM2 signaling in microglia and hence their engagement with amyloid plaques by cross-linking of TREM2.

In a further in vivo experiment, the anti-TREM2/Abeta protein antibodies according to the current invention were administered to APPswePS2 transgenic mice with MX04 treatment paradigm. In more detail, analysis of brain sections of mice from each of the three treatment groups (n=8; females; 8 months of age; 3x32 mg/kg i.p. for Abeta 8655, TREM2 4524 and 3x20 mg/kg i.p. for TREM2 4524; at 10 mL/kg; at day 1, 4 and 7; Methoxy-X04 treatment to all mice 16 hours before sampling, RoA: i.p. 5 mg/kg) was done at time points 168, 336 and 504 hours by immunofluorescence labeling with anti-human IgG-AF488 (hTREM2 Mab) +MOAB2-AF647 (Plaques) in Cortex, Hippocampus and Thalamus. The images were read-out by co-localization area and signal intensity. The uptake of amyloid into microglia was quantified by flow cytometry. As reference antibody a 2+2 anti- DP47/Abeta antibody (Abeta 8655) that lacks TREM2 binding and only binds Abeta was employed. Antibodies tested were two TREM2/Abeta protein bispecific antibodies, that are cross-reactive between human and mouse TREM2 (TREM20116 and TREM2 4524, respectively). Both bispecific anti-TREM2/ Abeta protein antibodies contain the paratope of TREM2 3295, whereas TREM2 0116 is in a 2+2 format and TREM2 4524 is in a 1+1 format. Consistent with the previous experiments using Fc-region effector function competent bivalent TREM2 binders, Fc-region effector function silent LALAPG variants also increased the mean percentage of microglia uptake of amyloid in the MX04 paradigm; unfortunately, due to the large variability of the model this did not reach statistical significance. Surprisingly and what was never observed with Fc-region effector function competent monospecific anti-TREM2 antibodies, bispecific anti-TREM2/Abeta protein antibodies lead to a significantly increased uptake of amyloid per microglia, as indicated by mean fluorescence intensity (MFI) of MX04 positive cells. The results are shown in Figure 11. Figure 11 contains the representative data from one out of two independent in vivo experiments.

Thus, it has been found that Fc-region effector function silent, bispecific anti- TREM2/Abeta protein antibodies, i.e. comprising the LALAPG mutation in the Fc- region, mediate an increased engagement of microglia with amyloid plaques in APPswePS2 transgenic mice. Surprisingly this results in more efficient uptake into microglia than achieved with monospecific anti-TREM2 antibodies that are Fc- region effector function competent. This mechanism is TREM2-dependent as the application of the monospecific anti-DP47/Abeta antibody does result in increased uptake.

In more detail, TREM24524 in 1+1 format and TREM2 0116 in 2+2 format induced a significantly increased number of ‘MX04 positive cells MFI’. As there was no significant difference between the TREM24524 and TREM20116 a similar potency at mass normalized dose can be assumed, however with potentially higher potency for the 1+1 format as it is monovalent for TREM2. The absence of such an increase in the Abeta 8655 group indicates a TREM2-mediated engagement of microglia on amyloid plaques.

Additionally it has been found that AUC of plaque decoration of antibodies in the 1+1 format is improved versus the 2+2 format.

Syk phosphorylation

Syk phosphorylation was determined. The results are shown in Figure 12.

In some experiments, total Syk was comparable between all conditions and test antibodies (data not shown) while an elevated baseline luminescence of pSyk at -1000 units was observed with medium and buffer controls (blue and red dashed lines for 100% for absence and presence of beads, respectively). This baseline elevation may be indicative of a tonic, low-level pSyk activation caused by a putative ligand in the cellular environment. Addition of Abeta coated beads only marginally elevated the baseline pSyk level. Incubation with various anti-TREM2 antibodies from the art (all at 50 nM) independent of the presence or absence of Abeta coated beads was leading to a robust pSyk upregulation for most of the tested antibodies. Surprisingly, the monospecific anti-TREM2 antibodies according to the current invention, i.e. without an Abeta binder (i.e., TREM2 3295, TREM2 3306; all at 50 nM) and the bispecific anti-TREM2/Abeta protein antibodies according to the current invention actually reduce the baseline elevated pSyk levels in the absence of Abeta coated beads. The same effect was observed for anti-TREM2 antibodies that do not have an Abeta binding site in the presence of Abeta coated beads. In strong contrast, however, in the presence of Abeta coated beads, the antibodies according to the current invention reversed the pSyk reduction and in some cases (i.e. TREM2 4483) the pSyk induction exceeded the baseline level.

In repetitions of the experiments where the baseline pSyk levels for medium and vehicle control was low (-500 units), the reduction of signal induced by the bispecific anti-TREM2/ Abeta protein antibodies according to the current invention was also less pronounced or barely detectable. Consistent with results described above, in the presence of Abeta beads the bispecific anti-TREM2/Abeta protein antibodies according to the current invention reversed (TREM2 2269) or even exceeded (TREM2 4483, TREM2 2267, TREM2 2270, TREM2 2272) the baseline signal level 2-4 fold. The results are shown in Figure 13.

Without being bound by this theory, the effects observed with the bispecific anti- TREM2/Abeta protein antibodies according to the current invention may be explainable by a multi-modal mechanism of the same bispecific anti-TREM2/ Abeta protein antibody: although acting as a functional agonist in the Abeta-free cellular migration assays in THP and iPSC-derived M0 cells, the antibodies appear to be antagonists of the pSyk pathway in the HEK TREM2/DAP12 cell system, which is in contrast to what is usually described for TREM2 antibodies in the art. Since the used bispecific anti-TREM2 paratopes all bind to the extracellular ligand binding domain of TREM2 (epitope regions 2 and 3), the hypothesis for that antagonistic effect could be a displacement of a putative ligand that normally induces a tonic low level TREM2 signal and thus leading to an interruption of the original trigger. In the presence of Abeta coated beads, multiple bispecific anti-TREM2/Abeta protein antibodies may decorate the beads via their Abeta paratope while their TREM2 paratope could enable a clustering of TREM2 on the surface of the cells and thus resulting in the induction of the pSyk cascade in an agonistic fashion.

Thus, the bispecific anti-TREM2/Abeta protein antibodies according to the current invention are functional agonists related to cellular migration and context-dependent antagonists and agonists of the pSyk pathway (i.e., antagonistic for the pSyk pathway in the absence of Abeta coated beads and agonistic with Abeta coated beads).

In order to show whether Abeta coating on beads was required to induce the agonistic effect or whether it is sufficient to provide Abeta stabilized by other means, Abeta was coated on the cell culture plate, incubated then with a bispecific anti- TREM2/Abeta protein antibody, i.e. TREM2 4483, washed and then cells were added for 30 minutes before harvesting to measure pSyk activation. In the same experiment also activation of ribosomal protein S6, which is further downstream of the Syk pathway, was monitored.

The results are shown in Figure 14.

Figure 14 shows a dose-dependent upregulation of pSyk and a similar effect for pS6 in the presence of Abeta. While the baseline pSyk appeared to be too low for further reducing the signal in the absence of Abeta, bispecific anti-TREM2/Abeta protein antibody TREM2 4483 shows a dose-dependent reduction of pS6 in the absence of Abeta. This finding replicates an antagonistic effect of this mAb in the absence of Abeta that is signaled further downstream into the pathway.

Whole Blood Assay

In a whole blood assay (WBA) a minor increase in TNF alpha release was observed for all tested antibodies with TREM2 2272 showing the highest release and wherein TREM2 2270 also triggered release of IL-8. The results are shown in the following Table.

Summary

From the data, when taken together, it can be seen that neither the 1+1 format nor the 2+2 format shows superiority over the other.

2+2 format: - approx. 3x higher potency than 1+1 in vivo and in vitro (mouse x-reactive candidates)

- longer plaque retention in APPswePS2 tg mice due to avidity

1+1 format:

- approx. 2x higher recycling efficiency in vivo and in vitro, leading to 2x lower clearance

- up to 2.7x higher brain uptake, in addition to effect of longer plasma halflife

- stronger initial plaque decoration compared with 2+2 format in APPswePS2 tg mice - at equimolar dose and despite 2x lower valency for the 1+1 vs. 2+2, both formats elicit similar levels of amyloid (MX04) uptake by microglia in APPswePS2 tg mice in vivo

Antibodies of both formats can be concentrated to 175 mg/mL or more.

Antibodies of both formats are expressed at high titers and with acceptable side product profiles.

Scientific Rationale

The mutation R47H in human TREM2 is genetically associated with an increased risk to develop Alzheimer’s Disease (LoF, enhanced neuritic dystrophy around plaques). TREM2 loss-of-function mutation results in FTD-like dementia (Nasu- Hakola disease). Furthermore, soluble TREM2 in CSF is associated with elevated tau and ptau levels. That is, CSF soluble TREM2 is gradually upregulated and accompanied by tau and NfL increase (neuronal injury) in prodromal AD and downregulated during cognitive decline during clinical AD (see, e.g., Jay et al., 2017, Mol. Neurodeg.; Ulland and Colonna, Nat Rev Neurol 2018; Deczkowska et al., Cell 2018).

TREM2-deficiency in mouse AD models results in reduced microglia survival, reduced microglia migration to the plaques and reduced plaque compaction. Controversial results on plaque number and density were observed.

TREM2 signaling on microglia and myeloid cells is induced by pathogen/damage- associated molecular patterns.

The microglia-TREM2 hypothesis in Alzheimer’s Disease supports the suitability of the antibody according to the current invention.

In more detail, a strong overall genetic and neuropathological rationale for a key role of innate immunity in Alzheimer’s Disease has been established in the art. TREM2 mutation R47H has been established as a genetic risk factor for Alzheimer’s Disease (proposed LoF, enhanced neuritic dystrophy around plaques resulting in loss of DAM phenotype). CSF sTREM2 is gradually upregulated and accompanied by tau and NfL increase (neuronal injury) in prodromal AD and downregulated during cognitive decline during clinical AD. Preclinical evidence of a mechanistic link between certain MS4A gene cluster variants and shedding of sTREM2 has been established. Certain MS4A gene cluster variants associated with increased CSF sTREM2 concentrations were also associated with reduced AD risk and delayed age at onset of disease.

That is, the available clinical and preclinical data point to a lost microglial functionality in Alzheimer’s Disease that is proposed to be restored by inducing proper TREM2 signaling; i.e., by agonistic anti-TREM2 monoclonal antibodies.

Without being bound by this theory, it is assumed that the bispecificity of the antibodies according to the current invention allows for a unique mode of action different from monospecific anti-TREM2 antibodies as well as monospecific anti- Abeta antibodies. This is achieved by enhancement of the non-inflammatory, protective phenotype of microglia at plaques, enhancement of the uptake of amyloid and cellular debris (phagocytosis / clearance function), dampening of inflammatory responses caused by inappropriate microglial activation as well as restriction of pathology / protection of neurons from toxic environment (barrier function). Additionally, it is assumed that TREM2 function of plaque distant microglia can be restored by induced motility and migration towards plaques and neuronal damage.

The before can be achieved according to teaching of the current invention by the molecule characteristics of the antibody according to the current invention, i.e. by Abeta binding for targeting microglia at amyloid plaques and local TREM2 oligomerization, by TREM2 engagement to induce protective phenotype, and by LALAPGto avoid inflammatory engagement of FcgRII and III and complement Clq activation.

Antibody Formats

General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

The term “activate” herein refers to the initiation or preservation of downstream signaling of TREM2 expressed at the cell surface leading to an increased cellular function or metabolism, e.g., in TREM2-expressing cells in healthy subjects or individuals with inflammatory or neurodegenerative diseases where proper TREM2- dependent cellular activities are impaired. This may be accomplished but is not limited to phosphorylation of TREM2 associated DAP 12 or DAP 10, leading via different intracellular signaling cascades to enhancement of Syk phosphorylation, phagocytosis, increased target-directed cellular motility (chemotaxis), increased cellular survival, modulating cytokine or chemokine release of cells expressing TREM2, increasing degradation of intracellular phagocytosed material, improved lipid metabolism, or changes in gene expression. Increased TREM2 dependent DAP12 or Syk phosphorylation or downstream signaling can be assessed by Western blot, ELISA or flow cytometry /FACS or specific reporter cell assays. Directed motility of cells, e.g., chemotaxis can be assessed by cellular bioassays. Modulation of cytokine release can be assessed by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), antibody arrays, or flow cytometry/FACS. Changes in gene expression can be assessed by quantitative RT-PCR, single cell RNA sequencing or spatialomics on the mRNA level or by Western blot, immunostaining or flow cytometry at the protein level.

An “anti-TREM2 antibody” or an “antibody that binds to TREM2” and similar phrases refer to an antibody that specifically binds to human TREM2 as defined herein.

An “anti-amyloid beta protein antibody” or an “antibody that binds to amyloid beta protein” and similar phrases refer to an antibody that specifically binds to human amyloid beta protein as defined herein.

An “anti-TREM2/amyloid beta protein antibody” or an “anti-TREM2/Abeta protein antibody” or an “antibody that binds to TREM2 and amyloid beta protein” and similar phrases refer to an antibody that specifically binds to TREM2 and amyloid beta protein as defined herein.

The term “agonist” as used herein refers to a substance, such as an antibody, that causes an increase in at least one activity or function of a molecule to which it binds, or otherwise activates or helps to activate the molecule. The term “antagonist” as used herein refers to a substance, such as an antibody, that causes a decrease in at least one activity or function of a molecule to which it binds, or that otherwise blocks or inhibits at least one activity or function of the molecule.

An “agonist anti-TREM2 antibody” or similar phrases herein, for example, refers to an antibody that induces luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2 and DAP12 and/or that induces SYK phosphorylation (p- SYK) in HEK or Jurkat-NFAT reporter cells expressing human TREM2 and DAP12, THP-1 cells, in human monocyte-derived macrophage (MDM) cells and/or in human induced pluripotent stem cell (iPSC)-derived macrophages, MO or microglia cells. Agonist anti-TREM2 antibodies may also have further activities, as described herein, such as enhancing survival of human iPSC-derived microglia in the absence of IL- 34 and CSF-1, and activating TREM2 signaling in human macrophages and microglia, among other activities, for example.

In some cases, the antibody according to the current invention is effector function competent. In some cases, the antibody has an Fc-region with reduced effector function. In one preferred embodiment of all aspects and embodiments of the current invention, the antibody according to the invention has an Fc-region that is effector function silent.

For example, the Fc-region of antibody therapeutics can bind to complement component Clq and Fc-gamma receptors (FcyR) to elicit cellular effector responses such as phagocytosis, cytokine release, and production of reactive oxygen species. See, e.g., X. Wang, et al., Protein & Cell 9: 63-73 (2018); S.B. Mkaddem et al., Frontiers in Immunology, available at https://doi.org/10.3389/fimmu.2019.00811 (2019). Hyperactivation of these pathways may be detrimental in the CNS, particularly in the context of pathology. See, e.g., D.J. DiSabato et al., J. Neurochemistry 139(S2): 136-153 (2016). Clinically, antibody therapeutics directed against amyloid beta that have intact effector function and bind to amyloid beta aggregates greatly increase incidence of a potentially harmful side-effect, ARIA (amyloid-related imaging abnormalities), whereas ARIA has not been observed from antibodies that bind only to monomeric forms of amyloid beta or antibodies that have reduced effector function. See, e.g., M. Filippi et al., JAMA Neurol. 79(3): 291-304 (2022). It has to be pointed out that at least one Fc-competent anti-TREM2 antibody caused ARIA in 19-71% of AD patients (depending on ApoE genotype) in a clinical trial setting (a Phase 2 study to evaluate efficacy and safety of AL002 in participants with early Alzheimer's Disease (INVOKE-2), NCT04592874). Without being bound by this theory, it can be assumed that Abeta binding by a therapeutic antibody alone may be less important to induce ARIA but that microglia or macrophage engagement in the brain perivascular space by TREM2 and FcgR could play a crucial role in this. In some embodiments, for instance as described in the Examples and figures herein, antibodies herein with reduced effector function (such comprising a human IgGl heavy chain constant region with either a LALAPG or an N297G mutation) were sufficient to elicit microglial responses in the mouse CNS via TREM2 stimulation.

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to full-length antibodies, monoclonal antibodies, monospecific, biparatopic antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies), and antibody-antibody fragment-fusions as well as combinations thereof as long as these have the desired antigen binding properties.

An “antibody conjugate” is an antibody or antibody fragment conjugated to one or more heterologous molecule(s), including but not limited to a label.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen (i.e. TREM2 or/and amyloid beta protein) to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. In more detail, a Fab fragment is defined by the cleavage of a full-length, native IgG antibody of the subclass IgGl by the enzyme papain. Papain cleaves in the hinge region sequence after the amino acid residue histidine (SEQ ID NO: 429; EPKSCDKTHJ.TCP; papain cleavage site indicated by a downward arrow). Thus, a Fab fragment comprises a first polypeptide formed by a variable domain and a constant domain, e.g. a VL and a CL, and a heavy chain Fab fragment comprising a variable domain, a constant domain and a fragment of the hinge region, e.g., a VH, a CHI and the hinge region fragment with the amino acid sequence EPKSCDKTH (amino acid residues 1 to 9 of SEQ ID NO: 429). For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23 : 1126-1136 (2005).

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The “class” of an antibody refers to the type of constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, wherein the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively. Some of these classes may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In certain embodiments, the antibody is of the IgGl isotype. In one preferred embodiment, the antibody is of the IgGl isotype with the P329G, L234A and L235A mutation to reduce Fc-region effector function (numbering according to Kabat). In certain embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve stability of IgG4 antibody. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

The light chain “constant domain” and the heavy chain “constant region” of an antibody refer to additional sequence portions outside of the FRs and CDRs and variable domains. Certain antibody fragments may lack all or some of the constant domain or/and region. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant heavy chain domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain.

The term “domain crossover” as used herein denotes that in a pair of an antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., in an antibody Fab (fragment antigen binding), the domain sequence deviates from the sequence in a native antibody in that at least one heavy chain domain is substituted by its corresponding light chain domain and vice versa. There are three general types of domain crossovers, (i) the crossover of the CHI and the CL domains, which leads by the domain crossover in the light chain to a VL-CH1 domain sequence and by the domain crossover in the heavy chain fragment to a VH-CL domain sequence (or a full length antibody heavy chain with a VH-CL-hinge-CH2- CH3 domain sequence), (ii) the domain crossover of the VH and the VL domains, which leads by the domain crossover in the light chain to a VH-CL domain sequence and by the domain crossover in the heavy chain fragment to a VL-CH1 domain sequence, and (iii) the domain crossover of the complete light chain (VL-CL) and the complete VH-CH1 heavy chain fragment (“Fab crossover”), which leads to by domain crossover to a light chain with a VH-CH1 domain sequence and by domain crossover to a heavy chain fragment with a VL-CL domain sequence (all aforementioned domain sequences are indicated in N-terminal to C-terminal direction).

“Effector functions” refer to those biological activities attributable to the Fc-region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include Clq binding and complement dependent cytotoxicity (CDC); Fc- receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (antibody-dependent cell-mediated phagocytosis, ADCP); down regulation of cell surface receptors (e.g., B cell receptor); and B-cell activation. Antibodies with “(intact) effector function”, for example, comprise a heavy chain constant region or Fc-region that possesses the native, intact effector functions of its particular isotype, such as a wild-type heavy chain constant region or Fc-region, or one that possesses modification that have not been shown to impact effector functions. In contrast, antibody heavy chain constant regions or Fc-regions may be modified in various ways, such as by amino acid substitution, insertion, or deletion, or by glycosylation modifications, to either reduce or enhance effector function, depending upon the use of an antibody. Herein, some antibodies may have “(intact) effector function” or are “effector function competent”, while others may be “effector function silent (effectorless)”, meaning that they do not show detectable CDC or ADCC activity or that they comprise a heavy chain constant region or Fc- region that has previously been shown to have no detectable CDC or ADCC activity. In other cases, antibodies may have a mutant Fc-region or heavy chain constant region with “reduced effector function” compared to the corresponding wild-type Fc-region or heavy chain constant region (e.g., that has previously been shown to have reduced effector function, or that has reduced effector function in the context of the antibodies here). Such antibodies with reduced effector function may, for example, show a lower degree of CDC and/or ADCC activity and/or FcyR binding activity compared to the corresponding wild-type Fc-region, and/or may retain some effector functions such as binding to particular FcgRs, but combined with low or no detectable CDC or ADCC, for example.

The term “full-length antibody” denotes an antibody having a structure substantially similar to that of a native antibody. A full-length antibody comprises two full length antibody light chains each comprising in N- to C-terminal direction a light chain variable region and a light chain constant domain, as well as two full length antibody heavy chains each comprising in N- to C-terminal direction a heavy chain variable domain, a first heavy chain constant domain, a hinge region, a second heavy chain constant domain and a third heavy chain constant domain. In contrast to a native antibody, a full-length antibody may comprise further immunoglobulin domains, such as e.g. one or more additional scFvs, or heavy or light chain Fab fragments, or scFabs conjugated to one or more of the termini of the different chains of the full length antibody, but only a single fragment to each terminus. These conjugates are also encompassed by the term full-length antibody. However, in a full-length antibody the heavy chain C-terminal lysine amino acid residue or the glycine-lysine dipeptide may be absent.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the amino acid residues in the CDRs correspond to those of a non-human antibody, and all or substantially all of the amino acid residues in the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Unless otherwise indicated, the CDRs are determined according to the sequence table herein, and the amino acid positions of regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The Kabat and Chothia CDRs differ in the CDR-H1 and CDR- H2 but are the same in the remaining four CDRs. One of skill in the art will understand that the CDR designations can also be determined according to McCallum, or any other scientifically accepted nomenclature system. See, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); MacCallum et al. J. Mol. Biol. 262: 732- 745 (1996)). Exemplary CDRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

“Framework” or “FR” refers to the residues of the variable domain of an antibody that are not part of the complementary determining regions (CDRs). The FR of a variable domain generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR- H3(CDR-L3)-FR4.

The term “heavy chain” refers to a polypeptide comprising at least an antibody variable domain and a hinge region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” refers to a polypeptide comprising a heavy chain variable domain (VH) and a heavy chain constant region (CHl-hinge- CH2-CH3), with or without a leader sequence, but including a domain exchange of the CHI domain with an antibody light chain constant domain (CL) or of the heavy chain variable domain with a light chain variable domain or both.

The term “heavy chain constant region” denotes the region of an immunoglobulin heavy chain that contains the constant domains, i.e., the CHI domain or the CL domain, the hinge region, the CH2 domain and the CH3 domain. In certain embodiments, a human IgG constant region extends from Alai 18 to the carboxyl- terminus of the heavy chain (numbering according to Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region or the C-terminal glycinelysine dipeptide (G446-Lys447) of the constant region may or may not be present (numbering according to Kabat EU index). The term “constant region” denotes a dimer comprising two heavy chain constant regions, which can be covalently linked to each other via the hinge region cysteine residues forming inter-chain disulfide bonds.

The term “heavy chain Fc-region” denotes the C-terminal region of an immunoglobulin heavy chain that contains at least a part of the hinge region (middle and lower hinge region), the CH2 domain and the CH3 domain. In certain embodiments, a human IgG heavy chain Fc-region extends from Asp221, or from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain (numbering according to Kabat EU index). Thus, a heavy chain Fc-region is smaller than a heavy chain constant region but essentially identical thereto with respect to the C-terminal portion. However, the C-terminal lysine (Lys447) or the C-terminal glycine-lysine dipeptide (G446-Lys447) of the heavy chain Fc-region may or may not be present (numbering according to Kabat EU index). The term “Fc-region” denotes a dimer comprising two heavy chain Fc-regions, which can be covalently linked to each other via the hinge region cysteine residues forming inter-chain disulfide bonds.

The Fc-region of an antibody (and likewise the constant region of an antibody) is directly involved in complement activation, Clq binding, C3 activation and Fc- receptor binding. While the influence of an antibody on the complement system is dependent on certain conditions, binding to Clq is caused by defined binding sites in the Fc-region. Such binding sites are known in the state of the art and described e.g., by Lukas, T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such binding sites include, e.g., the amino acid residues L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to Kabat EU index). Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation, Clq binding and C3 activation, whereas IgG4 do not activate the complement system, do not bind Clq and do not activate C3.

An “Fc-region of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.

A “humanized” antibody refers to an antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., the CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non- human antibody, refers to an antibody that has undergone humanization.

The term “light chain” refers to a polypeptide comprising at least a light chain variable domain, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full- length light chain” refers to a polypeptide comprising a light chain variable domain (VL) and a light chain constant domain (CL), with or without a leader sequence, but including a domain exchange of the CL domain with a CHI domain or of the light chain variable domain with a heavy chain variable domain or both.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, 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. For example, the monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to recombinant DNA methods, such methods and other exemplary methods for making monoclonal antibodies being described herein.

A "monospecific antibody" denotes an antibody that has a single binding specificity, i.e., specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2) or combinations thereof (e.g., full length antibody plus additional scFv or Fab fragments). A monospecific antibody does not need to be monovalent, i.e., a monospecific antibody may comprise more than one binding site specifically binding to the one antigen. A native antibody, for example, is monospecific but bivalent.

A "multispecific antibody" denotes an antibody that has binding specificities for at least two different epitopes on the same antigen (also denoted as biparatopic herein) or two different antigens. A “bispecific” antibody is an antibody that binds specifically to two antigens and a “trispecific” antibody is an antibody that binds specifically to three antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies) or combinations thereof (e.g., full length antibody plus additional scFv or Fab fragments). A multispecific antibody is at least bivalent, i.e., comprises two antigen binding sites. In addition, a multispecific antibody is at least bispecific. Thus, a bivalent, bispecific antibody is the simplest form of a multispecific antibody. Engineered antibodies with two, three or more (e.g., four) functional antigen binding sites have also been reported (see, e.g., US 2002/0004587).

The term "native antibody" denotes naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a heavy chain variable domain (VH) followed by three heavy chain constant domains (CHI, CH2, and CH3), whereby between the first and the second heavy chain constant domain a hinge region is located. Similarly, from N- to C- terminus, each light chain has a light chain variable domain (VL) followed by a light chain constant domain (CL). The light chain of a native antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

The term "recombinant antibody", as used herein, denotes all antibodies (chimeric, humanized and human) that are prepared, expressed, created or isolated by recombinant means, such as using a recombinant mammalian cell according to the current invention.

As used herein the term “replaced by each other” with respect to corresponding heavy and light chain domains refers to the aforementioned domain crossovers. As such, when CHI and CL domains are “replaced by each other” it is referred to the domain crossover mentioned under item (i) and the resulting heavy and light chain domain sequence. Accordingly, when VH and VL are “replaced by each other” it is referred to the domain crossover mentioned under item (ii); and when the CHI and CL domains are “replaced by each other” and the VH and VL domains are “replaced by each other” it is referred to the domain crossover mentioned under item (iii). Bispecific antibodies including domain crossovers are reported, e.g., in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254 and Schaefer, W., et al, Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192. Such antibodies are generally termed CrossMab.

The term “valent” or “valency” as used within the current application denotes the presence of a specified number of binding sites in an antibody. As such, the terms “bivalent”, “trivalent”, and “tetravalent” denote the presence of two binding sites, three binding sites, and four binding sites, respectively, in an antibody.

The term “variable domain” refers to the part of an antibody heavy or light chain that is involved in binding of the antibody to its antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4; and light chain (LC) CDR1-FR2- CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4. That is, a variable domain may lack a portion of FR1 and/or FR4 so long as it retains antigenbinding activity. A single VH or VL domain may be sufficient to confer antigenbinding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150 :880-887 (1993); Clarkson et al., Nature 352 :624-628 (1991)). It has to be pointed out that variable domains are herein defined by their amino acid sequence starting at the first residue after the signal peptide. However, the variable domain sequences given herein include variant sequences wherein the first N- terminal glutamine (Q) residue or the first N-terminal glutamic acid (E) residue is replaced by a pyroglutamic acid (pE) residue.

In some cases, the antibody may be bispecific or trispecific. In some cases, the antibody may be conjugated to another molecule, such as a label either directly or through a linker.

Multispecific Antibodies

In certain embodiments of all aspects and embodiments of the subject matter of the current invention, the antibody according to the invention is at least a bivalent, bispecific antibody. In certain embodiments, one of the binding specificities is for TREM2 and the other is for the amyloid beta protein.

Multispecific antibodies may be used to localize cytotoxic agents to cells, which express the one or more antigens.

Multispecific antibodies can be prepared as full-length antibodies, antibody-antibody fragment-fusions or antibody fragment-antibody fragment-fusions. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A., et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole” engineering (see, e.g., US 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.A., et al., J. Immunol. 148 (1992) 1547-1553); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431); using specific technology for making bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and preparing trispecific antibodies as described, e.g., in Tutt, A., et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792, and WO 2013/026831. The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting Fab” or “DAF” (see, e.g., US 2008/0069820 and WO 2015/095539).

Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover, i.e., by exchanging the VH/VL domains (see, e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see, e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-1020) in one or more binding arms of the same antigen specificity. In certain embodiments of all aspects and embodiments of the current invention, the cell according to the current invention expresses a multispecific antibody comprising a Cross-Fab fragment. The term “Cross-Fab fragment” denotes a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A Cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab heavy chain fragment and cognate light chain pairing. See, e g., WO 2016/172485.

The antibody or fragment may also be a multispecific antibody as described in WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, or WO 2010/145793.

The antibody or fragment thereof may also be a multispecific antibody as reported in WO 2012/163520.

Various further molecular formats for multispecific antibodies are known in the art and can be produced with the binding sites according to the current invention (see e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

In certain embodiments of all aspects and embodiments, the format of the multispecific antibody according to the current invention is selected from the group of antibody formats consisting of: a bivalent, bispecific full-length antibody with domain exchange

(i.e., a bivalent, bispecific full-length antibody comprising a first Fab fragment and a second Fab fragment and an Fc-region, wherein in the first Fab fragment a) (only) the CHI and CL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises a VL and a CHI domain and the heavy chain of the first Fab fragment comprises a VH and a CL domain); b) (only) the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises a VH and a CL domain and the heavy chain of the first Fab fragment comprises a VL and a CHI domain); or c) the CHI and CL domains are replaced by each other and the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises a VH and a CHI domain and the heavy chain of the first Fab fragment comprises a VL and a CL domain); wherein the second Fab fragment comprises a light chain comprising a VL and a CL domain and a heavy chain Fab fragment comprising a VH and a CHI domain; wherein the first Fab fragment specifically binds to a first antigen and the second Fab fragment specifically binds to a second antigen; and wherein the Fc-region comprises a first Fc-region polypeptide including a CH3 domain and a second Fc-region polypeptide including a CH3 domain, wherein both CH3 domains are engineered in a complementary manner by respective amino acid substitutions, in order to support heterodimerization of the first heavy chain comprising the first Fc-region polypeptide and the second heavy chain comprising the second Fc-region polypeptide, e.g., as reported in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, or WO 2013/096291 (incorporated herein by reference), preferably with the knob-into-hole mutations; a trivalent, bispecific antibody comprising a bivalent, full-length antibody and an additional monovalent heavy chain C-terminal binding site (BS) (i.e., a trivalent, bispecific antibody comprising a bivalent, full-length antibody and an addition monovalent (third) binding site conjugated to the C- terminus of one of the heavy chains of the bivalent, full-length antibody wherein a) the full-length antibody comprises two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding sites formed by each of the pairs of the full length heavy chain and the full length light chain specifically bind to a first antigen, and b) one additional binding site, wherein the additional binding site is conjugated to the C-terminus of one heavy chain of the full-length antibody, wherein the additional binding site specifically binds to a second antigen, preferably wherein the additional binding site is an additional Fab fragment specifically binding to the second antigen i) comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced by each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CHI) are replaced by each other, or ii) is a single chain Fab fragment); a trivalent, trispecific antibody comprising a bivalent, bispecific full-length antibody with domain exchange and an additional monovalent heavy chain C- terminal binding site (BS)

(i.e., a trivalent, trispecific antibody comprising a bispecific, bivalent, full- length antibody with domain exchange and an addition monovalent (third) binding site conjugated to the C-terminus of one of the heavy chains of the bivalent full-length antibody wherein a) the full-length antibody comprises a first Fab fragment, a second Fab fragment and an Fc-region, wherein in the first Fab fragment a) (only) the CHI and CL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises a VL and a CHI domain and the heavy chain of the first Fab fragment comprises a VH and a CL domain); b) (only) the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises a VH and a CL domain and the heavy chain of the first Fab fragment comprises a VL and a CHI domain); or c) the CHI and CL domains are replaced by each other and the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises a VH and a CHI domain and the heavy chain of the first Fab fragment comprises a VL and a CL domain); wherein the second Fab fragment comprises a light chain comprising a VL and a CL domain and a heavy chain Fab fragment comprising a VH and a CHI domain; wherein the first Fab fragment specifically binds to a first antigen and the second Fab fragment specifically binds to a second antigen; wherein the Fc-region comprises a first Fc-region polypeptide including a CH3 domain and a second Fc-region polypeptide including a CH3 domain, wherein both CH3 domains are engineered in a complementary manner by respective amino acid substitutions, in order to support heterodimerization of the first heavy chain comprising the first Fc-region polypeptide and the second heavy chain comprising the second Fc-region polypeptide, e.g., as reported in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, or WO 2013/096291 (incorporated herein by reference), preferably with the knob-into-hole mutations; b) one additional binding site, wherein the additional binding site is conjugated to the C-terminus of one heavy chain of the full-length antibody, wherein the additional binding site specifically binds to a third antigen, preferably wherein the additional binding site is an additional Fab fragment specifically binding to the third antigen i) comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced by each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CHI) are replaced by each other, or ii) is a single chain Fab fragment); a bivalent, bispecific one-armed single chain antibody

(i.e., an antibody comprising a first binding site that specifically binds to a first antigen and a second binding site that specifically binds to a second antigen, whereby the individual chains are as follows

- a light chain (comprising in N- to C-terminal direction a light chain variable domain and a light chain constant domain);

- a combined light-heavy chain (comprising in N- to C-terminal direction a light chain variable domain, a light chain constant domain, a peptidic linker, a heavy chain variable domain, and a heavy chain constant region with knob mutation in the CH3 domain); and

- a heavy chain (comprising in N- to C-terminal direction a heavy chain variable domain and a heavy chain constant region with hole mutations in the CH3 domain)); a bivalent, bispecific two-armed single chain antibody (i.e., an antibody comprising a first binding site that specifically binds to a first antigen and a second binding site that specifically binds to a second antigen, whereby the individual chains are as follows

- a first combined light-heavy chain (comprising in N-terminal to C- terminal direction a light chain variable domain, a light chain constant domain, a peptidic linker, a heavy chain variable domain and a heavy chain constant region with i) hole mutations or ii) knob mutation in the CH3 domain); and

- a second combined light-heavy chain (comprising in N- to C-terminal direction a light chain variable domain, a light chain constant domain, a peptidic linker, a variable heavy chain domain and a heavy chain constant region with i) knob mutation or ii) hole mutations in the CH3 domain)); a common light chain bispecific antibody

(i.e., an antibody comprising a first binding site that specifically binds to a first antigen and a second binding site that specifically binds to a second antigen, whereby the individual chains are as follows

- a light chain (comprising in N- to C-terminal direction a light chain variable domain and a light chain constant domain);

- a first heavy chain (comprising in N- to C-terminal direction a heavy chain variable domain and a heavy chain constant region with i) hole mutations or ii) knob mutation in the CH3 domain); and

- a second heavy chain (comprising in N- to C-terminal direction a heavy chain variable domain and a heavy chain constant region with i) knob mutation or ii) hole mutations in the CH3 domain)); a T-cell bispecific antibody (TCB)

(i.e., a bivalent, monospecific full-length antibody with additional, inserted heavy chain N-terminal binding site with domain exchange comprising

- a first and a second Fab fragment, wherein each binding site of the first and the second Fab fragment specifically bind to a first antigen,

- a third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to a second antigen, and wherein the third Fab fragment comprises a domain crossover such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced by each other, and

- an Fc-region comprising a first Fc-region polypeptide and a second Fc- region polypeptide, wherein the first and the second Fab fragment each comprise a heavy chain Fab fragment and a full-length light chain, wherein the C-terminus of the heavy chain Fab fragment of the first Fab fragment is fused to the N-terminus of the first Fc-region polypeptide, - I l l - wherein the C-terminus of the heavy chain Fab fragment of the second Fab fragment is fused to the N-terminus of the light chain variable domain of the third Fab fragment and the C-terminus of the CHI domain of the third Fab fragment is fused to the N-terminus of the second Fc-region polypeptide).

The “knobs into holes” dimerization modules and their use in antibody engineering are described in Carter P.; Ridgway

PrestaL.G.: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).

The CH3 domains in the heavy chains of an antibody can be altered by the “knob- into-hole” technology, which is described in detail with several examples in e.g., WO 96/027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621; and Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of these two CH3 domains and thereby of the polypeptide comprising them. Each of the two CH3 domains (of the two heavy chains) can be the “knob”, while the other is the “hole”. The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

The mutation T366W in the CH3 domain (of an antibody heavy chain) is denoted as “knob-mutation” or “mutation knob” and the mutations T366S, L368A, Y407V in the CH3 domain (of an antibody heavy chain) are denoted as “hole-mutations” or “mutations hole” (numbering according to Kabat EU index). An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681) e.g. by introducing a S354C mutation into the CH3 domain of the heavy chain with the “knob -mutation” (denotes as “knob- cys-mutations” or “mutations knob-cys”) and by introducing a Y349C mutation into the CH3 domain of the heavy chain with the “hole-mutations” (denotes as “hole-cys- mutations” or “mutations hole-cys”) (numbering according to Kabat EU index).

In any of the embodiments herein, the antibody may be an antibody fragment, such as an Fv, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2. In other embodiments, the antibody may be a whole antibody (i.e., comprising heavy and light chain constant regions). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgGl Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P/M252Y/S254T/T256E Fc region, a human IgGl N297G Fc region, a human IgGl LALAPG (L234A/L235A/P329G) Fc region, or a human IgGl N297G/M428L/N434S Fc region. If a murine IgG antibody, the antibody may be an mlgGl or mIgG2 or mIgG2 LALAPG antibody. An antibody may, in some cases, comprise a full-length heavy chain and/or a full-length light chain. In some cases, the antibody may lack the C-terminal Lys or the C-terminal Lys and Gly residues of the heavy chain constant region. In other cases, the antibody contains one or both of those C-terminal residues. anti-TREM2 antibodies

In one aspect, the invention provides antibodies that bind to human TREM2. In one aspect, provided are isolated antibodies that bind to human TREM2. In one aspect, the invention provides biparatopic antibodies that specifically bind to human TREM2.

Antibodies according to the current invention that bind or specifically bind to human TREM2 are referred to as anti-TREM2 antibodies.

In certain aspects, an anti-TREM2 antibody a) induces as bispecific anti-TREM2/Abeta protein antibody phagocytosis of amyloid plaques by macrophages, and/or b) induces as bispecific anti-TREM2/Abeta protein antibody microglial amyloid uptake in the absence of FcgRII as well as FcgRIII receptor engagement, and/or c) induces as bispecific anti-TREM2/Abeta protein antibody amyloid uptake in the absence of engagement of FcgR, and/or d) induces in Fc-effector function competent form acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, and/or e) induces as bispecific anti-TREM2/Abeta protein antibody without effector-function acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, preferably in the absence of Abeta and FcgR crosslinking, and/or f) induces migration of macrophages, preferably at a concentration of 0.14 to 34 nM in a migration assay as described herein, and/or g) increases LPC- and C5a stimulated cellular migration of THP-1 and iPSC-derived MO cells in the absence of any Abeta protein, and/or h) does not induce pSyk in the absence or presence of human Abeta protein, preferably the antibody is an antagonist of the Syk pathway, and/or i) does induce as bispecific anti-TREM2/Abeta protein antibody pSyk in the presence of human Abeta protein, and/or j) does not show a dose dependent Syk and S6 phosphorylation in DAP12 and TREM2 expressing HEK cells, and/or k) does not induce pSyk in TREM2/DAP12 overexpressing HEK or iPSC macrophages in the absence of cross-linking, and/or l) shows as bispecific anti-TREM2/Abeta protein antibody after peripheral administration in vivo enrichment at plaques in APPswePS2 transgenic mice, and/or m) modulates as bispecific anti-TREM2/Abeta protein antibody neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid, and/or n) has non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and/or o) provides as bispecific anti-TREM2/Abeta protein antibody for plaque retention and plaque-targeted brain exposures, preferably with higher local concentrations as an anti-TREM2 monospecific antibody, and/or p) does not induce TNFalpha, MIP-lalpha or IL-8 release from iPSC- derived macrophages, and/or q) blocks shedding of sTREM2, and/or r) stabilizes sTREM2 in biological fluids, and/or s) binds to the ECD of TREM2 and thereby results i the accumulation of sTREM2 in biological fluids, and/or t) induces as bispecific anti-TREM2/Abeta protein antibody the shift from homeostatic or less engaged towards activated microglia, and/or u) induces as bispecific anti-TREM2/Abeta protein antibody ARIA at a lower level than a monospecific antibody, preferably does not induce ARIA.

In one aspect, the invention provides an anti-TREM2 antibody comprising i) a first binding site binding to TREM2 comprising a CDR-H1, a CDR-H2, a CDR-H3 and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequence of

SEQ ID NO: 129-131 and 133-135, or

SEQ ID NO: 137-139 and 141-143, or

SEQ ID NO: 145-147 and 149-151, or

SEQ ID NO: 153-155 and 157-159, or

SEQ ID NO: 161-163 and 165-167, or

SEQ ID NO: 169-171 and 173-175, or

SEQ ID NO: 177-179 and 181-183, or

SEQ ID NO: 185-187 and 189-191, or SEQ ID NO: 193-195 and 197-199, or

SEQ ID NO: 201-203 and 205-207, or

SEQ ID NO: 209-211 and 213-215, or

SEQ ID NO: 217-219 and 221-223, or

SEQ ID NO: 225-227 and 229-231, or

SEQ ID NO: 233-235 and 237-239, or

SEQ ID NO: 241-243 and 245-247, or

SEQ ID NO: 249-251 and 253-255, or

SEQ ID NO: 257-259 and 261-263, or

SEQ ID NO: 265-267 and 269-271, or

SEQ ID NO: 273-275 and 277-279, or

SEQ ID NO: 281-283 and 285-287, or

SEQ ID NO: 289-291 and 293-295, or

SEQ ID NO: 297-299 and 301-303, or-

SEQ ID NO: 305-307 and 309-311, or

SEQ ID NO: 313-315 and 317-319, or

SEQ ID NO: 321-323 and 325-327, or

SEQ ID NO: 329-331 and 333-335, or

SEQ ID NO: 337-339 and 341-343, or

SEQ ID NO: 345-347 and 349-351.

In another aspect, an antibody of the invention comprises

(a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from

SEQ ID NO: 129-131, or

SEQ ID NO: 137-139, or

SEQ ID NO: 145-147, or

SEQ ID NO: 153-155, or

SEQ ID NO: 161-163, or

SEQ ID NO: 169-171, or

SEQ ID NO: 177-179, or

SEQ ID NO: 185-187, or SEQIDNO: 193-195, or

SEQIDNO: 201-203, or

SEQIDNO: 209-211, or

SEQIDNO: 217-219, or

SEQIDNO: 225-227, or

SEQIDNO: 233-235, or

SEQIDNO: 241-243, or

SEQIDNO: 249-251, or

SEQIDNO: 257-259, or

SEQIDNO: 265-267, or

SEQIDNO: 273-275, or

SEQIDNO: 281-283, or

SEQIDNO: 289-291, or

SEQIDNO: 297-299, or-

SEQIDNO: 305-307, or

SEQIDNO: 313-315, or

SEQIDNO: 321-323, or

SEQIDNO: 329-331, or

SEQIDNO: 337-339, or

SEQIDNO: 345-347. or (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from

SEQIDNO: 133-135, or

SEQIDNO: 141-143, or

SEQIDNO: 149-151, or

SEQIDNO: 157-159, or

SEQIDNO: 165-167, or

SEQIDNO: 173-175, or

SEQIDNO: 181-183, or

SEQIDNO: 189-191, or

SEQIDNO: 197-199, or

SEQIDNO: 205-207, or SEQIDNO: 213-215, or

SEQIDNO: 221-223, or

SEQIDNO: 229-231, or

SEQIDNO: 237-239, or

SEQIDNO: 245-247, or

SEQIDNO: 253-255, or

SEQIDNO: 261-263, or

SEQIDNO: 269-271, or

SEQIDNO: 277-279, or

SEQIDNO: 285-287, or

SEQIDNO: 293-295, or

SEQIDNO: 301-303, or-

SEQIDNO: 309-311, or

SEQIDNO: 317-319, or

SEQIDNO: 325-327, or

SEQIDNO: 333-335, or

SEQIDNO: 341-343, or

SEQIDNO: 349-351.

In any of the aspects provided herein, an anti-TREM2 antibody is humanized. In one aspect, an anti-TREM2 antibody further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-TREM2 antibody comprises one or more of the CDR sequences of the VH of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or

SEQ ID NO: 260, or

SEQIDNO: 268, or

SEQ ID NO: 276, or

SEQIDNO: 284, or

SEQ ID NO: 292, or

SEQIDNO: 300, or

SEQIDNO: 308, or

SEQIDNO: 316, or

SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQIDNO: 348.

In another embodiment, an anti-TREM2 antibody comprises one or more of the CDR sequences of the VL of

SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or

SEQIDNO: 352.

In another embodiment, an anti-TREM2 antibody comprises the CDR sequences of the VH of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or SEQIDNO: 228, or SEQIDNO: 236, or SEQ ID NO: 244, or SEQIDNO: 252, or SEQ ID NO: 260, or SEQIDNO: 268, or SEQ ID NO: 276, or SEQIDNO: 284, or SEQ ID NO: 292, or SEQIDNO: 300, or SEQIDNO: 308, or SEQIDNO: 316, or SEQIDNO: 324, or SEQIDNO: 323, or SEQIDNO: 340, or SEQIDNO: 348, and the CDR sequences of the VL of SEQIDNO: 136, or SEQIDNO: 144, or SEQIDNO: 152, or SEQIDNO: 160, or SEQIDNO: 168, or SEQIDNO: 176, or SEQIDNO: 184, or SEQIDNO: 192, or SEQ ID NO: 200, or SEQIDNO: 208, or SEQIDNO: 216, or SEQ ID NO: 224, or SEQIDNO: 232, or SEQ ID NO: 240, or SEQIDNO: 248, or SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or

SEQIDNO: 352.

In a further aspect, an anti-TREM2 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 308 or 332 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 312 or 336.

In one aspect, an anti-TREM2 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or SEQIDNO: 228, or SEQIDNO: 236, or SEQ ID NO: 244, or SEQIDNO: 252, or SEQ ID NO: 260, or SEQIDNO: 268, or SEQ ID NO: 276, or SEQIDNO: 284, or SEQ ID NO: 292, or SEQIDNO: 300, or SEQIDNO: 308, or SEQIDNO: 316, or SEQIDNO: 324, or SEQIDNO: 323, or SEQIDNO: 340, or SEQIDNO: 348 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or

SEQ ID NO: 260, or

SEQIDNO: 268, or

SEQ ID NO: 276, or

SEQIDNO: 284, or

SEQ ID NO: 292, or

SEQIDNO: 300, or

SEQIDNO: 308, or

SEQIDNO: 316, or

SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQIDNO: 348.

In one aspect, the anti-TREM2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of

SEQIDNO: 132, or

SEQ ID NO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or SEQIDNO: 252, or SEQ ID NO: 260, or SEQIDNO: 268, or SEQ ID NO: 276, or SEQIDNO: 284, or SEQ ID NO: 292, or SEQIDNO: 300, or SEQIDNO: 308, or SEQIDNO: 316, or SEQIDNO: 324, or SEQIDNO: 323, or SEQIDNO: 340, or SEQIDNO: 348 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or SEQ ID NO: 260, or

SEQIDNO: 268, or

SEQ ID NO: 276, or

SEQIDNO: 284, or

SEQ ID NO: 292, or

SEQIDNO: 300, or

SEQIDNO: 308, or

SEQIDNO: 316, or

SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQIDNO: 348.

In one aspect, the anti-TREM2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or

SEQ ID NO: 260, or

SEQIDNO: 268, or SEQ ID NO: 276, or

SEQIDNO: 284, or

SEQ ID NO: 292, or

SEQ ID NO: 300, or

SEQIDNO: 308, or

SEQIDNO: 316, or

SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQ ID NO: 348 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or

SEQ ID NO: 260, or

SEQIDNO: 268, or

SEQ ID NO: 276, or

SEQIDNO: 284, or SEQ ID NO: 292, or

SEQIDNO: 300, or

SEQ ID NO: 308, or

SEQIDNO: 316, or

SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQIDNO: 348.

In another aspect, the anti-TREM2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or

SEQ ID NO: 260, or

SEQIDNO: 268, or

SEQ ID NO: 276, or

SEQIDNO: 284, or

SEQ ID NO: 292, or

SEQIDNO: 300, or SEQIDNO: 308, or

SEQIDNO: 316, or

SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQIDNO: 348 and a framework of at least of at least 98% sequence identity to the framework amino acid sequence of the VH domain of

SEQIDNO: 132, or

SEQIDNO: 140, or

SEQIDNO: 148, or

SEQIDNO: 156, or

SEQIDNO: 164, or

SEQIDNO: 172, or

SEQIDNO: 180, or

SEQIDNO: 188, or

SEQIDNO: 196, or

SEQ ID NO: 204, or

SEQIDNO: 212, or

SEQ ID NO: 220, or

SEQIDNO: 228, or

SEQIDNO: 236, or

SEQ ID NO: 244, or

SEQIDNO: 252, or

SEQ ID NO: 260, or

SEQIDNO: 268, or

SEQ ID NO: 276, or

SEQIDNO: 284, or

SEQ ID NO: 292, or

SEQIDNO: 300, or

SEQIDNO: 308, or

SEQIDNO: 316, or SEQIDNO: 324, or

SEQIDNO: 323, or

SEQIDNO: 340, or

SEQIDNO: 348.

In one aspect, an anti-TREM2 antibody comprises one or more of the light chain

CDR amino acid sequences of the VL domain of

SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or

SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or SEQIDNO: 344, or

SEQIDNO: 352, and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of

SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or

SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or SEQIDNO: 352.

In one aspect, the anti-TREM2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of

SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or

SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or

SEQIDNO: 352 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of

SEQIDNO: 136, or SEQIDNO: 144, or SEQIDNO: 152, or SEQIDNO: 160, or SEQIDNO: 168, or SEQIDNO: 176, or SEQIDNO: 184, or SEQIDNO: 192, or SEQ ID NO: 200, or SEQIDNO: 208, or SEQIDNO: 216, or SEQ ID NO: 224, or SEQIDNO: 232, or SEQ ID NO: 240, or SEQIDNO: 248, or SEQIDNO: 256, or SEQ ID NO: 264, or SEQ ID NO: 272, or SEQIDNO: 280, or SEQIDNO: 288, or SEQ ID NO: 296, or SEQIDNO: 304, or SEQIDNO: 312, or SEQIDNO: 320, or SEQIDNO: 328, or SEQIDNO: 336, or SEQIDNO: 344, or SEQIDNO: 352. In one aspect, the anti-TREM2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of

SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or

SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or

SEQIDNO: 352 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or

SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or

SEQIDNO: 352.

In another aspect, the anti-TREM2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of

SEQIDNO: 136, or

SEQIDNO: 144, or SEQIDNO: 152, or

SEQIDNO: 160, or

SEQIDNO: 168, or

SEQIDNO: 176, or

SEQIDNO: 184, or

SEQIDNO: 192, or

SEQ ID NO: 200, or

SEQIDNO: 208, or

SEQIDNO: 216, or

SEQ ID NO: 224, or

SEQIDNO: 232, or

SEQ ID NO: 240, or

SEQIDNO: 248, or

SEQIDNO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQIDNO: 280, or

SEQIDNO: 288, or

SEQ ID NO: 296, or

SEQIDNO: 304, or

SEQIDNO: 312, or

SEQIDNO: 320, or

SEQIDNO: 328, or

SEQIDNO: 336, or

SEQIDNO: 344, or

SEQIDNO: 352 and a framework of at least particularly of at least 98% sequence identity to the framework amino acid sequence of the VH domain of

SEQIDNO: 136, or

SEQIDNO: 144, or

SEQIDNO: 152, or

SEQIDNO: 160, or SEQ ID NO: 168, or

SEQ ID NO: 176, or

SEQ ID NO: 184, or

SEQ ID NO: 192, or

SEQ ID NO: 200, or

SEQ ID NO: 208, or

SEQ ID NO: 216, or

SEQ ID NO: 224, or

SEQ ID NO: 232, or

SEQ ID NO: 240, or

SEQ ID NO: 248, or

SEQ ID NO: 256, or

SEQ ID NO: 264, or

SEQ ID NO: 272, or

SEQ ID NO: 280, or

SEQ ID NO: 288, or

SEQ ID NO: 296, or

SEQ ID NO: 304, or

SEQ ID NO: 312, or

SEQ ID NO: 320, or

SEQ ID NO: 328, or

SEQ ID NO: 336, or

SEQ ID NO: 344, or SEQ ID NO: 352.

In one aspect, the anti-TREM2 antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 305; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 306; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 307; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 309; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 310; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 311, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 308, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 312. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 308. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 312.

In one aspect, the anti-TREM2 antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 305; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 306; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 307; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 309; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 310; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 311, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 308, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 312, wherein the antibody specifically binds to TREM2. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 308. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 312.

In one aspect, the anti-TREM2 antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 329; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 330; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 331; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 333; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 334; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 335, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 332, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 336. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 332. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 336.

In one aspect, the anti-TREM2 antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 329; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 330; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 331; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 333; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 334; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 335, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 332, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 336; wherein the antibody specifically binds to TREM2. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 332. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 336.

In another aspect, an anti-TREM2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 308. In one aspect, an anti-TREM2 antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 308. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TREM2 antibody comprising that sequence retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 308. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-TREM2 antibody comprises the VH sequence of SEQ ID NO: 308, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 305, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 306, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 307. In another aspect, an anti-TREM2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 312. In one aspect, an anti-TREM2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 312. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TREM2 antibody comprising that sequence retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 312. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-TREM2 antibody comprises the VL sequence of SEQ ID NO: 312, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 309, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 310, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 311.

In another aspect, an anti-TREM2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 332. In one aspect, an anti-TREM2 antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 332. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TREM2 antibody comprising that sequence retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 332. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-TREM2 antibody comprises the VH sequence in SEQ ID NO: 332, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 329, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 330, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 331. In another aspect, an anti-TREM2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 336. In one aspect, an anti-TREM2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 336. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TREM2 antibody comprising that sequence retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 336. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-TREM2 antibody comprises the VL sequence of SEQ ID NO: 336, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 333, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 334, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 335.

In one preferred aspect, an anti-TREM2 antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 308 and SEQ ID NO: 312, respectively, including post-translational modifications of those sequences. In one embodiment, the heavy chain variable domain comprises in addition as first N-terminal amino acid residue a glutamic acid (E) residue or a pyroglutamic acid (pE) residue. In another preferred aspect, an anti-TREM2 antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 332 and SEQ ID NO: 336, respectively, including post-translational modifications of those sequences. In one embodiment, the heavy chain variable domain comprises in addition as first N- terminal amino acid residue a glutamic acid (E) residue or a pyroglutamic acid (pE) residue.

In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-TREM2 antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as TREM23295 or TREM23306. In certain aspects, an antibody is provided that binds to an epitope within a fragment of TREM2 consisting of amino acids 41-45 of SEQ ID NO: 424 (TREM2 3295) or amino acids 67-68 and 74-80 of SEQ ID NO: 424.

In a further aspect of the invention, an anti-TREM2 antibody according to any of the above aspects is a monoclonal antibody, including a chimeric, humanized or human antibody. In one aspect, an anti-TREM2 antibody is an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment that has the same paratope as of the full-length antibody.

In another aspect, the antibody is a full-length antibody, e.g., an intact, e.g., IgGl or IgGl LALAPG antibody or other antibody class or isotype as defined herein.

The terms “anti-TREM2 antibody” and “an antibody that binds to TREM2” refer to an antibody that is capable of binding human TREM2 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TREM2. In one aspect, the extent of binding of an anti-TREM2 antibody to an unrelated, non-TREM2 protein is less than about 10% of the binding of the antibody to TREM2 as measured, e.g., by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to TREM2 has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM (e.g., IE-8 M or less, e.g., from IE- 8 M to 10-11 M, e.g., from IE-9 M to IE-11 M). An antibody is said to “specifically bind” to TREM2 when the antibody has a KD of 1 pM or less.

In certain aspects, an antibody provided herein has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM (e.g., IE-8 M or less, e.g., from IE-8 M to 10-11 M, e.g., from IE-9 M to IE-11 M).

In one aspect, KD is measured using a BIACORE® surface plasmon resonance assay.

Anti-TREM2/Abeta protein antibodies

In one aspect, the invention provides bispecific antibodies that bind to human TREM2 and human amyloid beta protein. In one aspect, provided are isolated bispecific antibodies that bind to human TREM2 and human amyloid beta protein. In one aspect, the invention provides bispecific antibodies that specifically bind to human TREM2 and human amyloid beta protein.

A bispecific antibody according to the current invention that binds or specifically binds to human TREM2 and human amyloid beta protein (Abeta protein) is referred to as anti-TREM2/amyloid beta protein bispecific antibody.

In one preferred embodiment, human Abeta protein comprises the amino acid sequence of SEQ ID NO: 467.

The term “amyloid plaque” denotes aggregates of misfolded proteins that form in the spaces between nerve cells. These abnormally configured proteins are thought to play a central role in Alzheimer's disease. The amyloid plaques first develop in the areas of the brain concerned with memory and other cognitive functions.

The term “(human) Abeta protein binding site” refers to a binding site of an antibody that is capable of binding the human Abeta protein with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting human Abeta protein. It is of note that the human Abeta protein has several naturally occurring forms, whereby the human forms are referred to as Ap39, Ap40, Ap41, Ap42 and Ap43. The most prominent form, Ap42, has the amino acid sequence of SEQ ID NO: 467. In Ap41, Ap40, Ap39, the C-terminal amino acids A, IA and VIA are missing, respectively. In Ap43, an additional threonine residue is comprised at the C-terminus of SEQ ID NO: 467. In one preferred embodiment, the antibody according to the invention specifically binds to the human Abeta protein that has the amino acid sequence of SEQ ID NO: 467.

AMYLOID BETA PROTEIN

Amyloid beta (Ap or Abeta) denotes peptides of 36-43 amino acids that are the main component of the amyloid plaques found in the brains of people with Alzheimer's disease. The peptides derive from the amyloid-beta precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield Ap in a cholesteroldependent process and substrate presentation. Ap molecules can aggregate to form flexible soluble oligomers that may exist in several forms. It is now believed that certain misfolded oligomers (known as "seeds") can induce other Ap molecules to also take the misfolded oligomeric form, leading to a chain reaction akin to a prion infection. The oligomers are toxic to nerve cells. The other protein implicated in Alzheimer's disease, tau protein, also forms such prion-like misfolded oligomers, and there is some evidence that misfolded Ap can induce tau to misfold.

A study has suggested that APP and its amyloid potential is of ancient origins, dating as far back as early deuterostomes.

Ap is the main component of amyloid plaques, extracellular deposits found in the brains of people with Alzheimer's disease. Ap can also form the deposits that line cerebral blood vessels in cerebral amyloid angiopathy. The plaques are composed of a tangle of Ap oligomers and regularly ordered aggregates called amyloid fibrils, a protein fold shared by other peptides such as the prions associated with protein misfolding diseases. Research suggests that soluble oligomeric forms of the peptide may be causative agents in the development of Alzheimer's disease. It is generally believed that Ap oligomers are the most toxic. The ion channel hypothesis postulates that oligomers of soluble, non-fibrillar Ap form membrane ion channels allowing the unregulated calcium influx into neurons that underlies disrupted calcium ion homeostasis and apoptosis seen in Alzheimer's disease. Computational studies have demonstrated that also Ap peptides embedded into the membrane as monomers with predominant helical configuration, can oligomerize and eventually form channels whose stability and conformation are sensitively correlated to the concomitant presence and arrangement of cholesterol. A number of genetic, cell biology, biochemical and animal studies using experimental models support the concept that Ap plays a central role in the development of Alzheimer's disease pathology.

Brain Ap is elevated in people with sporadic Alzheimer's disease. Ap is the main constituent of brain parenchymal and vascular amyloid; it contributes to cerebrovascular lesions and is neurotoxic. It is unresolved how Ap accumulates in the central nervous system and subsequently initiates the disease of cells. Some researchers have found that the Ap oligomers induce some of the symptoms of Alzheimer's disease by competing with insulin for binding sites on the insulin receptor, thus impairing glucose metabolism in the brain. Significant efforts have been focused on the mechanisms responsible for Ap production, including the proteolytic enzymes gamma- and P-secretases, which generate Ap from its precursor protein, APP (amyloid precursor protein). Ap circulates in plasma, cerebrospinal fluid (CSF) and brain interstitial fluid (ISF) mainly as soluble Ap40 Amyloid plaques contain both Ap40 and Ap42, while vascular amyloid is predominantly the shorter Ap40. Several sequences of Ap were found in both lesions. Generation of Ap in the central nervous system may take place in the neuronal axonal membranes after APP- mediated axonal transport of P-secretase and presenilin-1.

Increases in either total Ap levels or the relative concentration of both Ap40 and Ap42 (where the former is more concentrated in cerebrovascular plaques and the latter in neuritic plaques) have been implicated in the pathogenesis of both familial and sporadic Alzheimer's disease. Due to its more hydrophobic nature, the Ap42 is the most amyloidogenic form of the peptide. However the central sequence KLVFFAE (SEQ ID NO: 466) is known to form amyloid on its own, and probably forms the core of the fibril. One study further correlated Ap42 levels in the brain not only with onset of Alzheimer's disease, but also reduced cerebrospinal fluid pressure, suggesting that a build-up or inability to clear Ap42 fragments may play a role in the pathology.

The "amyloid hypothesis" assumes that the plaques are responsible for the pathology of Alzheimer's disease, is accepted by the majority of researchers but is not conclusively established. An alternative hypothesis is that amyloid oligomers rather than plaques are responsible for the disease. Mice that are genetically engineered to express oligomers but not plaques (APPE693Q) develop the disease. Furthermore, mice that are in addition engineered to convert oligomers into plaques (APPE693Q X PS 1 AE9) are no more impaired than the oligomer only mice. Intra-cellular deposits of tau protein are also seen in the disease, and may also be implicated, as has aggregation of alpha synuclein.

In certain aspects, an anti-TREM2/Abeta protein bispecific antibody a) induces phagocytosis of amyloid plaques by macrophages, and/or b) induces microglial amyloid uptake in the absence of FcgRII as well as FcgRIII receptor engagement, and/or c) induces amyloid uptake in the absence of engagement of FcgR, and/or d) without effector-function induces acute uptake of MX04 labeled amyloid and Abeta protein in APPswePS2 transgenic mice, preferably in the absence of Abeta and FcgR crosslinking, and/or e) does induce pSyk in the presence of human Abeta protein, and/or f) shows after peripheral administration in vivo enrichment at plaques in APPswePS2 transgenic mice, and/or g) modulates neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid, and/or h) provides for plaque retention and plaque-targeted brain exposures, preferably with higher local concentrations as an anti-TREM2 monospecific antibody, and/or i) induces the shift from homeostatic or less engaged towards activated microglia, and/or j) induces ARIA at a lower level than a monospecific antibody, preferably does not induce ARIA.

In one aspect, the invention provides an anti-TREM2/Abeta protein bispecific antibody comprising i) a first binding site binding to TREM2 and ii) a second binding site binding to amyloid beta protein. In certain embodiments, the binding sites are specifically binding to their target.

In certain embodiments, the binding site binding to TREM2 comprises a CDR-H1, a CDR-H2, a CDR-H3 and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequence of

SEQ ID NO: 129-131 and 133-135, or

SEQ ID NO: 137-139 and 141-143, or

SEQ ID NO: 145-147 and 149-151, or

SEQ ID NO: 153-155 and 157-159, or

SEQ ID NO: 161-163 and 165-167, or

SEQ ID NO: 169-171 and 173-175, or

SEQ ID NO: 177-179 and 181-183, or

SEQ ID NO: 185-187 and 189-191, or

SEQ ID NO: 193-195 and 197-199, or

SEQ ID NO: 201-203 and 205-207, or

SEQ ID NO: 209-211 and 213-215, or

SEQ ID NO: 217-219 and 221-223, or

SEQ ID NO: 225-227 and 229-231, or SEQ ID NO: 233-235 and 237-239, or SEQ ID NO: 241-243 and 245-247, or SEQ ID NO: 249-251 and 253-255, or SEQ ID NO: 257-259 and 261-263, or SEQ ID NO: 265-267 and 269-271, or SEQ ID NO: 273-275 and 277-279, or SEQ ID NO: 281-283 and 285-287, or SEQ ID NO: 289-291 and 293-295, or SEQ ID NO: 297-299 and 301-303, or- SEQ ID NO: 305-307 and 309-311, or SEQ ID NO: 313-315 and 317-319, or SEQ ID NO: 321-323 and 325-327, or SEQ ID NO: 329-331 and 333-335, or SEQ ID NO: 337-339 and 341-343, or SEQ ID NO: 345-347 and 349-351.

In certain embodiments, the binding site binding to Abeta protein comprises a CDR- Hl, a CDR-H2, a CDR-H3 and a CDR-L1, a CDR-L2, a CDR-L3 comprising the amino acid sequence of

SEQ ID NO: 09-11 and 13-15.

In one aspect, the binding site binding to TREM2 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 305; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 306; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 307; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 309; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 310; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 311, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 308, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 312. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 308. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 312.

In one aspect, the binding site binding to TREM2 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 305; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 306; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 307; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 309; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 310; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 311, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 308, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 312, wherein the binding site specifically binds to TREM2. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 308. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 312.

In one aspect, the binding site binding to TREM2 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 329; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 330; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 331; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 333; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 334; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 335, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 332, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 336. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 332. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 336. In one aspect, the binding site binding to TREM2 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 329; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 330; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 331; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 333; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 334; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 335, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 332, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 336; wherein the binding site specifically binds to TREM2. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 332. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 336.

In another aspect, the binding site binding to TREM2 comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 308. In one aspect, the binding site binding to TREM2 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 308. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 308. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the binding site binding to TREM2 comprises the VH sequence in SEQ ID NO: 308, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR- Hl, comprising the amino acid sequence of SEQ ID NO: 305, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 306, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 307. In another aspect, a binding site binding to TREM2 is provided, wherein the binding site comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 312. In one aspect, the binding site binding to TREM2 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 312. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that binding site retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 312. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the binding site binding to TREM2 comprises the VL sequence in SEQ ID NO: 312, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO: 309, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 310, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 311.

In another aspect, the binding site binding to TREM2 comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 332. In one aspect, the binding site binding to TREM2 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 332. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that binding site retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 332. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the binding site binding to TREM2 comprises the VH sequence in SEQ ID NO: 332, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR- Hl, comprising the amino acid sequence of SEQ ID NO: 329, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 330, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 331. In another aspect, a binding site binding to TREM2 is provided, wherein the binding site comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 336. In one aspect, the binding site binding to TREM2 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 336. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that binding site retains the ability to bind to TREM2. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 336. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the binding site binding to TREM2 comprises the VL sequence of SEQ ID NO: 336, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO: 333, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 334, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 335.

In another aspect, a binding site binding to TREM2 is provided, wherein the binding site comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the binding site comprises the VH and VL sequences of SEQ ID NO: 308 and SEQ ID NO: 312, respectively, including post-translational modifications of those sequences.

In another aspect, a binding site binding to TREM2 is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the binding site comprises the VH and VL sequences of SEQ ID NO: 332 and SEQ ID NO: 336, respectively, including post-translational modifications of those sequences.

In one aspect, the anti-Abeta protein binding site comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16.

In one aspect, the anti-Abeta protein binding site comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16, wherein the binding site specifically binds to Abeta protein. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In another aspect, the Abeta protein binding site comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the anti-Abeta protein binding site comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 12. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that binding site retains the ability to bind to Abeta protein. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 12. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the Abeta binding site comprises the VH sequence of SEQ ID NO: 12, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 09, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 11. In another aspect, an Abeta binding site is provided, wherein the binding site comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the Abeta binding site comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that binding site retains the ability to bind to Abeta protein. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 16. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the Abeta binding site comprises the VL sequence in SEQ ID NO: 16, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 13, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 14, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, an Abeta binding site is provided, wherein the binding site comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the Abeta binding site comprises the VH and VL sequences of SEQ ID NO: 12 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences. In one aspect, the Abeta binding site comprises the VH and VL sequences of SEQ ID NO: 12 and SEQ ID NO: 16, respectively, wherein the heavy chain variable domain comprises as first N-terminal amino acid residue in addition a glutamine (Q) residue or a pyroglutamic acid (pE) residue.

In another aspect, the anti-TREM2/ Abeta protein antibody comprises a binding site binding to Abeta protein comprising: a VH of SEQ ID NO: 12 and a VL of SEQ ID NO: 16, or a VH of SEQ ID NO: 468 and a VL of SEQ ID NO: 469, or a VH of SEQ ID NO: 470 and a VL of SEQ ID NO: 471, or a VH of SEQ ID NO: 472 and a VL of SEQ ID NO: 473.

In another aspect, an anti-TREM2/ Abeta protein bispecific antibody comprises a first binding site comprising one or more of the CDR sequences of the VH of SEQ ID NO: 308 or 332 and a second binding site comprising one or more of the CDR sequences of the VH of SEQ ID NO: 12. In another embodiment, an anti- TREM2/ Abeta protein bispecific antibody comprises a first binding site comprising one or more of the CDR sequences of the VL of SEQ ID NO: 312 or 336 and a second binding site comprising one or more of the CDR sequences of the VL of SEQ ID NO: 16. In another embodiment, an anti-TREM2/ Abeta protein bispecific antibody comprises a first binding site with the CDR sequences of the VH of SEQ ID NO: 308 and the CDR sequences of the VL of SEQ ID NO: 312 and a second binding site with the CDR sequences of the VH of SEQ ID NO: 12 and the CDR sequences of the VL of SEQ ID NO: 16. In another embodiment, an anti- TREM2/Abeta protein bispecific antibody comprises a first binding site with the CDR sequences of the VH of SEQ ID NO: 332 and the CDR sequences of the VL of SEQ ID NO: 336 and a second binding site with the CDR sequences of the VH of SEQ ID NO: 12 and the CDR sequences of the VL of SEQ ID NO: 16.

In one preferred aspect of the current invention, the anti-TREM2/Abeta protein bispecific antibody comprises a first light chain comprising an amino acid sequence of SEQ ID NO: 474, a first heavy chain comprising an amino acid sequence of SEQ ID NO: 475, a second light chain comprising an amino acid sequence of SEQ ID NO: 476, and a second heavy chain comprising an amino acid sequence of SEQ ID NO: 477. In one embodiment, the first light chain comprises as first N-terminal amino acid residue a glutamine (Q) residue or a pyroglutamic acid (pE) residue and the second heavy chain comprises as first N-terminal amino acid residue a glutamic acid (E) residue or a pyroglutamic acid (pE) residue. In one embodiment, the first and the second heavy chain comprise independently of each other a lysine residue as last C-terminal amino acid residue.

In a further aspect of the invention, an anti-TREM2/Abeta protein bispecific antibody according to any of the above aspects is a monoclonal antibody, including a chimeric, humanized or human antibody. In one aspect, an anti-TREM2/Abeta protein bispecific antibody is an antibody fragment, e.g., an Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment that has the same paratopes as of the full-length antibody.

In another aspect, the antibody is a full-length antibody, e.g., an intact, e.g., IgGl or IgGl LALAPG antibody or other antibody class or isotype as defined herein.

In any of the aspects provided herein, an anti-TREM2/Abeta protein bispecific antibody according to the current invention is humanized. In one aspect, an anti- TREM2/Abeta protein bispecific antibody according to the current invention further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

Exemplary Antibody Variants, Fragments, and Constant Regions In a further aspect, an antibody according to the current invention may incorporate any of the features, singly or in combination, as described in the sections below.

Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, F(ab’)2, and (scFv)2 fragments, and other fragments as described below as long as these are at least bispecific. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and US 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US 5,869,046.

In another aspect, the antibody fragment is a diabody, a triabody or a tetrabody. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent and bispecific. See, for example, EP 404 097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a humanized single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells, as described herein. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in US 4,816,567; and Morrison et al., Proc. Natl. Acad. Set. USA, 81 :6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof as long as these bind to the respective targets.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Set. USA 86: 10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’ Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

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. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Pro c. 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)).

In some embodiments, the humanized antibodies may comprise a human IgGl heavy chain constant region.

Bispecific or Multispecific Antibodies

An antibody provided herein is a multispecific antibody, for example, a bispecific antibody or a trispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens or epitopes of the same antigen. In certain embodiments, one of the binding specificities is for human TREM2 and the other is for human amyloid beta protein. In certain embodiments, multispecific antibodies may bind to two different epitopes of TREM2. Bispecific antibodies may also be used to localize drugs such as cytotoxic agents or to localize detection labels to cells that express TREM2. In some embodiments, the multispecific antibody (e.g., bispecific antibody) comprises a first variable domain comprising the CDRs or variable regions as described herein. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., US 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 128(5): 1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444- 6448 (1993)); and using single-chain Fv (scFv) dimers ((scFv)?) see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576.

Further Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, 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. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table A under the heading of "preferred substitutions." More substantial changes are provided in Table A under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or increased or reduced ADCC or CDC activity.

TABLE A

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g, improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g, to improve antibody affinity. Such alterations may be made in HVR “hotspots”, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR- L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind its antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen contacting residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties. Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

Glycosylation variants

Alterations in glycosylation of an Fc-region, as well as certain Fc-region mutations, can impact the effector function of an antibody, by enhancing or reducing effector function, or in some cases may render an antibody effector function silent (effectorless).

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc-region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc-region. See, e.g., Wright et al. TIBTECH 15:26- 32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one aspect, antibodies may be modified to reduce or eliminate glycosylation at Asn297, such as by mutating that residue to a glycine or another amino acid (N297G). In other cases, other residues in an Fc-region may be modified to reduce ADCC activity and/or CDC activity or to reduce or modify Fc-gamma receptor binding.

In another embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc-region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc-region (EU numbering of Fc-region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, z.e., between positions 294 and 300 (EU numbering), due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO 2003/085107). In some embodiments, antibodies may have a human IgG heavy chain constant region, for example, comprising a mutation at Asn297 (EU numbering) to decrease fucosylation or alternatively, to eliminate glycosylation. In some embodiments, antibodies according to the invention may have an Asn297Ala or Asn297Gly mutation. Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc-region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; US 6,602,684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc-region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Fc-region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc-region of an antibody provided herein, thereby generating an Fc-region variant. The Fc-region variant may comprise a human Fc-region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions (i.e. to produce an antibody with reduced effector function or to produce an effector function silent antibody), which may make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc-receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR (FcgR) binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in US 5,500,362 (see, e.g., Hellstrom, I., et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I., et al., Proc. Nat ’I Acad. Sci. USA 82: 1499-1502 (1985); US 5,821,337 (see Bruggemann, M., et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that reported in Clynes et al. Proc. Nat ’I Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int. Immunol. 18(12): 1759- 1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc-region residues 234, 235, 238, 265, 269, 270, 297, 327 and 329 (see, e.g., US 6,737,056; EU numbering of residues). Such Fc-region mutants include Fc- region mutants with substitutions at two or more of amino acid positions 234, 235, 265, 269, 270, 297, 327, and 329 including the so-called “DANA” Fc-region mutant with substitution of residues 265 and 297 to alanine (US 7,332,581; EU numbering) and the so-called “PGLALA” Fc-region mutant with substitution of residues 329, 234 and 235 to glycine, alanine and alanine, respectively (WO 2012/130831). In some embodiments, the antibody comprises an engineered alanine at amino acid position 265 according to EU numbering convention. In some embodiments, the antibody comprises an engineered alanine at amino acid position 297 according to EU numbering convention. Certain antibody variants with improved or diminished binding to FcRs are described, e.g., in US 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions, which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region (EU numbering of residues).

In some embodiments, alterations are made in the Fc-region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US 2005/0014934. Those antibodies comprise an Fc-region with one or more substitutions therein which improve binding of the Fc-region to FcRn. Such Fc variants include those with substitutions at one or more of Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 (EU numbering), e.g., substitution of Fc-region residue 434 (US 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); US 5,648,260; US 5,624,821; and WO 94/29351 concerning other examples of Fc- region variants.

In some embodiments, the antibody may have a wild-type human IgGl Fc-region or a wild-type human IgG4 Fc-region, a human IgG4 S228P Fc-region, a human IgG4 S228P/M252Y/S254T/T256E Fc-region, a human IgGl N297G Fc-region, a human IgGl LALAPG (L234A/L235A/P329G) Fc-region, a human IgGl N297G/M428L/N434S Fc region, or a human IgGl LALAPG YTE (L234A/L235A/P329G/M252Y/S254T/T256E) Fc-region. (All positions are in EU numbering.) In one preferred embodiment of all aspects and embodiments comprises the antibody according to the current invention a human IgGl LALAPG (L234A/L235A/P329G) Fc-region. Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., “thioMAbs”, in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linkerdrug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc-region. Cysteine engineered antibodies may be generated as described, e.g., in US 7,521,541.

KTG engineered antibody variants

In certain embodiments, it may be desirable to create KTG engineered antibodies, in which after one or more residues of an antibody a Q-tag has been inserted. In particular embodiments, the inserted residues occur at accessible sites of the antibody. In certain embodiments, the Q-tags is selected from RYGQR (SEQ ID NO: 430), RWRQR (SEQ ID NO: 431), YRQRT (SEQ ID NO: 432), IRQRQ (both Q’s can be modified; SEQ ID NO: 433), FRYRQ (SEQ ID NO: 434) and YRYRQ (SEQ ID NO: 435), preferably YRYRQ (SEQ ID NO: 435). By inserting those residues a reactive group is thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linkerdrug moieties, to create an immunoconjugate, as described further herein using the transglutaminase of Kutzneria albida (KTG). In certain embodiments, the Q-tag may be inserted after any one or more of position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297) position 341 (HC341) and position 401 (HC401) of the heavy chain (numbering according to Kabat), preferably HC297. In certain embodiments, the recognition site(s) for the transglutaminase from Kutzneria albida (KTG) is(are) inserted interspaced between two flexible peptide linkers. In certain embodiments, the recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) has the amino acid sequence GGGSYRYRQGGGS (SEQ ID NO: 439).

KTG engineered antibodies may be generated as described, e.g., in WO 2023/118398.

Fc-region variants

The term “(human) Fc-region polypeptide” denotes an amino acid sequence that is identical to a “native” or “wild-type” (human) Fc-region polypeptide. The term “variant (human) Fc-region polypeptide” denotes an amino acid sequence, which is derived from a “native” or “wild-type” (human) Fc-region polypeptide by virtue of at least one “amino acid alteration”. A “variant (human) Fc-region” is consisting of two Fc-region polypeptides, whereby both can be variant (human) Fc-region polypeptides or one is a (human) Fc-region polypeptide and the other is a variant (human) Fc-region polypeptide.

In certain embodiments, the human Fc-region polypeptide has the amino acid sequence of a human IgGl Fc-region polypeptide or is a variant thereof, or of a human IgG2 Fc-region polypeptide or is a variant thereof, or of a human IgG3 Fc- region polypeptide or is a variant thereof, or of a human IgG4 Fc-region polypeptide or is a variant thereof. In certain embodiments, the Fc-region polypeptide is derived from an Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449 and has at least one amino acid mutation compared to the Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In certain embodiments, the Fc-region polypeptide comprises/has from about one to about ten amino acid mutations. In certain embodiments, the Fc-region polypeptide comprises/has from about one to about five amino acid mutations.

In certain embodiments, the Fc-region polypeptide has the amino acid sequence of a human IgGl Fc-region polypeptide with the LALAPG mutations and the knobmutation (SEQ ID NO: 444), wherein optionally the C-terminal lysine residue is deleted. In certain embodiments, the Fc-region polypeptide has the amino acid sequence of a human IgGl Fc-region with the LALAPG mutations and the knob-cys or hole-cys mutations (SEQ ID NO: 448 or SEQ ID NO: 447), wherein optionally the C-terminal lysine residue is deleted.

In certain embodiments, the Fc-region polypeptide has at least about 80 % sequence homology with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In certain embodiments, the Fc-region polypeptide has at least about 90 % sequence homology with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In certain embodiments, the Fc-region polypeptide has at least about 95 % sequence homology with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In one preferred embodiment, the Fc-region polypeptide has at least about 97.5 % homology with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449.

In certain embodiments, the Fc-region polypeptide has at least about 80 % sequence identity with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In certain embodiments, the Fc-region polypeptide has at least about 90 % sequence identity with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In certain embodiments, the Fc-region polypeptide has at least about 95 % sequence identity with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In one preferred embodiment, the Fc-region polypeptide has at least about 97.5 % identity with a human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449. In one preferred embodiment, the C-terminal lysine residue is deleted.

A variant Fc-region polypeptide derived from a parent (human) Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449 is further defined by the amino acid alterations that are contained compared to the parent or wild-type sequence. Thus, for example, the term P329G denotes an Fc-region polypeptide derived from a (human) Fc-region polypeptide with the mutation of proline to glycine at amino acid position 329 relative to the human Fc-region polypeptide of SEQ ID NO: 437 or SEQ ID NO: 449 (numbering according to Kabat).

A human IgGl Fc-region polypeptide has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 437), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with the mutations L234A, L235A (LALA mutations) has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 438) , optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with Y349C, T366S, L368A and Y407V mutations (knob-cys-mutations) has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 439), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with S354C, T366W (hole- cys-mutations) mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 440), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with L234A, L235A mutations and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 441), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with a L234A, L235A and S354C, T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 442), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with a P329G mutation (PG mutation) has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 443), optionally with an additional lysine residue (K) added to the C-terminus. A human IgGl Fc-region derived Fc-region polypeptide with L234A, L235A mutations and P329G mutation (LALAPG mutations) has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 444), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with a P329G mutation and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 445), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with a P329G mutation and S354C, T366W mutation has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 446), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with L234A, L235A, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 447), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgGl Fc-region derived Fc-region polypeptide with L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 448), optionally with an additional lysine residue (K) added to the C-terminus.

A human IgG4 Fc-region polypeptide has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 449).

A human IgG4 Fc-region derived Fc-region polypeptide with S228P and L235E mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 450). A human IgG4 Fc-region derived Fc-region polypeptide with S228P, L235E mutations and P329G mutation has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 451).

A human IgG4 Fc-region derived Fc-region polypeptide with S354C, T366W mutations has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 452).

A human IgG4 Fc-region derived Fc-region polypeptide with Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 453).

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E and S354C, T366W mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 454). A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 455).

A human IgG4 Fc-region derived Fc-region polypeptide with a P329G mutation has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 456).

A human IgG4 Fc-region derived Fc-region polypeptide with a P329G and Y349C,

T366S, L368A, Y407V mutations has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 457).

A human IgG4 Fc-region derived Fc-region polypeptide with a P329G and S354C,

T366W mutations has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 458). A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 459).

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E, P329G and S354C, T366W mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 460).

Antibody Derivatives and Conjugates

In certain embodiments, an antibody provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to 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, propropylene glycol homopolymers, proly propylene oxide/ethylene oxide copolymers, poly oxy ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. 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.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Set. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed.

In some embodiments, an anti-TREM2/amyloid beta protein antibody according to the invention is conjugated to a detection label and/or a drug. As used herein, a detection label is a moiety that facilitates detection of the antibody and/or facilitates detection of a molecule to which the antibody binds. Non-limiting exemplary detection labels include, but are not limited to, radioisotopes, fluorescent groups, enzymatic groups, chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc.

Nucleic Acids Encoding Antibodies

Nucleic acid molecules comprising polynucleotides that encode one or more chains of anti-TREM2 antibodies are also provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an anti-TREM2 antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of an anti-TREM2 antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain. In some such embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.

In some embodiments, a polynucleotide encoding a heavy chain or light chain of an anti-TREM2 antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.

Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

Vectors and Host Cells and Methods of Production

Vectors comprising polynucleotides that encode anti-TREM2 heavy chains and/or anti-TREM2 light chains are provided. Vectors comprising polynucleotides that encode anti-TREM2 heavy chains and/or anti-TREM2 light chains are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5: 1 and 1 :5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1 : 1 and 1 :5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1 :2 for the vector encoding the heavy chain and the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., BiotechnoL Prog. 20:880-889 (2004).

In some embodiments, a vector is chosen for in vivo expression of anti-TREM2 heavy chains and/or anti-TREM2 light chains in animals, including humans. In some such embodiments, expression of the polypeptide is under the control of a promoter that functions in a tissue-specific manner. For example, liver-specific promoters are described, e.g., in WO 2006/076288

For recombinant production of an anti-TREM2 antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such 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 host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies 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, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli2) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are 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. See 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 are also 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. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, 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. 23:243-251 (1980)); 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 3 A); 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. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Anti-TREM2 antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the TREM2 ECD and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an anti-TREM2 antibody. Hydrophobic interaction chromatography, for example, a butyl or phenyl column, may also be suitable for purifying some polypeptides. Many methods of purifying polypeptides are known in the art. In some embodiments, an anti-TREM2 antibody is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21 : 695-713 (2003).

Methods of Pharmaceutical Use

Herein are also reported methods of using anti-TREM2 antibodies herein, for example, in pharmaceutical treatments. For example, reported herein are methods of treating a condition associated with TREM2 loss of function in a subject. The current invention also encompasses methods of reducing the shedding of membrane-bound TREM2 leading to lower levels of sTREM2 in a subject. In addition, the invention also encompasses methods to increase sTREM2 levels by binding of a TREM2 antibody to the ECD of TREM2 leading to adaptation of the clearance of sTREM2 to the clearance of the bound antibody thereby accumulating sTREM2 in a respective biological fluid (i.e., blood, cerebrospinal fluid or brain interstitial fluid).

In some cases, the condition is a neuroinflammatory or neurodegenerative disease. Examples include, for instance, Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hakola disease, Guillain-Barre Syndrome (GBS), lysosomal storage disease, sphingomyelinlipidosis (Neimann-Pick C), mucopolysaccharidosis II/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuroBehcet’s disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some cases, the condition is Alzheimer’s disease. In some cases, the condition is MS. In some cases, the condition is Parkinson’s Disease. For example, pathological hallmarks of Alzheimer’s Disease (AD) include extracellular deposits of beta-amyloid peptides that form amyloid plaques and intracellular deposits of aggregated hyperphosphorylated tau called neurofibrillary tangles. These pathologies are accompanied by an increased activation of immune pathways in the brain, including inflammatory activation of astrocytes and microglia, and concomitant synaptic loss and neurodegeneration with neuronal loss. Familial forms of AD may be caused by mutations in presenilin 1/2 and amyloid precursor protein genes. Transgenic mice expressing these human mutations as well as a mutation in the Tau protein show age-dependent increases in Abeta pathology, hyperphosphorylated Tau and neurodegeneration that are in some aspects similar to what is observed in the human brain. Certain mutations causing a loss of TREM2 function result in irregular microglial compaction of plaque, elevated neuritic dystrophy surrounding the plaque, elevated abeta-induced tau pathology and neurodegeneration. Without wishing to be bound by theory, enhancing TREM2 activity may enhance microglia activity and facilitate the removal of toxic amyloidbeta proteins, by engulfment or compaction into less toxic amyloid plaques, thus treating AD.

Multiple sclerosis (MS) is a disease characterized by an autoimmune related demyelination in the CNS. In patients, the disease presents in episodes of symptoms such as ataxia, limb weakness, and optic neuritis among other neurological effects. Current treatments involve immune suppressing agents, and may have limited effectiveness, while no therapy effectively prevents or reverses the disease. Remyelinating therapies have been proposed as an approach for treating and possibly reversing MS. Compounds and therapies that promote oligodendrocyte differentiation from progenitors (OPCs) to mature, myelinating oligodendrocytes have been considered a possible route to a remyelinating therapy, as well as treatments, which modify glial cells. Specifically, microglia are thought to have a role in clearing myelin debris, promoting new myelin to form. TREM2 loss of function leads to hypomyelinating leukodystrophy, and TREM2 knockout mice have a profound impairment in remyelination and recovery in animal models of MS. Without wishing to be bound by theory, activation of the TREM2 pathway may accelerate remyelination, thus treating MS. In various embodiments, anti-TREM2 antibodies may be administered in vivo by various routes, including, but not limited to, oral, intravenous, subcutaneous, parenteral, intranasal, intramuscular, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation of cells expressing the antibody or by inhalation or by gene therapy. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. A nucleic acid molecule encoding an anti-TREM2 antibody may be administered, either directly or in a vector such as a viral vector. The appropriate formulation and route of administration may be selected according to the intended application.

In various embodiments, compositions comprising anti-TREM2 antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmac^y with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3^rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In various embodiments, compositions comprising anti-TREM2 antibodies may be formulated for injection or infusion, by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In various embodiments, the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. The compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers. A non-limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid polymer. A non-limiting exemplary non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods of making such formulations are described, for example, in EP 1 125 584 Al.

Pharmaceutical packs and kits comprising one or more containers, each containing one or more doses of an anti-TREM2 antibody are also provided. In some embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising an anti-TREM2 antibody, with or without one or more additional agents. In some embodiments, such a unit dosage is supplied in single-use prefilled syringe for injection. In various embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, a composition of the invention comprises heparin and/or a proteoglycan.

***

The following sequences, figures and examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Description of the Sequences

Numbers 01-423 in the following Tables refer to the respective SEQ ID NO.

Description of the Figures

Figure 1 Sketches of the different formats of the antibodies according to the current invention. Figure 2 Serum concentrations of exemplary antibodies according to the current invention in mouse.

Figures 3 and 4 Brain exposure of exemplary antibodies according to the current invention in APPswePS2 transgenic mice.

Figure 5 TMDD of exemplary antibodies according to the current invention. Figure 6 Non-specific uptake of exemplary antibodies according to the current invention.

Figure 7 Sketch of the migration assay setup. Figure 8 Results of the phagocytosis assay.

Figure 9 THP-1 phagocytosis data ranking based on % untreated.

Figure 10 In vivo amyloid uptake in the Methoxy-X04 paradigm with

APPswePS2 transgenic mice for exemplary antibodies according to the current invention.

Figure 11 Methoxy-X04 positive cells in APPswePS2 transgenic mice for exemplary antibodies according to the current invention.

Figure 12 Syk phosphorylation (left bars without Abeta beads; right bars with Abeta beads).

Figure 13 Syk phosphorylation (left bars without Abeta beads; right bars with Abeta beads).

Figure 14 pSyk and rpS6 activation.

Description of exemplary Experimental Work

Example 1

Antibody production

Antibody Formats

Mono- and bispecific antibodies were produced by transient transfection in HEK293 cells. For normal, monospecific, bivalent antibodies (normal IgGs) two expression plasmids were used, for bispecific, tetravalent antibodies (2+2 bispecific formats) three expression plasmids were used and for domain-exchanged, bispecific, bivalent antibodies (cross-mAbs) four expression plasmids were used for providing the coding sequences of the light and heavy chains. A normal IgG is formed by two identical pairs each comprising one light chain (VL-CL) and one heavy chain (VH- CHl-hinge-CH2-CH3). A Cross-mAb is formed by four different polypeptides, whereof one is a domain exchanged heavy chain with “cys-knob” mutations T366W and S354C (HC(k), VH(l)-CL-hinge-CH2-CH3(k)), one is a heavy chain with “cys- hole” mutations T366S, Y407V and L368A and Y349C (VH(2)-CHl-hinge-CH2- CH3(h)), one is a light chain (VL-CL) and one is a domain-exchanged light chain (VL-CH1) (see, e.g., Schaefer, W., et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192). A 2+2 bispecific antibody is formed by two identical pairs each comprising a heavy chain to which at its C-terminus a light chain variable domain followed by a heavy chain constant domain 1 is genetically fused via a (GGSGG)2 linker (SEQ ID NO: 436) or a (GGSGG)₄ linker (SEQ ID NO: 461) (VH(l)-CHl- hinge-CH2-CH3 -linker- VL(2)-CH1), a light chain (VL(l)-CL) and a domain- exchanged light chain (VH(2)-CL).

Transient Antibody Production

The antibodies were produced by transient transfection of human embryonic kidney 293-F cells using the FreeStyle™ 293 Expression System according to the manufacturer's instruction (Invitrogen, USA). Briefly, suspension FreeStyle™ 293- F cells were cultivated in FreeStyle™ 293 Expression medium at 37 °C/8 % CO2 and seeded in fresh medium at a density of 1-2 x 1E6 viable cells/mL on the day of transfection. DNA293fectin™ complexes were prepared in Opti-MEM I medium (Invitrogen, USA) using 325 pL of 293fectin™ (Invitrogen, Germany) and a total of 250 pg of the respective plasmid DNA in a 1 : 1, 1 : 1 : 1 or 1 : 1 : 1 : 1 molar ratio for a 250 mL final transfection volume. Antibody containing cell culture supernatants were harvested 7 days after transfection

Purification of Recombinant Antibodies

The antibody-containing cell culture supernatant was filtered and purified by two chromatographic steps. Antibodies were captured by affinity chromatography using HiTrap MabSelect™ SuRe™ (Cytiva) equilibrated with PBS (equilibration buffer, 1 mM KH2PO4, 10 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KC1, pH 7.4). Unbound proteins were removed by washing the chromatography material with equilibration buffer. The antibody was recovered by elution with a solution comprising 100 mM Na- Acetate, pH 3.0. Immediately after elution, the eluted solution was adjusted to pH 6.0 with 2 M Tris-base, pH 9.0. Size exclusion chromatography on Superdex 200™ (Cytiva) was used as the second purification step. The size exclusion chromatography was performed using 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Antibody-containing solutions were stored at -80° C. Example 2

THP-1 Abeta-beads engagement and uptake assay (also known as “Abeta beads phagocytosis assay”)

In order to assess the potential of the antibody constructs to mimic plaque decoration and engagement of target cells with Abeta plaques, microbeads were coated with Abeta protein in order to represent an artificial plaque. Such Abeta-coated beads were then co-incubated with THP-1 cells, which have been shown to express TREM2 and also react with migration upon stimulation with certain TREM2 monoclonal antibodies (mAbs). As the size of the microbeads also allows for their uptake by the THP-1 cells, the Abeta coated beads were labeled with a pH sensitive dye, which becomes fluorescent in the acidic pH of the lysosome. Thus, the degree of bead uptake could be and was quantified and used as an indicator of engagement of THP-1 that is mediated by the respective tested antibody.

Preparation of pHRodo-labeled Abeta-coated beads

Amidine latex beads (100 pL, 1.0 pm; ThermoFisher) were diluted in 1 mL PBS, centrifuged (5 sec., 16,000 x g) and, after discarding the fluid, resuspended in 50 pL PBS. A 1 mg/mL solution (100 pL) of Abeta 42 peptide (Anaspec; dissolved in PBS according to the manufacturer’s instructions) was added and the suspension was incubated o/n at 37 °C in the incubator without shaking. After washing once with PBS, the beads were resuspended in 100 pL of PBS, pH 8.3, and 20 pL of a 1 mg/mL solution of pHRodo Red succinimidyl ester (ThermoFisher) was added, followed by an incubation for 1 h at RT in the dark, followed by a further wash with PBS and resuspended in 1 mL PBS.

Abeta-bead engagement and uptake assay

THP-1 cells are cultured in RPMI/10 % FBS/50 pM 2-mercaptoethanol at cell densities between 0.5 and 1 x 1E6 cells/mL. At the day of the experiment, cells were harvested and resuspended in fresh medium at 0.5 x 1E6 cells/mL. An aliquot of this suspension (70 pL) was added into each well of a flat-bottom 96-well plate (Greiner), followed by 25 pL of test antibody solution at the respective concentration as well as 5 pL bead suspension. The plate was then shaken on a plate shaker (900-1200 rpm) for 20 sec., spun for 5 min. at 120 g and incubated in an IncuCyte S3 analyzer for 16 h. Phase and red fluorescent channel images were acquired every 4 h under 20x magnification. Relative microbeads uptake was quantified using the IncuCyte cell-by-cell algorithm, setting the fluorescence intensity gate on untreated cells in such a way that 90% of cells are negative, 10% positive.

Example 3

Syk and S6 phosphorylation assay using HEK/DAP12/TREM2

Transient transfection of HEK cells with human TREM2/DAP12

HEK293 A cells expressing DAP12 and TREM2 were generated by transfection with the insertion vector containing the sequence of human DAP12-2A-TREM2 and the vector containing the sequence for the piggybac transposase. After electroporation cells were cultured in DMEM containing GlutaMax-I (Gibco, 31966), supplemented with 10 % FCS (Gibco, 10082), 1 mM Sodium Pyruvate (Gibco, 11360), 100 U/ml Penicillin/Streptomycin (Gibco, 14140). Forty-eight hours post transfection selection was started by addition of 1 pg/ml Puromycin. After 5 days in selection, cells were detached with trypsin and single cell clones were generated by limiting dilution seeding 0.5 cells/well in a 384 well plate. Clonal growth was monitored using IncuCyte whole well scans and wells with only one colony were picked for further characterization.

Cell preparation

For each experiment, the cells were thawed and passaged once in DMEM containing GlutaMax-I (Gibco, 31966), supplemented with 10 % FCS (Gibco, 10082), 1 mM Sodium Pyruvate (Gibco, 11360), 100 U/ml Penicillin/Streptomycin (Gibco, 14140) and 1 pg/ml of Puromycin (Gibco, 10131).

Opsonization of amidine latex beads with AB 1-42

The Api-42 stock solution was prepared following manufacturer’s instructions as follows: 1 mg of lyophilized Api-42 (Anaspec, AS-20276) was reconstituted in 1 % ammonium hydroxide (Anaspec, AS-61322) and immediately diluted in PBS to a stock concentration of 1.25 mg/ml. Api-42 was aliquoted and stored at -80 °C.

Amidine latex beads (4 mg; ThermoFisher, A37322) were washed with PBS and incubated overnight at 37 °C with 125 pg of Api-42. The Ap coated beads were washed once with PBS and resuspended in 1 ml of PBS pH 7.2 (Gibco, 20012). The Ap beads were stored at 4 °C.

AB beads engagement assay

To keep the endogenous pSyk level low and to avoid interference of the phenol red in the AlphaLISA assay, all the dilutions were done in FCS deprived DMEM media containing no phenol red (Gibco, 31053) and supplemented with 1% GlutaMax (Gibco, 35050), 1 mM Sodium Pyruvate, 100 U/ml Penicillin/Streptomycin and 1 pg/ml of Puromycin. This medium will be mentioned in the following as assay medium.

A 8x concentrated antibody/isotype control stock solution was mixed at 1: 1 ratio (v:v) with Ap-beads, diluted 1 to 8 in assay medium in a round bottom 96 well plate and incubated for two hours at RT with shaking, resulting in a 250 nM and/or 50 nM antibody concentration in the assay. Three technical replicates were done.

Forty-five minutes prior to the end of the two hours antibody -beads mix incubation time, the cells were harvested and resuspended in assay medium for counting. The cells were centrifuged and resuspended in assay medium at 3.33 million cells per ml. 10 pl of antibody- Ap beads mix were transferred to a well of a flat bottom 96 wellplate and 1E5 cells/well were added. The plate was briefly shaken and thereafter centrifuged. The cells together with antibody-beads mix were incubated 30 min. at 37 °C. The cells were then lysed for 30 min. at 4 °C, with shaking and immediately analyzed.

AB coated plate stimulation assay

Collagen I coated 96 well-plates (Greiner, 655956 or Invitrogen, Al 1428-03) were coated 20 h+/-4 h at 37 °C with 5 and 15 pg/ml of Ap42. A 3 fold serial dilution of the antibodies were prepared in assay medium as follows: 2000 nM down to 0.3 nM for TREM2 antibodies and 200 nM down to 0.03 nM for the isotype controls. After removal of the Ap solution, antibodies were added to the plate and incubated for two hours at RT with shaking. Three technical replicates were done.

Forty-five minutes prior to the end of the two hour antibody -beads incubation time, the cells were harvested and the cell pellet was resuspended in assay medium for counting. The cells were centrifuged and resuspended in assay media at 3.33 million cells per ml. The plates were washed twice with assay media and 1E5 cells/well were added. The plates were briefly shaken, centrifuged, and then incubated for 30 min. at 37 °C. The cells were lysed for 30 min. at 4 °C with shaking and immediately analyzed.

AlphaLISA

Phospho(Tyr525/526) Syk and total Syk levels were measured using Alpha SureFire® Ultra™ Multiplex phospho/total SYK Assay Kit (Perkin Elmer, MPSU- PTSYK K10K) on an EnVision plate reader. In this assay, two dual emission wavelengths were measured sequentially: 615 nm signal (Europium) corresponding to phospho(Tyr525/526) Syk and 545 nm signal (Terbium) corresponding to total Syk. S6 and pS6 were measured with Alpha SureFire Ultra Multiplex phospho(Ser235/236)/Total Ribosomal Protein S6 Assay Kit (Perkin Elmer, MPSU- PTRPS6-B10K).

The assays were performed following manufacturer’s instructions.

Briefly, 10 pl of undiluted cell lysate was transferred to a well of a white opaque 384 Optiplate and 5 pl of acceptor beads mix were added per well. The plate was sealed with aluminum foil and incubated for one hour at RT with shaking. Thereafter the donor beads mix (5 pl) was added per well in reduced light exposure, the plate was sealed and incubated for one hour at RT with shaking.

Syk or S6 activation in HEK293A cells expressing TREM2 and DAP12 was determined as the ratio, in percent, of phosphorylated Syk or S6 over total Syk or S6. Based on handling of TREM2/DAP12 transiently transfected HEK cells, it has been observed that the cells expressed different levels of baseline pSyk levels. This allowed for testing effects of antibodies on top of a tonic TREM2-mediated pSyk signal (i.e., elevated baseline pSyk, presumably due to a putative activating TREM2 ligand in the culture condition) or at low close to detection limit pSyk to observe effects on induction of the inactive TREM2/pSyk pathway.

Claims

Patent Claims

1. An anti-TREM2 antibody specifically binding to human TREM2 comprising a) in the heavy chain variable domain a HVR-H1 of SEQ ID NO: 305, (DYAMS) a HVR-H2 of SEQ ID NO: 306 (IIGDSGDNTYYADSVKG) and a HVR-H3 of SEQ ID NO: 307 (YDIDV), and in the light chain variable domain a HVR-L1 of SEQ ID NO: 309 (RASQSISSYLN), a HVR-L2 of SEQ ID NO: 310 (AASDLQS) and a HVR-L3 of SEQ ID NO: 311 (QQANSFPPT) (TREM2 3295), or b) in the heavy chain variable domain a HVR-H1 of SEQ ID NO: 329 (SYAMN), a HVR-H2 of SEQ ID NO: 330 (TMSGSGGDTFYADSVKG) and a HVR-H3 of SEQ ID NO: 331 (EGGTVFDN), and in the light chain variable domain a HVR-L1 of SEQ ID NO: 333 (RASQDISNDLG), a HVR-L2 of SEQ ID NO: 334 (AASFLQS) and a HVR-L3 of SEQ ID NO: 335 (LQDYNLPFT) (TREM2 3306).

2. The anti-TREM2 antibody according to claim 1, wherein the antibody comprises a) a heavy chain variable domain of SEQ ID NO: 308 (VQLVESGGGLVQPGRSLRLSCAASGFTFGDYAMSWFRQAP GKGLEWVSIIGDSGDNTYYADSVKGRFAISRDNSKNTLYLQ MNSLRAEDTAVYYCMNYDIDVWGQGTTVTVSS) and a light chain variable domain of SEQ ID NO: 312 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKRLIYAASDLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATY YCQQANSFPPTFGGGTKVEIK), or b) a heavy chain variable domain of SEQ ID NO: 332 (VQLLESGGGLVQPGGSLRLSCVASGFIFNSYAMNWVRQAPG KGLEWVSTMSGSGGDTFYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAIYYCAKEGGTVFDNWGQGTLVTVSS) and a light chain variable domain of SEQ ID NO: 336 (AIQMTQSPSSLSTSVGDRVTITCRASQDISNDLGWYQQKPGK APKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCLQDYNLPFTFGPGTKVDFK).

3. The anti-TREM2 antibody according to any one of claims 1 to 2 wherein the antibody binds to human TREM2 with an affinity of less than 1 nM.

4. The anti-TREM2 antibody according to any one of claims 1 to 3, wherein the antibodies induce migration in vitro at a concentration of 5-15 nM.

5. The anti-TREM2 antibody according to any one of claims 1 to 4, wherein the antibody is a bivalent, monospecific antibody.

6. The anti-TREM2 antibody according to any one of claims 1 to 4, wherein the antibody is a multispecific antibody.

7. The anti-TREM2 antibody according to claim 6, wherein the antibody is a bivalent, bispecific antibody, or a trivalent, bispecific antibody, or a trivalent, trispecific antibody, or a tetravalent, bispecific antibody.

8. The anti-TREM2 antibody according to any one of claims 6 to 7, wherein the antibody comprises a first binding site binding to human TREM2 and at least one further binding site binding to a different epitope on human TREM2 than the first binding site, or binding to human A-beta protein, or binding to human transferrin receptor 1, or binding to human tau protein, or binding to human alpha-synuclein, or binding to human TDP-43, or binding to human huntingtin protein, or binding to human apolipoprotein E, or binding to delipidated human apolipoprotein E, or binding to human myelin basic protein.

9. The anti-TREM2 antibody according to any one of claims 6 to 8, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising i) a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 305, a HVR-H2 of SEQ ID NO: 306 and a HVR-H3 of SEQ ID NO: 307, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 309, a HVR-L2 of SEQ ID NO: 310 and a HVR-L3 of SEQ ID NO: 311 (TREM2 3295), or ii) a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 329, a HVR-H2 of SEQ ID NO: 330 and a HVR-H3 of SEQ ID NO: 331, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 333, a HVR-L2 of SEQ ID NO: 334 and a HVR-L3 of SEQ ID NO: 335 (TREM2 3306), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 09, a HVR-H2 of SEQ ID NO: 10 and a HVR-H3 of SEQ ID NO: 11, and in the light chain variable domain a HVR-L1 of SEQ ID NO: 13, a HVR-L2 of SEQ ID NO: 14 and a HVR-L3 of SEQ ID NO: 15 (Abeta 8675).

10. The anti-TREM2 antibody according to any one of claims 6 to 8, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising i) a heavy chain variable domain of SEQ ID NO: 308 and a light chain variable domain of SEQ ID NO: 312 (TREM2 3295), or ii) a heavy chain variable domain of SEQ ID NO: 332 and a light chain variable domain of SEQ ID NO: 336 (TREM2 3306), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain of SEQ ID NO: 12 (VELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVSAINATGTRTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYC ARGKGS SGYVRYFD VWGQGTL VTVS S) and in the light chain variable domain of SEQ ID NO: 16 (DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPG QAPRLLIYGAS SRATGVPARF SGSGSGTDFTLTIS SLEPEDF AT YYCLQIYNMPITFGQGTKVEIK) (Abeta 8675).

11. The anti-TREM2 antibody according to any one of claims 6 to 8, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 305, a HVR-H2 of SEQ ID NO: 306 and a HVR-H3 of SEQ ID NO: 307, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 309, a HVR-L2 of SEQ ID NO: 310 and a HVR-L3 of SEQ ID NO: 311 (TREM2 3295), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain comprising a HVR-H1 of SEQ ID NO: 329, a HVR-H2 of SEQ ID NO: 330 and a HVR-H3 of SEQ ID NO: 331, and a light chain variable domain comprising a HVR-L1 of SEQ ID NO: 333, a HVR-L2 of SEQ ID NO: 334 and a HVR-L3 of SEQ ID NO: 335 (TREM2 3306).

12. The anti-TREM2 antibody according to any one of claims 6 to 8, wherein the antibody comprises a) a first binding site binding to human TREM2 comprising a heavy chain variable domain of SEQ ID NO: 308 and a light chain variable domain of SEQ ID NO: 312 (TREM2 3295), and b) a second binding site binding to human Abeta protein comprising a heavy chain variable domain of SEQ ID NO: 332 and a light chain variable domain of SEQ ID NO: 336 (TREM2 3306).

13. The anti-TREM2 antibody according to any one of claims 6 to 8, wherein the antibody comprises a fist light chain of SEQ ID NO: 462

(AIQMTQSPSSLSTSVGDRVTITCRASQDISNDLGWYQQKPGKAPKLLI YAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNLPFT FGPGTKVDFKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC), a second light chain of SEQ ID NO: 463

(QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSAINATGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARGKGSSGYVRYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), a first heavy chain of SEQ ID NO: 464

(DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL LIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPI TFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG), and a second heavy chain of SEQ ID NO: 465 (EVQLLESGGGLVQPGGSLRLSCVASGFIFNSYAMNWVRQAPGKGLE WVSTMSGSGGDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAI YYCAKEGGTVFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGOL VEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG).

14. The anti-TREM2 antibody according to any one of claims 1 to 13, wherein the antibody is mouse cross-reactive.

15. The anti-TREM2 antibody according to any one of claims 1 to 13, wherein the antibody is not mouse cross-reactive.

16. The anti-TREM2 antibody according to any one of claims 1 to 15, wherein the antibody is a) a full-length antibody of the human subclass IgGl, b) a full-length antibody of the human subclass IgG4, c) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A and P329G, d) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S,

L368A, Y407V and Y349C in the respective other heavy chain, e) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, f) a full-length antibody of the human subclass IgG4 with the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, g) a full-length antibody of the human subclass IgG4 with the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, h) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and H435A in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, i) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and H435A in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, j) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, k) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, or l) a full-length antibody of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A in both heavy chains and the mutations i) T366W, and ii) S354C or Y349C, in one heavy chain and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other heavy chain, or m) one of a) to 1) without the C-terminal lysine residue.

17. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. The anti-TREM2 antibody according to any one of claims 1 to 16 for use as a medicament.

19. The anti-TREM2 antibody according to any one of claims 1 to 16 or the medicament according to claim 18 for use in the treatment of a disease.

20. A method of treating a condition associated with TREM2 loss of function in a subject in need thereof, comprising administering an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 to the subject.

21. A method of treating a condition associated with toxic gain of TREM2 function in a subject in need thereof, comprising administering an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 to the subject.

22. A method of treating a condition associated with hyperactivated microglia in a subject in need thereof, comprising administering an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17 to the subject.

23. A method of reducing levels of amyloid plaques in a subject in need thereof, comprising administering an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 to the subject.

24. A method of reducing slowing cognitive and functional decline in a subject in need thereof by targeted promotion and enrichment of beneficial and neuroprotective microglial functions at amyloid plaques or the brain vasculature covered by amyloid, comprising administering an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 to the subject.

25. A method of modulating neuroprotective activity of microglia in the vicinity of amyloid plaques or brain vasculature covered by amyloid in a subject in need thereof, comprising administering an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 to the subject.

26. An antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 for use in treating a condition associated with TREM2 loss of function in a subject in need thereof.

27. An antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 for use in treating a condition associated with toxic gain of TREM2 function in a subject in need thereof.

28. An antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 for use in treating a condition associated with hyperactivated microglia in a subject in need thereof.

29. An antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 for use in reducing levels of amyloid plaques in a subject in need thereof.

30. An antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 in the preparation of a medicament for treating a condition in a subject in need thereof.

31. The use of an antibody according to any one of claims 1 to 16 or a pharmaceutical composition according claim 17 in the preparation of a medicament for reducing levels of amyloid plaques in a subject in need thereof.

32. The anti-TREM2 antibody or method or use according to any one of claims 18 to 31, wherein the disease or the condition is, or the subject suffers from, a neuroinflammatory or neurodegenerative disease.

33. The anti-TREM2 antibody or method or use according to claim 32, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hakola disease, Guillain- Barre Syndrome (GBS), lysosomal storage disease, sphingomyelinlipidosis (Neimann-Pick C), mucopolysaccharidosis II/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet’s disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury.

34. The anti-TREM2 antibody or method or use according to claim 33, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer’s disease.

35. The anti-TREM2 antibody or method or use according to any one of claims 33 to 34, wherein the neuroinflammatory or neurodegenerative disease is early Alzheimer’s disease.

36. The anti-TREM2 antibody or method or use according to any one of claims 33 to 35, wherein the subject is amyloid positive.

37. The anti-TREM2 antibody or method or use according to claim 32, wherein the neuroinflammatory or neurodegenerative disease is MS.

38. The anti-TREM2 antibody or method or use according to any one of claims 18 to 32, wherein the disease or the condition is, or the subject suffers from, Dementia with Lewy bodies.

39. The anti-TREM2 antibody or method or use according to any one of claims 18 to 32, wherein the disease or the condition is, or the subject suffers from,

Parkinson’s disease with Dementia.