Monospecific and multispecific anti-TREM2 antibodies, methods and uses thereof

Abstract

Herein is reported an anti-TREM2 antibody that specifically binds to human TREM2, the antibody comprising: HVR-H1 of SEQ ID NO: 305 in the heavy chain variable domain, HVR-H2 of SEQ ID NO: 306 and HVR-H3 of SEQ ID NO: 307, and HVR-L1 of SEQ ID NO: 309 in the light chain variable domain, HVR-L2 of SEQ ID NO: 310 and HVR-L3 of SEQ ID NO: 311 (TREM 23295), or HVR-H1 of SEQ ID NO: 329 in the heavy chain variable domain, HVR-H2 of SEQ ID NO: 330 and HVR-L3 of SEQ ID NO: 331 in the light chain variable domain, HVR-L2 of SEQ ID NO: 334 and HVR-L3 of SEQ ID NO: 335 ( wherein pSyk is not induced in the absence or in the presence of human A [beta] protein.

Description

[0001] This invention belongs to the field of monospecific and multispecific antibodies. Novel monospecific, bispecific, and trispecific anti-TREM2 antibodies with improved properties are reported in more detail herein. Background Technology

[0002] The trigger receptor (TREM2), expressed on myeloid cells 2, is expressed on the cell surface of myeloid-derived cells such as monocytes / macrophages, microglia, osteoclasts, neutrophils, and dendritic cells. It is a transmembrane receptor of the innate immune system.

[0003] Without being bound by any specific theory, it is believed that upon certain ligand bindings, TREM2 forms a signaling complex with the transmembrane adaptor protein DNAX activator protein 12 (DAP12), which is then phosphorylated by the protein kinase SRC. The activated TREM2 / DAP12 signaling complex is believed to mediate intracellular signaling by recruiting and phosphorylating kinases such as syk kinase. TREM2 / DAP12 signaling regulates cellular activities such as phagocytosis, cell growth and survival, cytokine secretion, and cell migration (such as microglia and macrophages). TREM2 can also activate phosphatidylinositol 3-kinase (PI3K) in the TREM2 / DAP12 / DAP10 heterodimer complex, which can then be inhibited by inositol phosphatase-1 (SHIP1), which contains the Src homology 2 (SH2) domain. TREM2 undergoes regulated proteolysis, in which the membrane-associated full-length TREM2 is cleaved at the H157-S158 peptide bond by α-secretases, integrins, and the metalloproteinase-containing protein 17 (ADAM17) and ADAM10, into a detachable sTREM2 portion and a membrane-retaining C-terminal fragment that is further degraded by γ-secretases. Altered sTREM2 levels have been reported in the cerebrospinal fluid of patients with frontotemporal dementia or those with certain mutations in TREM2 during the prodromal stage of Alzheimer's disease (AD) and after AD diagnosis. Some mutations are associated with a higher genetic risk of developing Alzheimer's disease. Additionally, certain mutations in TREM2 are associated with functional alterations such as impaired phagocytosis and reduced microglial function (see, for example, WO 2020 / 172450).

[0004] TREM2 has been found to localize to plaques and microglia surrounding neurons in the brains of TgCRND8 mice (see, for example, US 2015 / 0065567), and to be enriched on the surface of those microglia in areas that come into contact with amyloid plaques or neuronal debris (see, for example, WO 2020 / 079580). Knockdown of TREM2 or DAP12 in microglia leads to reduced phagocytosis of apoptotic neurons, while overexpression of TREM2 leads to increased phagocytosis of dead neurons in microglia, and similarly increases phagocytosis of other myeloid cells (see, for example, WO 2014 / 074942; WO 2016 / 023019; WO2019 / 118513; Kleinberger et al., Sci. Transl. Med. 6 (2014) 243ra86, pp. 1–12).

[0005] Cells expressing high levels of TREM2 are thought to be involved in immune surveillance, cell-cell interactions, tissue debris clearance, and resolution of potential inflammatory responses (see, for example, WO 2019 / 118513).

[0006] However, the mechanisms by which TREM2 contributes to neurodegeneration remain unclear. Furthermore, studies investigating the effects of TREM2 signaling on inflammatory responses have yielded conflicting results indicating either anti-inflammatory or pro-inflammatory effects of 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 roles for TREM2 in microglial survival (Wang et al., Cell160:1061-1071 (2015)) and in regulating energy metabolism (Ulland et al., Cell 170:649-663 (2017)). Several studies have indicated the role of 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 other studies have not observed this role (e.g., Wang et al., Cell 160:1061-1071 (2015)) (see, for example, WO 2020 / 121195).

[0007] WO 2019 / 055841 reported on anti-TREM2 antibodies and their usage.

[0008] Green, K. reported a new approach to targeting TREM2, raising the question of whether to upregulate or downregulate (ALZFORUM, July 29, 2021).

[0009] Fassler, M. et al. reported that conjugation of TREM2 with a novel monoclonal antibody induces microglial activation and improves cognitive function in an Alzheimer's disease model (J. Neuroinfl.18 (2021) 19).

[0010] Van Lengerich, B. et al. reported that TREM2-activating antibodies containing blood-brain barrier transport mediators enhanced microglia metabolism in an Alzheimer's disease model (Nat. Neurosci. 26 (2023) 416-429). Summary of the Invention

[0011] This article reports monospecific and multispecific antibodies that bind to human TREM2. The antibodies according to the invention possess improved properties. Among other things, these improved properties are particularly relevant to improved therapeutic properties. For example, the antibodies according to the invention induce migration and phagocytosis. Therefore, TREM2-specific antibodies according to the invention can be advantageously used for the treatment of brain diseases.

[0012] The antibody according to the invention binds to different epitopes in the extracellular domain of human TREM2, which is known in the art.

[0013] The antibody according to the present invention is a mono- or bi-complementary anti-TREM2 antibody.

[0014] The antibodies according to the invention have the ability to induce cell migration and phagocytosis in vitro. That is, the antibodies according to the invention do not induce pSyk, but increase LPC- and C5a-stimulated cell migration. More specifically, the antibodies according to the invention induce the migration of M0 cells derived from THP-1 and iPSCs in vitro in the absence of any Aβ, but rather as an amplifier of existing inflammatory or damaging signals (C5a or lysoPC, respectively) that induce elevated baseline migration. This indicates that the anti-TREM2 antibody according to the invention can also modulate TREM2 on microglia in certain disease settings that avoid Aβ plaques.

[0015] In one embodiment, the antibody binds to the extracellular domain of TREM2 and / or the human transferrin receptor.

[0016] In some embodiments, in bead-based phagocytosis assays, the antibody according to the invention induces phagocytosis.

[0017] In some embodiments, the antibody according to the invention comprises a human IgG1 Fc region containing L234A, L235A, and P329G substitutions (LALAPG substitutions) (according to Kabat numbering).

[0018] In some embodiments, the antibody according to the invention is covalently or non-covalently conjugated with at least one other molecule. In some embodiments, the antibody is covalently or non-covalently conjugated with at least one other molecule, wherein the at least one other molecule includes a detection marker and / or a drug.

[0019] One aspect of the invention is a group of isolated nucleic acids or two or more nucleic acids encoding antibodies according to the invention.

[0020] One aspect of the present invention is a vector comprising one or more isolated nucleic acids encoding heavy and light chains of an antibody according to the present invention.

[0021] One aspect of the present invention is a mammalian cell containing a nucleic acid or vector according to the present invention.

[0022] One aspect of the invention is a method for producing an antibody according to the invention, the method comprising culturing mammalian cells according to the invention under conditions suitable for expressing the antibody according to the invention, and recovering the antibody according to the invention from the mammalian cells or culture medium. One aspect is an antibody produced by this method.

[0023] One aspect of the present invention is a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable carrier.

[0024] One aspect of the present invention is an antibody according to the invention, which is used as a drug.

[0025] One aspect of the present invention is an antibody according to the invention, which is used to treat diseases.

[0026] According to one aspect of the invention is a method for treating a subject with symptoms associated with loss of TREM2 function, the method comprising administering to the subject an antibody according to the invention or a pharmaceutical composition according to the invention.

[0027] According to one aspect of the invention is a method for treating symptoms associated with toxic gain in TREM2 function in a subject with such need, the method comprising administering to the subject an antibody according to the invention or a pharmaceutical composition according to the invention.

[0028] According to one aspect of the invention is a method for treating symptom-associated hyperactivity of microglia in a subject with this need, the method comprising administering to the subject an antibody according to the invention or a pharmaceutical composition according to the invention.

[0029] According to one aspect of the invention, there is a method for reducing amyloid plaque levels in subjects who require this, the method comprising administering to the subject an antibody according to the invention or a pharmaceutical composition according to the invention.

[0030] According to one aspect of the invention, there is an antibody or pharmaceutical composition according to the invention for treating symptoms associated with loss of TREM2 function in a subject with this need.

[0031] One aspect of the invention is an antibody or pharmaceutical composition according to the invention, which is used to reduce amyloid plaque levels in subjects who require this.

[0032] One aspect of the invention is the use of the antibody or pharmaceutical composition according to the invention in the preparation of a medicament for treating the condition of a subject in need.

[0033] One aspect of the invention is the use of the antibody or pharmaceutical composition according to the invention in the preparation of a medicament for reducing amyloid plaque levels in subjects who require it.

[0034] In all aspects and embodiments of the invention, the disease or symptom is or the subject suffers from a neuroinflammatory disease or a neurodegenerative disease. In some embodiments, the neuroinflammatory disease or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hacola disease, Guillain-Barré syndrome (GBS), lysosomal storage disease, sphingomyelin storage disease (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neurobehçet'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.

[0035] Therefore, the present invention covers at least the following embodiments:

[0036] 1. An anti-TREM2 antibody having one or more of the following properties:

[0037] a) This antibody, as a bispecific anti-TREM2 / Aβ antibody, induces macrophage phagocytosis of amyloid plaques, and / or

[0038] b) This antibody, as a bispecific anti-TREM2 / Aβ antibody, induces amyloid uptake in microglia in the absence of FcgRII and FcgRIII receptor binding, and / or

[0039] c) This antibody, as a bispecific anti-TREM2 / Aβ antibody, induces amyloid uptake in the absence of FcgR binding, and / or

[0040] d) The antibody, in a form with Fc effector function, induced acute uptake of MX04-labeled amyloid and Aβ proteins in APPswePS2 transgenic mice, and / or

[0041] e) This antibody, as a non-effectoral bispecific anti-TREM2 / Aβ antibody, preferably induces acute uptake of MX04-labeled amyloid and Aβ proteins in APPswePS2 transgenic mice in the absence of Aβ and FcgR crosslinking, and / or

[0042] f) The antibody preferably induces macrophage migration in a migration assay as described herein at a concentration of 0.14 to 34 nM, and / or

[0043] g) This antibody increases LPC- and C5a-stimulated cell migration in THP-1 and iPSC-derived M0 cells in the absence of any Aβ protein, and / or

[0044] h) The antibody does not induce pSyk in the absence or presence of human Aβ protein, preferably the antibody is an antagonist of the Syk pathway, and / or

[0045] i) This antibody, as a bispecific anti-TREM2 / Aβ antibody, does indeed induce pSyk in the presence of human Aβ protein, and / or

[0046] j) This antibody did not show dose-dependent phosphorylation of Syk and S6 in HEK cells expressing DAP12 and TREM2, and / or

[0047] k) This antibody does not induce pSyk in TREM2 / DAP12-overexpressing HEK or iPSC macrophages in the absence of cross-linking, and / or

[0048] l) This antibody, as a bispecific anti-TREM2 / Aβ antibody, showed in vivo enrichment at plaque sites in APPswePS2 transgenic mice after peripheral administration, and / or

[0049] m) This antibody, as a bispecific anti-TREM2 / Aβ antibody, regulates the neuroprotective activity of microglia near amyloid plaques covered by amyloid or the cerebral vascular system, and / or

[0050] n) This antibody possesses non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and / or

[0051] o) This antibody, as a bispecific anti-TREM2 / Aβ antibody, provides plaque retention and plaque-targeted brain exposure, preferably as an anti-TREM2 monospecific antibody with higher local concentrations, and / or

[0052] p) This antibody does not induce the release of TNFα, MIP-1α, or IL-8 from iPSC-derived macrophages, and / or

[0053] q) This antibody blocks the shedding of sTREM2, and / or

[0054] r) This antibody stabilizes sTREM2 in the biological fluid, and / or

[0055] s) The antibody binds to the ECD of TREM2, thereby leading to the accumulation of sTREM2 in the biological fluid, and / or

[0056] t) This antibody, as a bispecific anti-TREM2 / Aβ antibody, induces the conversion of homeostatic or less conjugated microglia into activated microglia, and / or

[0057] u) This antibody, as a bispecific anti-TREM2 / Aβ antibody, induces ARIA at a lower level than that of monospecific antibodies, and preferably does not induce ARIA.

[0058] 2. An anti-TREM2 antibody that binds to the same epitope as anti-TREM2 antibody TREM2 3295 or TREM2 3306.

[0059] 3. An anti-TREM2 antibody that specifically binds to human TREM2, the antibody comprising...

[0060] a) HVR-H1 of SEQ ID NO: 305 (DYAMS), HVR-H2 of SEQ ID NO: 306 (IIGDSGDNTYYADSVKG), and HVR-H3 of SEQ ID NO: 307 (YDIDV) in the heavy chain variable structural domain, and HVR-L1 of SEQ ID NO: 309 (RASQSISSYLN), HVR-L2 of SEQ ID NO: 310 (AASDLQS), and HVR-L3 of SEQ ID NO: 311 (QQANSFPPT) (TREM2 3295) in the light chain variable structural domain.

[0061] or

[0062] b) HVR-H1 of SEQ ID NO: 329 (SYAMN), HVR-H2 of SEQ ID NO: 330 (TMSGSGGDTFYADSVKG), and HVR-H3 of SEQ ID NO: 331 (EGGTVFDN) in the heavy chain variable structural domain, and HVR-L1 of SEQ ID NO: 333 (RASQDISNDLG), HVR-L2 of SEQ ID NO: 334 (AASFLQS), and HVR-L3 of SEQ ID NO: 335 (LQDYNLPFT) in the light chain variable structural domain (TREM2 3306).

[0063] 4. The anti-TREM2 antibody according to any one of Examples 2 to 3, wherein the antibody has one or more of the following characteristics:

[0064] a) This antibody, as a dual complementary anti-TREM2 antibody, induces macrophage phagocytosis of amyloid plaques, and / or

[0065] b) This antibody, as a dual-complementary anti-TREM2 antibody, induces amyloid uptake in microglia in the presence of FcgRII and FcgRIII receptor binding, and / or

[0066] b) This antibody induced acute uptake of MX04-labeled amyloid and Aβ proteins in APPswePS2 transgenic mice, and / or

[0067] c) The antibody preferably induces macrophage migration in the migration assay as described herein at a concentration of 0.14 to 34 nM, more preferably at a concentration of 15 nM or less, and / or

[0068] d) This antibody increases LPC- and C5a-stimulated cell migration in THP-1 and iPSC-derived M0 cells in the absence of any Aβ protein, and / or

[0069] e) The antibody does not induce pSyk in the absence or presence of human Aβ protein, preferably the antibody is an antagonist of the Syk pathway, and / or

[0070] f) This antibody did not show dose-dependent phosphorylation of Syk and S6 in HEK cells expressing DAP12 and TREM2, and / or

[0071] g) This antibody does not induce pSyk in TREM2 / DAP12-overexpressing HEK or iPSC macrophages in the absence of cross-linking, and / or

[0072] h) This antibody possesses non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and / or

[0073] i) This antibody does not induce the release of TNFα, MIP-1α, or IL-8 from iPSC-derived macrophages, and / or

[0074] j) This antibody blocks the shedding of sTREM2, and / or

[0075] k) This antibody stabilizes sTREM2 in the biological fluid, and / or

[0076] l) The antibody binds to the ECD of TREM2, thereby leading to the accumulation of sTREM2 in the biological fluid.

[0077] 5. The anti-TREM2 antibody according to any one of Examples 2 to 4, wherein the antibody comprises

[0078] a) The heavy-chain variable structural domains of SEQ ID NO: 308 (VQLVESGGGLVQPGRSLRLSCAASGFTFGDYAMSWFRQAPGKGLEWVSIIGDSGDNTYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCMNYDIDVWGQGTTVTVSS) and the light-chain variable structural domains of SEQ ID NO: 312 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKRLIYAASDLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK),

[0079] or

[0080] b) The heavy chain variable domain of SEQ ID NO: 332 (VQLLESGGGLVQPGGSLRLSCVASGFIFNSYAMNWVRQAPGKGLEWVSTMSGSGGDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAKEGGTVFDNWGQGTLVTVSS) and the light chain variable domain of SEQ ID NO: 336 (AIQMTQSPSSLSTSVGDRVTITCRASQDISNDLGWYQQKPGKAPKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNLPFTFGPGTKVDFK).

[0081] 6. The anti-TREM2 antibody according to any one of Examples 2 to 5, wherein the antibody binds to human TREM2 with an affinity of less than 1 nM.

[0082] 7. The anti-TREM2 antibody according to any one of Examples 2 to 6, wherein the antibody is induced to migrate in vitro at a concentration of 5 to 15 nM.

[0083] 8. The anti-TREM2 antibody according to any one of Examples 2 to 7, wherein the antibody is a bivalent monospecific antibody.

[0084] 9. The anti-TREM2 antibody according to any one of Examples 2 to 7, wherein the antibody is a multispecific antibody.

[0085] 10. The anti-TREM2 antibody according to Example 9, wherein the antibody is a bivalent bispecific antibody, or a trivalent bispecific antibody, or a trivalent trispecific antibody, or a quadrivalent bispecific antibody.

[0086] 11. The anti-TREM2 antibody according to any one of Examples 9 to 10, wherein the antibody comprises a first binding site for binding to human TREM2 and at least one additional binding site, the at least one additional binding site binding to an epitope on human TREM2 that is different from the first binding site, or binding to human A-β protein, or binding to human transferrin receptor 1, or binding to human tau protein, or binding to human α-synuclein, or binding to human TDP-43, or binding to human huntingtin protein, or binding to human apolipoprotein E, or binding to defatted human apolipoprotein E, or binding to human myelin basic protein.

[0087] 12. The anti-TREM2 antibody according to any one of Examples 9 to 11, wherein the antibody comprises

[0088] The first binding site for human TREM2, the first binding site containing

[0089] i) Heavy chain variable domains comprising HVR-H1 (SEQ ID NO: 305), HVR-H2 (SEQ ID NO: 306), and HVR-H3 (SEQ ID NO: 307), and light chain variable domains comprising HVR-L1 (SEQ ID NO: 309), HVR-L2 (SEQ ID NO: 310), and HVR-L3 (SEQ ID NO: 311) (TREM2 3295),

[0090] or

[0091] ii) Heavy chain variable domains comprising HVR-H1 of SEQ ID NO: 329, HVR-H2 of SEQ ID NO: 330, and HVR-H3 of SEQ ID NO: 331, and light chain variable domains comprising HVR-L1 of SEQ ID NO: 333, HVR-L2 of SEQ ID NO: 334, and HVR-L3 of SEQ ID NO: 335 (TREM2 3306).

[0092] 13. The anti-TREM2 antibody according to any one of Examples 9 to 11, wherein the antibody comprises

[0093] The first binding site for human TREM2, the first binding site containing

[0094] i) The heavy chain variable domain of SEQ ID NO: 308 and the light chain variable domain of SEQ ID NO: 312 (TREM2 3295),

[0095] or

[0096] ii) The heavy chain variable domain of SEQ ID NO: 332 and the light chain variable domain of SEQ ID NO: 336 (TREM2 3306).

[0097] 14. The anti-TREM2 antibody according to any one of Examples 9 to 11, wherein the antibody comprises

[0098] a) A first binding site for binding to human TREM2, comprising a heavy chain variable domain containing HVR-H1 (SEQ ID NO: 305), HVR-H2 (SEQ ID NO: 306), and HVR-H3 (SEQ ID NO: 307), and a light chain variable domain containing HVR-L1 (SEQ ID NO: 309), HVR-L2 (SEQ ID NO: 310), and HVR-L3 (SEQ ID NO: 311) (TREM2 3295).

[0099] as well as

[0100] b) A second binding site for binding to human TREM2, comprising a heavy chain variable domain containing HVR-H1 of SEQ ID NO: 329, HVR-H2 of SEQ ID NO: 330, and HVR-H3 of SEQ ID NO: 331, and a light chain variable domain containing HVR-L1 of SEQ ID NO: 333, HVR-L2 of SEQ ID NO: 334, and HVR-L3 of SEQ ID NO: 335 (TREM2 3306).

[0101] 15. The anti-TREM2 antibody according to any one of Examples 9 to 11, wherein the antibody comprises

[0102] a) A first binding site for binding to human TREM2, comprising the heavy chain variable domain of SEQ ID NO: 308 and the light chain variable domain of SEQ ID NO: 312 (TREM2 3295).

[0103] as well as

[0104] b) A second binding site that binds to human TREM2, the second binding site comprising the heavy chain variable domain of SEQ ID NO: 332 and the light chain variable domain of SEQ ID NO: 336 (TREM2 3306).

[0105] 16. The anti-TREM2 antibody according to any one of Examples 2 to 15, wherein the antibody is cross-reactive in mice.

[0106] 17. The anti-TREM2 antibody according to any one of Examples 2 to 15, wherein the antibody is not cross-reactive in mice.

[0107] 18. The anti-TREM2 antibody according to any one of Examples 2 to 17, wherein the antibody is

[0108] a) Full-length antibody against the human IgG1 subclass.

[0109] b) Full-length antibody against the human IgG4 subclass.

[0110] c) Full-length antibodies against human IgG1 subclasses with mutations L234A, L235A, and P329G.

[0111] d) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, and P329G in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0112] e) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, and P329G in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain.

[0113] f) Full-length antibodies against the human IgG4 subclass containing mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0114] g) Full-length antibodies against the human IgG4 subclass containing mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain.

[0115] h) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, I253A, H310A, and H435A in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0116] i) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, I253A, H310A, and H435A in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain.

[0117] j) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0118] k) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain, or

[0119] l) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, H310A, H433A, and Y436A in both heavy chains, and mutations i) T366W and ii) S354C or Y349C in one heavy chain and mutations i) T366S, L368A, and Y407V and ii) Y349C or S354C in the corresponding other heavy chain, or

[0120] One of m)a) to l) that does not contain a C-terminal lysine residue.

[0121] 19. A pharmaceutical composition comprising an antibody according to any one of Examples 1 to 18 and a pharmaceutically acceptable carrier.

[0122] 20. The anti-TREM2 antibody according to any one of Examples 2 to 18, which is used as a drug.

[0123] 21. The anti-TREM2 antibody according to any one of Examples 2 to 18 or the medicament according to Example 20, for the treatment of a disease.

[0124] 22. A method of treating a subject with symptoms associated with loss of TREM2 function, the method comprising administering to the subject an antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19.

[0125] 23. A method of treating a subject with symptoms associated with toxic gain in TREM2 function, the method comprising administering to the subject an antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19.

[0126] 24. A method of treating a subject with symptoms associated with overactivated microglia, the method comprising administering to the subject an antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19.

[0127] 25. A method for reducing the level of amyloid plaques in a subject with such need, the method comprising administering to the subject an antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19.

[0128] 26. A method for reducing slow cognitive and functional decline in a subject in need by specifically promoting and enriching the function of beneficial and neuroprotective microglia in amyloid plaques or the cerebral vascular system covered by amyloid, the method comprising administering to the subject an antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19.

[0129] 27. A method for modulating the neuroprotective activity of amyloid plaques or microglia near the amyloid-covered cerebral vascular system in a subject with such need, the method comprising administering to the subject an antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19.

[0130] 28. An antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19, for treating symptoms associated with loss of TREM2 function in a subject with this need.

[0131] 29. An antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19, for treating symptoms associated with toxic gain in TREM2 function in a subject with this need.

[0132] 30. An antibody according to any one of Examples 2 to 18 or a pharmaceutical composition according to Example 19, for treating symptoms associated with overactive microglia in a subject in need.

[0133] 31. The antibody according to any one of Examples 2 to 18 or the pharmaceutical composition according to Example 19, for reducing the level of amyloid plaques in a subject with this need.

[0134] 32. The antibody according to any one of Examples 2 to 18 or the pharmaceutical composition according to Example 19, used to prepare a medicament for treating the condition of a subject in need.

[0135] 33. Use of the antibody according to any one of Examples 2 to 18 or the pharmaceutical composition according to Example 19 in the preparation of a medicament for reducing the level of amyloid plaques in subjects with such need.

[0136] 34. The anti-TREM2 antibody or method or use according to any one of Examples 20 to 33, wherein the disease or condition is or the subject suffers from a neuroinflammatory disease or a neurodegenerative disease.

[0137] 35. The anti-TREM2 antibody or method or use according to Example 34, wherein the neuroinflammatory disease or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hacola disease, Guillain-Barré syndrome (GBS), lysosomal storage disease, sphingomyelin storage disease (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neurobehçet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury or spinal cord injury.

[0138] 36. The anti-TREM2 antibody or method or use according to any one of Examples 34 to 35, wherein the neuroinflammatory disease or neurodegenerative disease is Alzheimer's disease.

[0139] 37. The anti-TREM2 antibody or method or use according to any one of Examples 34 to 36, wherein the neuroinflammatory disease or neurodegenerative disease is early Alzheimer's disease.

[0140] 38. The anti-TREM2 antibody or method or use according to any one of Examples 34 to 37, wherein the subject is amyloid-positive.

[0141] 39. The anti-TREM2 antibody or method or use according to Example 34, wherein the neuroinflammatory disease or neurodegenerative disease is multiple sclerosis (MS).

[0142] 40. The anti-TREM2 antibody or method or use according to any one of Examples 20 to 34, wherein the disease or symptom is or the subject suffers from Lewy body dementia.

[0143] 41. The anti-TREM2 antibody or method or use according to any one of Examples 20 to 34, wherein the disease or condition is or the subject has Parkinson's disease with dementia.

[0144] Further objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. These objects and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and explanatory and are not intended to limit the claims. For example, in addition to the various embodiments depicted and claimed herein, the subject matter of the invention relates to other embodiments having other combinations of the features reported and claimed herein. Thus, the specific features presented herein, especially as aspects or embodiments, can be combined with each other in other ways within the scope of the subject matter of the invention, such that the invention includes any suitable combination of the features reported herein. For illustrative and descriptive purposes, a description of specific embodiments of the subject matter of the invention has been presented. It is not intended to be exhaustive or to limit the subject matter of the invention verbatim to the reported embodiments. Detailed Implementation

[0145] Compared to molecules known in the art (34 nM), the anti-TREM2 antibody according to the invention induces the migration of THP-1 or iPSC-derived macrophages in vitro at surprisingly low concentrations (5 to 15 nM).

[0146] The antibody according to the invention has sub-nM TREM2 binding and sufficient affinity to induce effective amyloid uptake of Aβ in vitro and in vivo.

[0147] The anti-TREM2 antibody according to the present invention differs from known anti-TREM2 antibodies in the art because the antibody according to the present invention, as a molecule with Fc region effector function, does not induce or even downregulate pSyk in TREM2 / DAP12 overexpressing HEK or iPSC M0 cells. In contrast, known anti-TREM2 antibodies in the art upregulate pSyk in both the presence and absence of Aβ beads.

[0148] The antibody according to the invention has the potential to be used as an adjunct to anti-amyloid antibodies in early to moderate AD, and can also be used in combination therapy with other amyloid-reducing molecules or non-amyloid-targeting therapy in Alzheimer's disease.

[0149] The anti-TREM2 antibody according to the present invention can be used in combination with the anti-Aβ antibody.

[0150] Therefore, the present invention is based at least in part on the following findings

[0151] - Anti-TREM2 monospecific antibodies do not induce significant Aβ-bead uptake in THP-1 Aβ-bead binding and uptake assays (Example 3, Figures 5 and 6),7 which is independent of the Fc region isotype (wt or LALAPG); however, the dual complementary site antibody TREM2 3737, as a molecule with Fc region effector function, induces significant Aβ-bead uptake;

[0152] - In the THP-1 LPC-induced migration assay, all monospecific anti-TREM2 antibodies (TREM2 3306, TREM2 3308, TREM2 3292, TREM2 3297, TREM2 3293, TREM2 3295, TREM2 0819) of epitope regions 2 and 3 according to the invention, their Fc effector-silenced LALAPG variants and forms containing the double complementary sites of TREM2 3306, 3295 or TREM2 0885 from the prior art as a reference (but not an anti-Aβ monospecific mAb) induce migration;

[0153] - In human iPSC M0 C5a-induced migration assays, anti-TREM2 double complementary site antibodies containing TREM2 3306 increased migration, similar to or superior to existing technology TREM2 0885 from the effector-functional silenced Fc region (LALAPG);

[0154] - Compared with anti-TREM2 antibodies (Alector, Denali, Cognyxx, WashU) from the art, the anti-TREM2 monoclonal antibodies according to the present invention do not have the effector function ability to induce the release of TNFα / MIP-1α / IL-8 from iPSC-derived macrophages in the Fc region;

[0155] - Neither the anti-TREM2 antibody nor TREM2 0885 according to the present invention showed dose-dependent phosphorylation of Syk and S6 in HEK / DAP12 / TREM2 cells, while anti-TREM2 antibodies known in the art (Alector, Denali, Cognyxx, WashU, Amgen) induce pSyk or pS6;

[0156] - The anti-TREM2 monoclonal antibody according to the present invention does not induce pSyk or pS6 in the presence of Aβ beads or Aβ coating on the plate;

[0157] - The anti-TREM2 antibodies (TREM2 3306, TREM2 3295) according to the invention bind to epitopes in the ligand-binding Ig-like domain of the ECD of TREM2; the anti-TREM2 antibodies (TREM2 0819, TREM2 3292 and TREM2 3295) according to the invention have epitopes in the same region as the prior art TREM2 0885 (mAb21), while the anti-TREM2 antibodies (TREM2 3297 and TREM2 3306) do not overlap with any antibodies known in the art; antibodies in the art (i.e., those from Denali and Alector) bind near the ADAM cleavage site (the antibodies disclosed by Denali and Alector clearly bind to the C-terminal sTREM2 portion).

[0158] - The anti-TREM2 antibody TREM2 3306 is not cross-reactive to mouse TREM2 (the same as prior art antibodies from Denali and Alector), but prior art anti-TREM2 antibodies TREM2 3295 and TREM2 0885, which have cross-reactive Fc effector capabilities in mice, induce acute uptake of MX04-labeled amyloid and Aβ proteins in APPswePS2 tg mice.

[0159] General definition

[0160] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0161] Methods and techniques applicable to carrying out the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I through III (1997); Glover, ND and Hames, BD (eds.), DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture – a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, 2nd ed., CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J. (ed.), Cell Biology, 2nd ed., Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd ed., Alan R. Liss, Inc. NY (1987). The contents of these references are incorporated into this paper by reference.

[0162] Recombinant DNA technology can be used to generate derivatives of nucleic acids. Such derivatives can be modified, for example, at one or more nucleotide positions by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be readily performed by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD and Higgins, SG, Nucleic acid hybridization – a practical approach (1985) IRL Press, Oxford, England).

[0163] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a,” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0164] The term "about" indicates a range of + / - 20% of the following value. In some embodiments, the term "about" indicates a range of + / - 10% of the following value. In some embodiments, the term "about" indicates a range of + / - 5% of the following value.

[0165] As used herein, the term “activity” in relation to protein activity means any activity of a protein, including but not limited to enzyme activity, ligand binding, drug transport, ion transport, protein localization, receptor binding, and / or structural activity.

[0166] As used herein, the terms “comprising,” “including,” “having,” “possessing,” “may,” “containing,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term “comprising” also covers the term “comprising…”. This document also contemplates other embodiments that, whether or not explicitly stated, “include” the embodiments or elements presented herein, “consist of,” and “substantially constitute” them.

[0167] The term "effective amount" in this article refers to an amount sufficient to produce the desired outcome, such as treatment, inhibition, or reduction as described above.

[0168] As used herein, the term "exogenous" refers to a nucleotide sequence or nucleic acid that is not derived from a specific cell but is introduced into the cell via DNA delivery methods (e.g., transfection, electroporation, or transformation). Thus, exogenous nucleic acids are artificial elements, where artificiality can originate from, for example, a combination of portions of nucleic acids from different sources (e.g., a combination of a recombinase recognition sequence having an SV40 promoter and a coding sequence for a green fluorescent protein is an artificial nucleic acid) or from the deletion of portions of nucleic acids (e.g., sequences encoding only the extracellular domain of a membrane-binding receptor or cDNA), or nucleobase mutations. The term "endogenous" refers to a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but wherein the "exogenous" sequence is introduced into the cell, for example, via recombinant DNA technology.

[0169] As used herein, the terms “expression” and “expresses” refer to transcription and translation that occur within a cell. The expression level of nucleic acids in a cell can be determined based on the amount of corresponding mRNA present in the cell or the amount of nucleic acid-encoded proteins produced by the cell. For example, mRNA transcribed from nucleic acids is ideally quantified by northern hybridization. (Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3–7.57 (Cold Spring Harbor Laboratory Press, 1989)). Proteins encoded by nucleic acids can be quantified by a variety of methods, such as by measuring the protein’s biological activity or by employing assays unrelated to that activity, such as Western blotting or radioimmunoassay using antibodies that react with the protein. (Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1–18.88 (Cold Spring Harbor Laboratory Press, 1989)).

[0170] As used herein, the term "heterogeneous" refers to a polypeptide that is not derived from a specific cell, but rather that the corresponding coding nucleic acid has been introduced into the cell via DNA delivery methods (e.g., transfection, electroporation, or transformation). Therefore, a heterologous polypeptide is an artificial polypeptide relative to the cell that expresses it, regardless of whether the polypeptide is a naturally occurring polypeptide from a different cell / organism or a synthetic polypeptide.

[0171] The term "integration site" refers to the location in a cell's genome where a foreign nucleic acid is inserted / can be inserted / has been inserted. In some embodiments, the integration site is inserted between two adjacent nucleotides in the cell's genome. In some embodiments, the integration site comprises a segment of nucleotides. In some embodiments, the integration site is located within a specific locus in the genome of a mammalian cell. In some embodiments, the integration site is located within an endogenous gene in the mammalian cell.

[0172] "Isolated" nucleic acids refer to nucleic acids that have been isolated from components of their natural environment. Isolated nucleic acids include those contained in cells that normally contain nucleic acids, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0173] "Isolated nucleic acid encoding antibody" refers to one or more nucleic acids encoding the heavy and light chains (or fragments thereof) of the antibody according to the present invention. Such one or more nucleic acids include nucleic acids in a single vector or a separate vector, and such one or more nucleic acids are present at one or more locations in the host cell.

[0174] The terms “(mammal) cell” and “(mammal) cell line” are used interchangeably in this document to refer to cells in which one or more exogenous nucleic acids have been introduced, including progeny of such cells.

[0175] "Mammalian cells containing exogenous nucleotide sequences" and "recombinant mammalian cells" are both "transformed cells." This term includes primary transformed cells and their progeny, regardless of passage number. Progeny may not, for example, be completely identical to the nucleic acid contents of the parent cells, but may contain mutations. It also encompasses mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0176] The term "nucleic acid" or "polynucleotide" includes any molecule and / or compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of one base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), one sugar (i.e., deoxyribose or ribose), and one phosphate ester group. Nucleic acids are typically described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid. The base sequence is typically indicated in a 5'-to-3'- orientation, i.e., from the 5'-end to the 3'-end. In this document, the term nucleic acid encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, as well as synthetic forms of DNA. Nucleic acids can be linear or circular. Furthermore, the term nucleic acid includes both sense and antisense strands, and single-stranded and double-stranded forms. Additionally, the nucleic acids described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA molecules suitable as vectors for the direct expression of antibodies used in this 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.

[0177] As used herein, the term "operably linked" refers to the juxtaposition of two or more components in a manner that allows them to function in a desired way. For example, if a promoter and / or enhancer are used to regulate transcription of a coding sequence, then the promoter and / or enhancer are operably linked to the coding sequence. In some embodiments, the nucleic acid sequences "operably linked" are linked and adjacent on a single chromosome. In some embodiments, for example, when two protein-coding regions (such as a secretory leader region and a polypeptide) must be joined, these sequences are linked, adjacent, and within the same reading frame. In some embodiments, the operably linked promoter is located upstream of and adjacent to the coding sequence. In some embodiments, for example, with respect to enhancer sequences regulating coding sequence expression, the two components are operably linked but not adjacent. If the enhancer increases transcription of the coding sequence, then the enhancer is operably linked to the coding sequence. Operatically linked enhancers can be located upstream, inside, or downstream of the coding sequence, and can be located at a considerable distance from the promoter of the coding sequence. Operable ligation can be accomplished using recombination methods known in the art, such as PCR and / or by ligation at a convenient restriction site. If a convenient restriction site is not available, synthetic oligonucleotide adaptors or linkers can be used according to standard practice. The internal ribosome entry site (IRES) is operablely ligated to the open reading frame (ORF) if it allows for the initiation of ORF translation at an internal location in a manner independent of the 5' end.

[0178] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, and for alignment purposes without considering any conserved substitutions as part of sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted with the user documentation to the US Copyright Office, Washington DC, 20559, registered under US Copyright Registry No. TXU510087 and described in WO 2001 / 007611.

[0179] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or carrier conventional in the art, used in conjunction with a therapeutic agent, collectively constituting a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the receptor at the dose and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used. 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, ideally, the carrier is not sensitive to the skin and does not cause an injection site reaction.

[0180] As used herein, the term "recombinant mammalian cell" refers to a mammalian cell containing a foreign nucleotide sequence capable of expressing a polypeptide. Such recombinant mammalian cells are cells in which one or more foreign nucleic acids have been introduced, including progeny of such cells. Therefore, the term "mammalian cell containing nucleic acid encoding an antibody" refers to a cell containing a foreign nucleic acid integrated into the mammalian cell genome and capable of expressing an antibody. In some embodiments, a mammalian cell containing a foreign nucleic acid is a cell containing a foreign nucleic acid integrated at a single site within a locus in the mammalian cell genome, wherein the foreign nucleic acid contains a first recombinant recognition sequence and a second recombinant recognition sequence side-mounted with at least one first selection marker, and a third recombinant recognition nucleic acid located between the first and second recombinant recognition sequences, and all recombinant recognition sequences are distinct. In this case, integration is achieved via recombinase-mediated cassette exchange (RMCE).

[0181] As used herein, the term "selection marker" refers to a nucleic acid that allows for the specific selection or exclusion of cells carrying that nucleic acid in the presence of a suitable selective reagent. For example, but not as a limitation, a selection marker can allow for the positive selection of mammalian cells transformed with the nucleic acid of that selection marker in the presence of a suitable selective reagent (selective culture conditions); untransformed mammalian cells will not be able to grow or survive under those selective culture conditions. Selection markers can be positive, negative, or bifunctional. Positive selection markers allow for the selection of cells carrying the marker, while negative selection markers allow for the selective elimination of cells carrying the marker. Selection markers can confer resistance to drugs or compensate for metabolic or catabolistic defects in host cells. Resistance genes that can be used as selection markers in eukaryotic cells include, but are not limited to, genes targeting aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, cymoxanil, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143. In addition to aiding selection in the presence of appropriate selective reagents, selection markers can alternatively encode molecules not normally present in cells, such as 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 molecules can be distinguished from cells lacking the corresponding nucleic acid, for example, by detecting the fluorescence emitted by the encoded polypeptide or by the absence of such fluorescence.

[0182] As used herein, the term "shedding" refers to the process by which soluble TREM2 is produced from membrane-bound TREM2 through stem-region proteolytic cleavage of the protein. In vivo, "shedding" can occur at the cell surface, for example, due to the cleavage of TREM2 on the cell membrane by metalloproteinases or other enzymes. In some cases, cleavage occurs between H157 and S158 (sequentially numbered; SEQ ID NO: 416) of the protein, forming sTREM2 containing extracellular domain residues 19 to 157 of SEQ ID NO: 416.

[0183] The term "signal sequence" or "lead sequence" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates the secretion of the polypeptide from mammalian cells. The leader sequence can be cleaved as the polypeptide is exported from mammalian cells, forming a mature protein. Leader sequences can be natural or synthetic, and they may be heterologous or homologous to the protein they are linked to. Non-limiting exemplary leader sequences also include leader sequences derived from heterologous proteins. In some embodiments, the antibody lacks a leader sequence. In some embodiments, the antibody comprises at least one leader sequence, which may be selected from both a natural antibody leader sequence and a heterologous leader sequence.

[0184] The terms “subject” and “patient” are used interchangeably in this document to refer to a person. In some embodiments, methods for treating other mammals are also provided, including but not limited to rodents, apes, cats, dogs, horses, cattle, pigs, sheep, goats, laboratory mammals, farm mammals, sporting mammals, and pet mammals.

[0185] As used herein, the term "treatment" covers any application or administration of a therapeutic agent for a disease of a human or other mammal, and includes inhibiting the disease or its progression, suppressing or slowing the disease or its progression, preventing or slowing its development, suppressing, reducing or slowing the development of at least one symptom of the disease, slowing the onset of the disease, preventing the onset of at least one symptom of the disease, slowing the onset of at least one symptom of the disease, partially or completely alleviating the disease, or curing the disease, for example by inducing regression, or restoring or repairing lost, missing or defective functions; or stimulating inefficient processes. The terms "inhibition" or "inhibit" mean the reduction or cessation of any symptom or phenotypic feature, or the reduction or cessation of the incidence, severity or likelihood of such symptom or feature.

[0186] Unless otherwise explicitly stated (i.e., mouse TREM2 or cynomolgus monkey TREM2, etc.), the term "trigger receptor expressed on myeloid-2" and its abbreviation "TREM2" as used herein refer to the human TREM2 protein ("hTREM2"). An exemplary hTREM2 amino acid sequence (including the signal sequence of amino acids 1 to 18) is shown in SEQ ID NO: 416, while an exemplary sequence without the signal sequence is shown in SEQ ID NO: 417. TREM2 including the signal sequence may also be referred to as the "pre-protein" or "pre-protoprotein" 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 comprises a type V immunoglobulin (Ig) domain (at amino acids 19-128), followed by a TREM2 “stem” domain (at amino acids 129-174; SEQ ID NO: 416), which together form the protein’s “extracellular domain,” followed by a transmembrane domain (residues 175-197) and a cytosol domain (residues 198-230). The soluble form of the protein described herein, “soluble TREM2” or “sTREM2,” can be produced in vivo by cleavage in the stem domain or by selective splicing. For example, a protease can cleave the protein between amino acid residues H157 and S158 (sequentially numbered; SEQ ID NO: 416) to produce soluble TREM2 comprising the N-terminal portion of the protein up to H157 (e.g., residues 19-157). The portion of the mature TREM2 protein that can be cleaved to form sTREM2 is also called the “extracellular domain” and contains residues 19 to 157 of SEQ ID NO: 416. In general, as used herein, the term “TREM2” refers to the mature form of the protein. Furthermore, hTREM2 comprises several isoforms or alleles whose native sequences or splices may differ from those shown in SEQ ID NO: 416. The term TREM2 encompasses all these native forms of TREM2 unless a specific isoform or sequence is mentioned.

[0187] As used herein, the term "vector" refers to a nucleic acid capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of mammalian cells into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0188] Recombination generation method

[0189] Antibodies can be generated using recombinant methods and compositions, such as those described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0190] In one aspect of the invention, a method for generating multispecific antibodies is provided, wherein the method comprises culturing recombinant mammalian cells containing one or more nucleic acids encoding antibodies according to the invention under conditions suitable for antibody expression, recovering the antibody from the cells (or cell culture medium), and optionally purifying the multispecific antibody using one or more chromatographic steps, thereby generating multispecific antibodies.

[0191] expression carrier

[0192] For the recombinant production of antibodies, the nucleic acid encoding the antibody is generated or isolated and inserted into one or more vectors for further cloning and / or expression in mammalian cells. Such nucleic acids can be readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to the nucleic acids encoding the heavy and light chains of the antibody), or obtained through recombinant methods or chemical synthesis.

[0193] Typically, for the large-scale recombinant production of, for example, therapeutic antibodies, mammalian cells that stably express and secrete said antibodies are required. These cells are called "recombinant cells" or "recombinant production cells," and the process for producing such cells is called "cell line development."

[0194] In the first step of the cell line development process, suitable mammalian cells (such as, for example, CHO cells) are transfected with nucleic acids suitable for expressing the antibody. In the second step, cells that stably express the antibody are selected based on the co-expression of selection markers that have been co-transfected with nucleic acids encoding the antibody.

[0195] Nucleic acids encoding the amino acid sequence of an antibody chain, or coding nucleic acids, are purely informational, and their expression requires additional regulatory elements. Therefore, coding nucleic acids are typically integrated into so-called expression cassettes. The minimum regulatory elements required for an expression cassette to function in mammalian cells are the promoter, which functions in the mammalian cell, located upstream of the coding nucleic acid (5'), and the polyadenylation signal sequence, which functions in the mammalian cell, located downstream of the coding nucleic acid (3'). The promoter, coding nucleic acid, and polyadenylation signal sequence are arranged in an operatively linked manner.

[0196] In the case of heteropolymeric proteins (such as antibodies) whose target polypeptide is composed of different (monomer) polypeptides, what is needed is not just a single expression cassette, but multiple expression cassettes that are different from each other at least in terms of the encoded nucleic acids contained therein. That is, at least one expression cassette is needed for each of the different (monomer) polypeptides of the heteropolymeric protein.

[0197] For example, a full-length monospecific antibody is a heteropolymeric protein containing two copies of the light chain and two copies of the heavy chain. Therefore, a full-length antibody is a tetrapolymer composed of two different polypeptides. Consequently, expression of a full-length antibody requires at least two expression cassettes: one for the light chain and another for the heavy chain.

[0198] For example, if a full-length antibody is a bispecific antibody, meaning it contains two different binding sites that specifically bind to two different antigens, then the two light chains and the two heavy chains are also different from each other. Therefore, such a bispecific full-length antibody is composed of four different polypeptides and thus requires four expression cassettes.

[0199] The antibody expression cassette is then integrated into one or more so-called "expression vectors." An "expression vector" is a nucleic acid that provides all the necessary elements for amplifying the vector in prokaryotic cells and for expressing one or more encoded nucleic acids contained therein in mammalian cells. Typically, an expression vector contains a prokaryotic plasmid proliferation unit, such as a prokaryotic plasmid proliferation unit for *E. coli*, which includes an origin of replication and prokaryotic selection markers, as well as eukaryotic selection markers, and further expression cassettes required for the expression of one or more encoded nucleic acids of the antibody. An "expression vector" is a transporter for introducing the expression cassette into mammalian cells to produce recombinant mammalian cells.

[0200] As outlined in the preceding paragraphs, the more complex the antibody, the higher the number of different expression cassettes required. Inherently, as the number of expression cassettes increases, the total size of the nucleic acid integrated into the host cell genome (i.e., the number of base pairs (bp)) also increases. The size of the expression vector also increases accordingly. However, the practical upper limit for vector size is in the range of approximately 15 kbp; beyond this range, processing and handling efficiency decreases significantly. This problem can be addressed by using two or more expression vectors. Therefore, expression cassettes are divided into different expression vectors, each containing only a portion of the expression cassette, thus reducing the size (bp) of each vector.

[0201] Cell line development

[0202] Cell line development (CLD) for producing recombinant mammalian cells expressing antibodies employs one or more nucleic acids that are randomly integrated (RI) or targeted integrated (TI), containing the appropriate expression cassette required for antibody expression and production.

[0203] Using RI, generally speaking, several vectors or fragments thereof are integrated into the cell's genome at the same or different loci.

[0204] Typically, using TI (Transgenic Integrator) involves integrating a single copy of a transgene containing different expression cassettes into a predetermined "hotspot" in the host cell genome.

[0205] Targeted integration

[0206] Targeted integration (TI) allows the integration of exogenous nucleic acids into predetermined sites in the mammalian cell genome.

[0207] In some embodiments, TI mammalian cells are used in the method according to the invention for introducing exogenous nucleic acids encoding antibodies. Using such recombinant TI mammalian cells provides robust, stable cell culture performance and reduces the risk of sequence variation in the resulting recombinant antibodies. TI cells and strategies for their use are described in detail, for example, in US2021 / 0002669, the contents of which are incorporated herein by reference in their entirety.

[0208] In some embodiments employing targeted integration, exogenous nucleic acids are integrated into specific loci within the genome of TI cells.

[0209] In targeted integration, site-specific recombination is used to introduce exogenous nucleic acids into specific loci in the genome of mammalian TI cells. This is an enzymatic process in which the sequence at the integration site in the genome is exchanged for the exogenous nucleic acid. One system used to achieve this type of nucleic acid exchange is the Cre-lox system. The enzyme catalyzing the exchange is Cre recombinase. The sequence to be exchanged is defined by the positions of two lox(P) sites in the genome and the exogenous nucleic acid. These lox(P) sites are recognized by Cre recombinase. No further steps are required, i.e., no ATP is needed. The Cre-lox system was initially discovered in bacteriophage P1.

[0210] In all aspects and certain embodiments of the invention, the cells used in the method according to the invention have undergone targeted integration of one or more nucleic acids encoding antibodies.

[0211] In all aspects and embodiments of the invention, targeted integration is mediated by a recombinase that recognizes one or more recombinant recognition sequences (RRS) present in the genome of a mammalian cell and in a foreign nucleotide sequence to be integrated into the genome of the mammalian cell.

[0212] In all aspects and certain embodiments of the invention, targeted integration is mediated by homologous recombination.

[0213] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by recombinases and is both necessary and sufficient for recombinase-mediated recombination events. RRS can be used to define the location in a nucleotide sequence where a recombination event will occur.

[0214] In all aspects and some embodiments of the invention, RRS is recognized by Cre recombinase.

[0215] In all aspects and certain embodiments of the invention, RRS is the LoxP site and Cre recombinase targets integration via recombination-mediated integration.

[0216] In all aspects and some embodiments of the invention, the RRS is recognized by the FLP recombinase.

[0217] In all aspects and certain embodiments of the invention, RRS is an FRT site and the FLP recombinase targets and integrates via recombination-mediated integration.

[0218] In all aspects and some embodiments of the invention, the RRS is recognized by the Bxb1 integrase.

[0219] In all aspects and certain embodiments of the invention, the RRS is a Bxb1 attP or Bxb1attB site and the Bxb1 integrase mediates targeted integration via recombination.

[0220] In all aspects and some embodiments of the invention, the RRS is recognized by the φC31 integrase.

[0221] In all aspects and certain embodiments of the invention, the RRS is a φC31 attP or φC31attB site and the φC31 integrase is targeted integration mediated by recombination.

[0222] Recombinases can be introduced into cells using expression vectors containing the enzyme or as the coding sequence of a protein or mRNA.

[0223] According to the present invention, any known or future mammalian cell, including landing sites as described herein, at a single site within a locus integrated into the genome, can be used in the present invention with respect to TI. Such cells are referred to as mammalian TI cells.

[0224] In all aspects and embodiments of the invention, the mammalian TI host cell is a hamster cell, human cell, rat cell, or mouse cell containing a landing site as described herein. In a preferred embodiment, the mammalian TI cell is a CHO cell. In some embodiments, the mammalian TI CHO cell is a CHO K1 cell, CHO K1SV cell, CHOG44 cell, CHO DUKXB-11 cell, CHO K1S cell, or CHO K1M cell containing a landing site as described herein at a single site integrated within a genomic locus.

[0225] In all aspects and certain embodiments of the invention, mammalian TI cells include an integrated landing site, wherein the landing site contains one or more recombination recognition sequences (RRS). The RRS may be recognized by a recombinase (e.g., Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase). One or more RRS may be independently selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence, and φC31 attB sequence. If multiple RRSs must exist, the choice of each of these sequences depends on another sequence within the limits of choosing different RRSs.

[0226] In some embodiments of all aspects and examples of the invention, the landing site includes one or more recombinant recognition sequences (RRSs), wherein the RRS can be recognized by a recombinase. In some embodiments, the integrated landing site includes at least two RRSs. In some embodiments, the integrated landing site includes three RRSs, wherein a third RRS is located between a first RRS and a second RRS. In some preferred embodiments, all three RRSs are different. In some embodiments, the landing site includes a first RRS, a second RRS, and a third RRS, and at least one selection marker located between the first RRS and the second RRS, and the third RRS is different from the first RRS and / or the second RRS. In some embodiments, the landing site also includes a second selection marker, and the first and second selection markers are different. In some embodiments, the landing site also includes a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operatively linked to the third selection marker. The third selection marker may be different from the first or second selection marker.

[0227] An exemplary mammalian TI cell suitable for the method according to the invention is a CHO cell, which carries a landing site at a single site within a locus integrated into its genome, wherein the landing site contains three xenogeneic-specific loxP sites for Cre recombinase-mediated DNA recombination.

[0228] In some embodiments, the xenogeneic-specific loxP site is L3 (SEQ ID NO: 418), LoxFas (SEQ ID NO: 419), and 2L (SEQ ID NO: 420) (see, for example, Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), wherein L3 and 2L are laterally attached to the landing site at the 5' and 3' ends, respectively, and LoxFas is located between the L3 and 2L sites. The landing site also contains a bicistronic unit that links the expression of the selection marker to the expression of the fluorescent GFP protein via IRES, thereby allowing for positive selection to stabilize the landing site, as well as selection for the absence of the site after transfection and Cre recombination (negative selection). Green fluorescent protein (GFP) is used to monitor the RMCE response.

[0229] This configuration of the landing site, as outlined in the previous paragraph, allows for the simultaneous integration of two vectors, such as a so-called pro-vector carrying L3 and LoxFas sites, and a post-vector containing LoxFas and 2L sites. Functional elements of the selection marker gene, distinct from those present at the landing site, can be distributed between the two vectors: the promoter and start codon can be located on the pro-vector, while the coding region and polyA signaling site are located on the post-vector. Only integration mediated by the correct recombinase from both vectors induces resistance to the corresponding selector.

[0230] Generally speaking, a mammalian TI cell is a mammalian cell containing a landing site at a single locus integrated within the mammalian cell genome, wherein the landing site contains a first recombination recognition sequence and a second recombination recognition sequence flanked by at least one first selection marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and all recombination recognition sequences are distinct.

[0231] The selected biomarkers can be chosen from groups containing the following: aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, cymoxanil, chloramphenicol, zeocin, and mycophenolic acid. The chosen marker can also be a fluorescent protein selected from the group consisting of: 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, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

[0232] Exogenous nucleic acids are nucleic acids that are not derived from specific cells but are introduced into the cells via DNA delivery methods (such as transfection, electroporation, or transformation). In some embodiments, mammalian TI cells contain at least one landing site at one or more integration sites integrated into the mammalian cell genome. In some embodiments, the landing site is integrated at one or more integration sites within a specific locus of the mammalian cell genome.

[0233] In some embodiments of all aspects and embodiments of the invention, the integration site includes at least one selection marker. In some embodiments, the integrated landing site includes a first RRS, a second RRS, and a third RRS, and at least one selection marker. In some embodiments, the selection marker is located between the first RRS and the second RRS. In some embodiments, the two RRSs are side-attached to at least one selection marker, i.e., the first RRS is located at the 5' (upstream) of the selection marker and the second RRS is located at the 3' (downstream) of the selection marker. In some embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker. In some embodiments, the landing site includes a first RRS, a second RRS, and a third RRS, and at least one selection marker located between the first RRS and the third RRS.

[0234] In some embodiments of all aspects and embodiments of the invention, at the integrated landing site, the selection marker is located between a first RRS and a second RRS, and these two side-mounted RRSs are different. In some preferred embodiments, the first side-mounted RRS is a LoxP L3 sequence, and the second side-mounted RRS is a LoxP 2L sequence. In some embodiments, the LoxP L3 sequence is located at the 5' end of the selection marker, and the LoxP 2L sequence is located at the 3' end of the selection marker. In some embodiments, the first side-mounted RRS is a wild-type FRT sequence, and the second side-mounted RRS is a mutant FRT sequence. In some embodiments, the first side-mounted RRS is a Bxb1 attP sequence, and the second side-mounted RRS is a Bxb1 attB sequence.

[0235] In some embodiments, the first side-connected RRS is a φC31 attP sequence, and the second side-connected RRS is a φC31 attB sequence. In some embodiments, the two RRSs are positioned in the same orientation. In some embodiments, both RRSs are in either a forward or reverse orientation. In some embodiments, the two RRSs are positioned in opposite orientations.

[0236] In some embodiments of the invention, the integrated landing site comprises a first selection marker and a second selection marker lateralized to two RRSs, wherein the first selection marker is different from the second selection marker. In some embodiments, both selection markers are independently selected from the group consisting of: glutamine synthetase selection marker, thymidine kinase selection marker, HYG selection marker, and puromycin resistance selection marker. In some embodiments, the integrated landing site comprises a thymidine kinase selection marker and a HYG selection marker. In some embodiments, the first selectable marker is selected from the group consisting of: aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, cyprodinil, chloramphenicol, zeocin, and mycophenolic acid; and the second selectable marker is selected from the group consisting of: GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire fluorescent proteins. In some embodiments, the first selection marker is a glutamine synthase selection marker, and the second selection marker is a GFP fluorescent protein. In some embodiments, the two RRSs of the two selection markers are different.

[0237] In some embodiments, the selection marker is operatively linked to a promoter sequence. In some embodiments, the selection marker is operatively linked to the SV40 promoter. In some embodiments, the selection marker is operatively linked to the human cytomegalovirus (CMV) promoter.

[0238] In some embodiments of all aspects and examples of the invention, one or more exogenous nucleic acids encoding antibodies have been integrated into mammalian TI cells via single or double recombinase-mediated cassette exchange (RMCE). This yields recombinant mammalian cells, such as recombinant CHO cells, in which a defined and specific expression cassette sequence has been integrated into a single locus in the genome, thereby leading to efficient expression and production of the antibody.

[0239] 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 a 34 bp LoxP sequence. Cre recombinase originates from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp non-palindromic core region and two flanking 13 bp inverted repeat sequences. Cre recombinase binds to the 13 bp repeat sequences, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and requires no additional host factors. If two LoxP sequences are placed in the same nucleotide sequence with the same orientation, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences, resulting in a covalently closed loop. If two LoxP sequences are placed in the same nucleotide sequence in reverse order, Cre recombinase-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. If the two LoxP sequences are on two different DNA molecules, and if one DNA molecule is circular, Cre recombinase-mediated recombination will cause integration of the circular DNA sequence.

[0240] The term "matching RRS" indicates that recombination has occurred between two RRSs. In some embodiments, the two matching RRSs are identical. In some embodiments, both RRSs are wild-type LoxP sequences. In some embodiments, both RRSs are mutant LoxP sequences. In some embodiments, both RRSs are wild-type FRT sequences. In some embodiments, both RRSs are mutant FRT sequences. In some embodiments, the two matching RRSs are different sequences, but can be recognized by the same recombinase. In some embodiments, the first matching RRS is a Bxb1 attP sequence, and the second matching RRS is a Bxb1 attB sequence. In some embodiments, the first matching RRS is a φC31 attB sequence, and the second matching RRS is a φC31 attB sequence.

[0241] When using a dual-vector combination, a “dual plasmid RMCE” strategy or “dual RMCE” is employed in the method according to the invention. For example, but not as a limitation, the integrated landing site may contain three RRSs, such as in the following arrangement: wherein a third RRS (“RRS3”) exists between a first RRS (“RRS1”) and a second RRS (“RRS2”), and the first vector contains two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector contains two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence.

[0242] The dual-plasmid RMCE strategy involves using three RRS sites to implement two independent RMCEs. Therefore, the landing site in mammalian TI cells using the dual-plasmid RMCE strategy includes a third RRS site (RRS3), which has no cross-activity with either the first RRS site (RRS1) or the second RRS site (RRS2). Both target plasmids require identical flanking RRS sites for efficient targeting; one plasmid (pre-plasmid) flanks RRS1 and RRS3, and the other expression plasmid (post-plasmid) flanks RRS3 and RRS2. Two selection markers are also required in this dual-plasmid RMCE. One selection marker expression cassette is divided into two parts. The pre-plasmid will contain the promoter, followed by the start codon and the RRS3 sequence. The post-plasmid will 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 (i.e., operational linking) of the fusion protein. The complete expression cassette of the selection marker will only be assembled, thus equipping the cell with resistance to the corresponding selectant, if both plasmids are correctly inserted.

[0243] Dual-plasmid RMCE involves a double recombination crossover event between two xenogeneic-specific RRSs and donor DNA molecules within a target genomic locus, catalyzed by a recombinase. Dual-plasmid RMCE is programmed to introduce copies of DNA sequences from both the combined pre- and post-vectors into a predetermined locus in the mammalian TI cell genome. RMCE can prevent the introduction of prokaryotic vector sequences into the mammalian TI cell genome, thereby reducing and / or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0244] In some embodiments of all aspects and examples of the invention, targeted integration is achieved through two RMCEs, wherein two different DNA sequences are integrated into predetermined sites in the genome of an RRS-matched mammalian TI cell, wherein each DNA sequence contains at least one expression cassette encoding a portion of a multispecific antibody according to the invention and / or at least one selection marker or a portion thereof flanked by two xenogeneic specific RRSs. In some embodiments, targeted integration is achieved through multiple RMCEs, wherein DNA sequences from multiple vectors are all integrated into predetermined sites in the genome of a mammalian TI cell, wherein each DNA sequence contains at least one expression cassette encoding a portion of a heteropolymeric polypeptide and / or at least one selection marker or a portion thereof flanked by two xenogeneic specific RRSs. In some embodiments, the selection marker may be partially encoded on a first vector and partially encoded on a second vector, such that the selection marker can only be expressed by proper integration of both via dual RMCEs.

[0245] cell lines

[0246] Suitable mammalian cells for expressing antibodies are typically derived from multicellular organisms (such as, for example, vertebrates).

[0247] Examples of mammalian cells that can be used in the methods of the present invention include human amniotic fluid 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 with SV40 (COS-7); human embryonic kidney cells (e.g., HEK293 or HEK293T cells as described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); and Buffalo rat hepatocytes (BRL). 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, for example, in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells.

[0248] Particularly useful mammalian cells for use in the methods according to the 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 K1 cells, as well as human embryonic kidney cells (HEK293 cells). For a review of certain mammalian cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totoa, NJ (2004), pp. 255-268.

[0249] In all aspects and certain embodiments of the invention, the mammalian cells used in the methods or uses according to the invention are Chinese hamster ovary (CHO) cells (e.g., CHO K1, CHO DG44, etc.) or human embryonic kidney (HEK) cells.

[0250] The antibodies and their properties according to the present invention

[0251] It has been found that the anti-TREM2 antibody according to the invention provides advantageous properties, including the induction of microglia migration.

[0252] The anti-TREM2 antibody according to the present invention has sub-nM (two-digit pM) to low nM affinity for human TREM2.

[0253] In particular, the antibody exhibits good concentration / viscosity results, reaching 20 cP at 131 to 183 mg / ml at 20°C.

[0254] Therefore, it has been found that the antibody according to the invention does not induce the release of harmful soluble factors in iPSC-derived M0 macrophages.

[0255] Antibody

[0256] Antibodies against human TREM2 (SEQ ID NO: 416) have been generated by immunizing wild-type rabbits, transgenic rabbits (tg rabbits) based on the human IgG locus, transgenic rats (tg rats) based on the human IgG locus, and by phage display. The resulting antibodies have been characterized and tested in different forms using different combinations of complementary sites. The respective monospecific single-complementary-site and double-complementary-site antibodies are listed in the table below. Schematic diagrams of the corresponding forms are shown in Figure 1.

[0257]

[0258] Antibodies generated through immunization have been characterized in terms of their activity in cell migration assays, phagocytosis assays, pSyk activation assays, epitope binding assays, and human and mouse TREM2 monomer binding affinity assays.

[0259] Epitope

[0260] In the following comparison, the epitopes of different anti-TREM2 antibodies are shown in the sequence of human TREM2 (SEQ ID NO: 416).

[0261] s: signal peptide / sequence

[0262] 1: TREM2 0885

[0263] 2: TREM2 0886

[0264] 3: TREM2 3306 (conformational epitope)

[0265] 4: TREM2 3297 (conformational epitope)

[0266] 5: TREM2 2903, 3247-3259, 3164, 3263, 3264

[0267] 6: TREM2 2900

[0268] 7: TREM2 2904

[0269] 8: Cleavage site soluble TREM2

[0270] 9: TREM2 0705 (conformational epitope)

[0271] A: TREM2 0702

[0272] B: TREM2 8010

[0273] C: TREM2 3295

[0274]

[0275] It can be seen that the epitopes of the antibodies according to the present invention are different from those of anti-TREM2 antibodies known in the art. This can be further seen from the properties of the anti-TREM2 antibodies according to the present invention, which are different from those known in the art. Binding to the same epitope will result in the same properties.

[0276] Pharmacokinetics

[0277] Pharmacokinetic (PK) parameters were determined in human FcRn transgenic wild-type mice and human FcRn transgenic SCID mice.

[0278] The results are shown in the table below.

[0279]

[0280] Compared to TREM2 3306, Aβ 8655 has a higher clearance rate.

[0281] Development

[0282] All antibodies can be produced in good yield.

[0283] Thermal stability was analyzed by storage at 40°C for 2 weeks (pH 6, His / NaCl buffer, 1 mg / mL; denaturing conditions) and at 37°C for 4 weeks (pH 7.4, PBS, 10 mg / mL; natural conditions simulating in vivo conditions).

[0284] All candidates showed LMW formation after 4 weeks of PBS stress.

[0285] Nevertheless, under the conditions of use, the untested format is unstable, indicating good shelf-life stability.

[0286] Targeted-mediated drug delivery (TMDD)

[0287] mice

[0288] The anti-TREM2 antibody bound to the cell surface of mouse macrophages. Both M1 and M2 macrophages showed TREM2 expression on their cell surface. Compared to M1 macrophages, M2 macrophages showed approximately 2-fold TREM2 expression.

[0289] TREM2 binders exhibited characteristic TMDD-like behavior in the internalization of M1 and M2 macrophages.

[0290] people

[0291] The same phenomena observed with human M2 macrophages were observed as with mouse macrophages.

[0292] The bivalent TREM2 binder exhibited saturation kinetics, while the monovalent TREM2 binder showed mild TMDD-like behavior. The Aβ antibody showed linear uptake. More specifically, the bivalent TREM2 binder TREM2 3306 showed significantly higher v compared to the monovalent binder. max and lower K m See the table below and Figure 2.

[0293]

[0294] In human M2 macrophages, nonspecific uptake was significantly higher (approximately 3 to 5 times) compared to mouse hFcRn Tg32+ / + M2 macrophages (4 to 15 mAb / cell / min / nM), but the magnitude was similar. The bivalent monospecific anti-Aβ antibody exhibited the lowest linear uptake. The bivalent monocomplementary anti-TREM2 antibody TREM2 3306 and the reference antibody movizumab showed even higher nonspecific uptake (see Figure 3).

[0295]

[0296] Migration assays were performed using macrophages (M0 cells) derived from THP-1 or iPSCs.

[0297] M0 cells derived from THP-1 and iPSCs were cultured in a transwell assay, exposed to a chemokine gradient (lysoPC or complement C5a, respectively), and co-incubated 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 schematic diagram of this assay is shown in Figure 4.

[0298] In the first set of experiments, the Fc-active version of TREM2 at epitope regions 2 and 3 was tested in parallel with the reference antibody TREM20885 (mAb21) at 10 µg / ml (approximately EC50 in the THP-1 migration assay). The antibodies listed in the table below achieved maximum migration before 10 µg / ml. Most antibodies showed strong migration induction with increases of 2.0 to 3.6 times (expressed as a percentage change compared to the medium) compared to the medium control. The reference antibody reached a plateau at 0.015 µg / ml (based on dose-response curves), while the antibody according to the invention reached maximum response at 0.02 to 0.62 µg / ml. The maximum response of the reference antibody was set at 100%. The antibody according to the invention showed a range of 49% to 117% of this response. There was no significant difference in potency between the different epitope regions.

[0299]

[0300] Negative data indicates a potential reduction in migration.

[0301] Similarly, antibody migration was tested in iPSC-derived macrophages, which showed similar effects to those in THP-1 cells, as shown in the table below.

[0302]

[0303] Complementary sites of TREM2 3295 and TREM2 3306 are used to engineer further antibody formats, such as... Figure 1 Different 1+1, 2+1, and 2+2 formats were shown, along with varying specificities such as monospecific, monocomplementary, or monospecific, bicomplementary, or bispecific. Additionally, different linker lengths and Fc region variations were tested. However, all antibody forms contained the LALAPG mutation (L234A / L235A / P329G mutation) to reduce Fc region binding to FcgR and complement C1q. Without being bound by this theory, it is hypothesized that the risk of Fc region-mediated induced inflammation by this antibody is reduced or even eliminated.

[0304] Different forms and specific antibodies were tested in the M0 migration assays derived from THP-1 and iPSCs and compared to the LALAPG version of the reference antibody mAb21 (TREM2 2907). It must be noted that the reference antibody with the LALAPG mutation (TREM2 2907) exhibited a migration index that was typically only about 66% of that of the reference antibody without the LALAPG mutation (TREM2 0885), suggesting that the reference antibody is most likely to exert some of its efficacy via FcgR binding. As a negative control, an anti-Aβ antibody (Aβ 8653) was also included, which never showed any migration induction.

[0305] Equally surprising is that antibodies known in the art sometimes induce minimal migration above baseline only within the same dose range of 0.14 to 34 nM, and some baseline migration is reduced at concentrations > 1 nM, indicating functional inhibition. The antibodies according to the invention do not list reduced baseline migration at any tested concentration.

[0306]

[0307] Phagocytosis assay

[0308] Further testing has been conducted to determine whether the anti-TREM2 antibody according to the invention can induce the phagocytosis of amyloid particles.

[0309] To assess this, an assay was performed using Aβ-coated beads labeled with pHRodo. Upon uptake of these coated beads and transport to lysosomes, the pH-sensitive dye becomes detectable and can be quantitatively absorbed.

[0310] Data were generated by co-incubating phagocytic THP-1 cells with pHRodo-labeled beads coated with Aβ42 for 16 hours. The baseline uptake of these cells was approximately 10%, which was subtracted from the mean. Antibodies were compared using the percentage of cells with amyloid-coated beads that did not undergo mAb adjustment. Most antibodies known in the art remain below 5% of uptake or are undetectable. The monospecific anti-TREM2 antibody of the present invention, which does not contain the LALAPG mutation, also did not show any uptake of Aβ beads (data not shown).

[0311] The Fc region effector function of the monospecific anti-Aβ antibody was silenced, and the LALAPG construct was found to lack uptake of Aβ-coated beads.

[0312] The corresponding results are shown in Figure 5 and the table below.

[0313]

[0314] Some of the monospecific, dual complementary anti-TREM2 antibodies according to the present invention can exhibit unexpected effects, namely, antibody-induced uptake of labeled Aβ beads.

[0315] The monospecific dual complementary antibody with the standard IgG format (TREM2 3737) performed best. Most surprisingly, bead uptake of TREM2 3737 occurred even in the absence of Aβ binders and LALAPG molecules.

[0316] Without being bound by this theory, it is assumed that the monospecific, dual complementary anti-TREM2 antibody according to the present invention can be used to treat Aβ-dependent diseases, such as Alzheimer's disease, with a reduced or even no risk of Aβ-targeting-related adverse events (such as ARIA).

[0317] Different forms of dual complementary anti-TREM2 antibodies are TREM2 3737, TREM2 3738, TREM2 3846, TREM2 3857, TREM2 3847, TREM2 3858, TREM2 3739, TREM2 2727 and TREM2 3859.

[0318] SPR and FACS data showed that monospecific, dual-complementary anti-TREM2 antibodies exhibited enhanced binding compared to their original conjugates (e.g., TREM2 3737, TREM2 3738, TREM2 3739). TREM2 3737 even showed activation of phagocytosis in the absence of FcgR or Aβ binding. This data is shown in the table below.

[0319]

[0320] Based on EC50 and AUC values, all antibodies were strong phagocytic inducers. The introduction of the YTE mutation appeared to have no effect. No effect was observed in TREM2 knockout THP-1 cells.

[0321] Amyloid uptake in vivo in APPswePS2 transgenic mice within the methoxy-X04 paradigm

[0322] In APP transgenic mice, amyloid plaques were labeled with the methoxy-X04 (MX04) paradigm, followed by in vitro analysis of isolated microglia by flow cytometry, adapted from the literature (Bolmont et al., J Neurosci, 2008; Heneka et al., Nature 2013; Kummer et al., EMBO Journal 2021). In short, APPswePS2 transgenic mice were aged to form amyloid plaques and intravenously injected with either the antibody according to the invention or an inert IgG control. One week after antibody injection, the mice were injected with MX04, and the brains were collected the following day for microglia isolation and flow cytometry analysis. In the first set of experiments (Experiments 1 and 2), the anti-TREM2 antibody according to the invention, which previously showed positive responses in migration and Aβ-coated bead uptake assays and possesses Fc region effector function, was 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 a moderate to strong increase in amyloid-to-microglia expression, some of which reached statistical significance. The results are shown in Figure 7.

[0323] Experiments have shown that anti-TREM2 antibodies with Fc region effector function can induce increased uptake of amyloid protein by microglia. As an alternative to monospecificity, dual-complementary-site anti-TREM2 antibodies can be used. Without being bound by this theory, it can be hypothesized that this approach optimizes TREM2 signaling in microglia, thus enabling them to bind to amyloid plaques via TREM2 cross-linking.

[0324] Surprisingly, uptake into microglia can be achieved using a monospecific anti-TREM2 antibody with Fc region effector capabilities. This mechanism is TREM2-dependent, as application of the monospecific anti-DP47 / Aβ antibody does indeed lead to increased uptake (data not shown).

[0325] Syk phosphorylation

[0326] Syk phosphorylation was determined. The results are shown in Figure 8.

[0327] In some experiments, total Syk was comparable across all conditions and test antibodies (data not shown), while a baseline luminescent increase of approximately 1000 units of pSyk was observed with culture medium and buffer controls (blue and red dashed lines represent 100% in the absence and presence of beads, respectively). This baseline increase may indicate tetanic low-level pSyk activation induced by a presumed ligand in the cellular environment. Addition of Aβ-coated beads only slightly increased baseline pSyk levels. Incubation with various anti-TREM2 antibodies (all 50 nM) from the art, regardless of the presence or absence of Aβ-coated beads, resulted in robust upregulation of pSyk for most tested antibodies. Surprisingly, the monospecific anti-TREM2 antibodies according to the invention (i.e., TREM2 3295, TREM2 3306; both 50 nM) actually reduced the baseline-elevated pSyk levels in the absence of Aβ-coated beads. Importantly, this effect was observed in anti-TREM2 antibodies that do not have an Aβ binding site in the presence of Aβ-coated beads.

[0328] In replicates of experiments with low baseline pSyk levels (approximately 500 units) in both the culture medium and the medium control, the results obtained were consistent with those described above. The results are shown in Figure 9.

[0329] The bispecific anti-TREM2 complement sites used all bind to the extracellular ligand-binding domains of TREM2 (eptopes 2 and 3). The hypothesis for this antagonism is that the substitution of the putative ligand, which normally induces low levels of tetanic TREM2 signaling, leads to the interruption of the original triggering. The TREM2 complement sites can cause TREM2 to cluster on the cell surface and thus induce the pSyk cascade in an agitative manner.

[0330] Scientific principles

[0331] The R47H mutation in human TREM2 is genetically associated with an increased risk of developing Alzheimer's disease (LoF, characterized by enhanced periplasmic neurotrophic dystrophism). Loss-of-function mutations in TREM2 lead to FTD-like dementia (Nasu-Hacola disease). Furthermore, soluble TREM2 in CSF is associated with elevated levels of tau and pTau. That is, CSF-soluble TREM2 is progressively upregulated in prodromal AD with increased tau and NfL (neuronal damage), and downregulated during cognitive decline in clinical AD (see, for example, Jay et al., 2017, Mol. Neurodeg.; Ulland and Colonna, Nat Rev Neurol 2018; Deczkowska et al., Cell 2018).

[0332] TREM2 deficiency in a mouse AD model leads to reduced microglia survival, decreased microglia migration to plaques, and reduced plaque compaction. Controversial results were observed regarding plaque number and density.

[0333] TREM2 signaling on microglia and myeloid cells is induced by a pathogen / damage-associated molecular pattern.

[0334] The microglia-TREM2 hypothesis in Alzheimer's disease supports the applicability of the antibodies according to the present invention.

[0335] More specifically, a robust, holistic genetic and neuropathological framework has been established in this field for the crucial role of innate immunity in Alzheimer's disease. The TREM2 mutation R47H has been identified as a genetic risk factor for Alzheimer's disease (proposed LoF, enhanced periplasmic neurotrophic dystrophy leading to loss of the DAM phenotype). CSF sTREM2 is progressively upregulated in prodromal AD with increased tau and NfL (neuronal damage) and downregulated during cognitive decline in clinical AD. Preclinical evidence has been established for a mechanistic link between certain MS4A gene cluster variants and sTREM2 shedding. Certain MS4A gene cluster variants associated with elevated CSF sTREM2 concentrations are also associated with reduced AD risk and delayed age of onset.

[0336] In other words, available clinical and preclinical data point to the loss of microglial function in Alzheimer's disease, which has been proposed to be restored by inducing appropriate TREM2 signaling; that is, by stimulating anti-TREM2 monoclonal antibodies.

[0337] Without being bound by this theory, it is hypothesized that the bispecificity of the antibody according to the invention allows for a unique mode of action distinct from both monospecific anti-TREM2 antibodies and monospecific anti-Aβ antibodies. This is achieved by enhancing the non-inflammatory protective phenotype of microglia at the plaque site, enhancing the uptake of amyloid protein and cellular debris (phagocytosis / clearance function), inhibiting the inflammatory response induced by inappropriate microglia activation, and limiting pathological / protecting neurons from a toxic environment (barrier function). Furthermore, it is hypothesized that TREM2 function of microglia distal to the plaque can be restored by inducing movement and migration to the plaque, as well as neuronal damage.

[0338] According to the teachings of the present invention, the above objectives can be achieved by the molecular characteristics of the antibodies according to the present invention, namely by targeting Aβ binding and local TREM2 oligomerization of microglia at amyloid plaques, inducing a protective phenotype through TREM2 binding, and by avoiding inflammatory binding of FcgRII and III and complement C1q activation through LALAPG.

[0339] antibody form

[0340] General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0341] The term "activation" in this article refers to the initiation or preservation of downstream signaling of TREM2 expressed on the cell surface, resulting in increased cellular function or metabolism, such as in TREM2-expressing cells in healthy subjects or individuals with inflammatory or neurodegenerative diseases, where appropriate TREM2-dependent cellular activity is impaired. This can be achieved, but is not limited to, phosphorylation of TREM2-associated DAP12 or DAP10, leading to enhanced Syk phosphorylation, phagocytosis, increased targeted cell motility (chemotaxis), increased cell survival, regulation of cytokine or chemokine release from TREM2-expressing cells, increased degradation of intracellular phagocytosed material, improved lipid metabolism, or changes in gene expression via various intracellular signaling cascades. Increased TREM2-dependent DAP12 or Syk phosphorylation or downstream signaling can be assessed by Western blotting, ELISA, or flow cytometry / FACS or specific reporter cell assays. Targeted cell motility (e.g., chemotaxis) can be assessed by cell bioassays. Regulation of cytokine release can be assessed using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), antibody arrays, or flow cytometry / FACS. Changes in gene expression can be assessed using quantitative RT-PCR at the mRNA level, single-cell RNA sequencing or spatial omics, or Western blotting at the protein level, immunostaining, or flow cytometry.

[0342] "Anti-TREM2 antibody" or "antibody that binds to TREM2" and similar phrases refer to antibodies that specifically bind to human TREM2 as defined herein.

[0343] "Anti-amyloid β protein antibody" or "antibody that binds to amyloid β protein" and similar phrases refer to antibodies that specifically bind to human amyloid β protein as defined herein.

[0344] "Anti-TREM2 / Amyloid β antibody" or "anti-TREM2 / β antibody" or "antibody that binds to TREM2 and amyloid" and similar phrases refer to antibodies that specifically bind to TREM2 and amyloid as defined herein.

[0345] As used herein, the term "agonist" refers to a substance that causes an increase in the activity or function of at least one molecule it binds to, or otherwise activates or assists in the activation of the molecule, such as an antibody. As used herein, the term "antagonist" refers to a substance that causes a decrease in the activity or function of at least one molecule it binds to, or otherwise blocks or inhibits at least one activity or function of the molecule, such as an antibody.

[0346] For example, the term "agonist anti-TREM2 antibody" or similar phrases used herein refer to antibodies that induce luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2 and DAP12 and / or induce SYK phosphorylation (p-SYK) in HEK or Jurkat-NFAT reporter cells, THP-1 cells, human monocyte-derived macrophages (MDM) cells, and / or human induced pluripotent stem cell (iPSC)-derived macrophages, M0 cells, or microglia. As described herein, agonist anti-TREM2 antibodies may also possess additional activities, such as enhancing the survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1, and, among other activities, particularly activating TREM2 signaling in human macrophages and microglia.

[0347] In some cases, the antibody according to the invention has effector function. In some cases, the antibody has an Fc region with reduced effector function. In a preferred embodiment of all aspects and embodiments of the invention, the antibody according to the invention has an Fc region with silenced effector function.

[0348] For example, the Fc region of antibody therapeutics can bind to complement component C1q and the Fc-γ receptor (FcγR) to trigger cellular effector responses such as phagocytosis, cytokine release, and reactive oxygen species production. See, for example, X. Wang et al., Protein & Cell 9: 63-73 (2018); SBMkaddem et al., Frontiers in Immunology, available at https: / / doi.org / 10.3389 / fimmu.2019.00811 (2019). Overactivation of these pathways can be detrimental in the CNS, especially in a pathological context. See, for example, DJDiSabato et al., J. Neurochemistry 139(S2): 136-153 (2016). Clinically, antibody therapies targeting amyloid-β with full effector function and binding to amyloid-β aggregates significantly increase the incidence of potentially harmful side effects such as ARIA (amyloid-associated imaging abnormalities), which has not been observed in antibodies that bind only to the monomeric form of amyloid-β or have reduced effector function. See, for example, M. Filippi et al., JAMA Neurol. 79(3): 291-304 (2022). It must be noted that in clinical trial settings, at least one anti-TREM2 antibody with Fc activity has caused ARIA in 19% to 71% of AD patients (depending on ApoE genotype) (a phase 2 study evaluating the 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 Aβ binding by therapeutic antibodies alone may be less important in inducing ARIA, but binding by TREM2 and FcgR to microglia or macrophages in the perivascular space of the brain may play a crucial role in this regard. In some embodiments, such as those described in the examples and figures herein, antibodies with reduced effector function (such as those containing the human IgG1 heavy chain constant region with LALAPG or N297G mutations) are sufficient to elicit a microglial response in the mouse CNS via TREM2 stimulation.

[0349] The term “antibody” is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to full-length antibodies, monoclonal antibodies, monospecific dual complementary site antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies), and antibody-antibody fragment fusions and combinations thereof, provided that they possess the desired antigen-binding properties.

[0350] "Antibody conjugate" is an antibody or antibody fragment conjugated to one or more heterologous molecules (including but not limited to labels).

[0351] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody (i.e., TREM2 and / or amyloid β protein). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. More specifically, the Fab fragment is defined by the cleavage of a full-length natural IgG antibody of the IgG1 subclass by the enzyme papain. Papain cleaves the IgG1 subclass in the hinge region sequence (SEQ ID NO: 421; EPKSCDKTH↓TCP; the papain cleavage site is indicated by a down arrow) after the histidine residue of the amino acid. Therefore, a Fab fragment comprises a first polypeptide consisting of a variable domain and a constant domain (e.g., VL and CL); a heavy chain Fab fragment comprising a variable domain, a constant domain, and a hinge region, such as VH and CH1; and a hinge region fragment having the amino acid sequence EPKSCDKTH (amino acid residues 1 to 9 of SEQ ID NO: 421). For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0352] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0353] An antibody's "class" 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, with the constant heavy chain domains corresponding to different classes of immunoglobulins designated as α, δ, ε, γ, and μ, respectively. Some of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments, the antibody is the IgG1 isotype. In a preferred embodiment, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations (according to Kabat numbering) that reduce Fc region effector function. In some embodiments, the antibody is the IgG2 isotype. In some embodiments, the antibody is the IgG4 isotype with an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The light chain of an antibody, based on the amino acid sequence of its constant structural domain, can be classified into one of two types, known as Kappa (κ) and Lambda (λ).

[0354] The "constant regions" of an antibody's light and heavy chains refer to additional sequence portions beyond the FR and CDR domains and the variable domains. Some antibody fragments may lack all or part of the constant domains and / or regions. Each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain.

[0355] As used herein, the term “domain crossover” means that in the antibody heavy chain VH-CH1 fragment and its corresponding homologous antibody light chain pair, i.e. in antibody Fab (fragment antigen binding), the domain sequence deviates from the sequence in the native antibody because at least one heavy chain domain is replaced by its corresponding light chain domain, and vice versa. There are three common types of domain crossings: (i) the crossing of the CH1 and CL domains, which results in a VL-CH1 domain sequence due to domain crossing in the light chain and a VH-CL domain sequence due to domain crossing in the heavy chain (or a full-length antibody heavy chain with a VH-CL-hinge-CH2-CH3 domain sequence); (ii) the crossing of the VH and VL domains, which results in a VH-CL domain sequence due to domain crossing in the light chain and a VL-CH1 domain sequence due to domain crossing in the heavy chain; and (iii) the crossing of the complete light chain (VL-CL) and the complete VH-CH1 heavy chain (“Fab crossing”), which results in a light chain with a VH-CH1 domain sequence due to domain crossing and a heavy chain with a VL-CL domain sequence due to domain crossing (all of the aforementioned domain sequences are represented in the direction from the N-terminus to the C-terminus).

[0356] "Effective function" refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (antibody-dependent cell-mediated phagocytosis, ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. For example, antibodies with "(intact) effector function" contain a heavy chain constant region or Fc region with the native intact effector function of their specific isotype, such as the wild-type heavy chain constant region or Fc region, or a heavy chain constant region or Fc region with modifications that have not yet shown to affect effector function. In contrast, antibody heavy chain constant regions or Fc regions can be modified in various ways, such as by amino acid substitution, insertion, or deletion, or by glycosylation, to reduce or enhance effector function, depending on the antibody used. In this document, some antibodies may possess “(full) effector function” or “effector capability,” while others may be “effector silence (effectless),” meaning they do not exhibit detectable CDC or ADCC activity, or they contain heavy chain constant regions or Fc regions that have previously been shown to lack detectable CDC or ADCC activity. In other cases, antibodies may have mutant Fc regions or heavy chain constant regions with “reduced effector function” compared to the corresponding wild-type Fc regions or heavy chain constant regions (e.g., previously shown to have reduced effector function, or have reduced effector function in the context of this antibody). Such antibodies with reduced effector function compared to the corresponding wild-type Fc regions may, for example, exhibit lower levels of CDC and / or ADCC activity and / or FcγR binding activity, and / or may, for example, retain some effector function, such as binding to a specific FcgR, but with low or undetectable binding to CDC or ADCC.

[0357] The term "full-length antibody" refers to an antibody with a structure substantially similar to that of a natural antibody. A full-length antibody comprises two full-length antibody light chains and two full-length antibody heavy chains. Each full-length antibody light chain contains a light chain variable domain and a light chain constant domain in the N-terminal to C-terminal direction. Each full-length antibody heavy chain contains 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 the N-terminal to C-terminal direction. Unlike natural antibodies, full-length antibodies may contain additional immunoglobulin domains, such as one or more additional scFvs, or heavy chain or light chain Fab fragments, or scFab, conjugated to one or more ends of different chains of the full-length antibody, but only a single fragment is conjugated to each end. These conjugates are also covered by the term full-length antibody. However, in full-length antibodies, the C-terminal lysine amino acid residue of the heavy chain or the glycine-lysine dipeptide may be absent.

[0358] "Humanized" antibodies refer to chimeric antibodies that comprise amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, the humanized antibody will substantially comprise at least one of these variable domains, typically two variable domains, wherein all or substantially all of the amino acid residues in the CDR correspond to amino acid residues in the CDR of the non-human antibody, and all or substantially all of the amino acid residues in the FR correspond to amino acid residues in the FR of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. "Humanized form" antibodies, such as non-human antibodies, refer to antibodies that have undergone humanization.

[0359] As used herein, the term "hypervariant region" or "HVR" refers to the individual regions within the variable domains of an antibody that are hypervariable in sequence and determine antigen-binding specificity, such as "complementarity-determining regions" ("CDRs"). Typically, an antibody contains six CDRs: three in the VH domain (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3) and three in the VL domain (CDR-L1, CDR-L2, CDR-L3). Unless otherwise indicated, CDRs are identified according to the sequence listing herein, and the amino acid positions of the regions and domains in the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The Kabat and Chothia CDRs differ in CDR-H1 and CDR-H2 but are identical in the remaining four CDRs. Those skilled in the art will understand that the CDR name can also be determined according to the method described by McCallum or any other scientifically accepted naming system. See, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996).

[0360] Exemplary CDRs in this document include:

[0361] (a) Hyperchromatic rings present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987));

[0362] (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 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

[0363] (c) Antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)).

[0364] "Frame" or "FR" refers to residues of a variable domain of an antibody that are not part of the complementarity-determining region (CDR). The FR of a variable domain typically consists of four FRs: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0365] The term "heavy chain" refers to a polypeptide comprising at least one antibody variable domain and a hinge region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of the 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 (CH1-hinge-CH2-CH3), with or without a leader sequence, but including domain exchange between the CH1 domain and the antibody light chain constant domain (CL), or between the heavy chain variable domain and the light chain variable domain, or both.

[0366] The term "heavy chain constant region" refers to a region in the immunoglobulin heavy chain containing constant structural domains, namely the CH1 or CL domain, hinge region, CH2 domain, and CH3 domain. In some embodiments, the human IgG constant region extends from Ala118 to the C-terminus of the heavy chain (according to the Kabat EU index number). However, the C-terminal lysine residue (Lys447) or the C-terminal glycine-lysine dipeptide (G446-Lys447) of the constant region may or may not be present (according to the Kabat EU index number). The term "constant region" also refers to a dimer containing two heavy chain constant regions that can be covalently linked to each other via cysteine ​​residues in the hinge region, forming an interchain disulfide bond.

[0367] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a hinge region (neutral and lower hinge region), a CH2 domain, and a CH3 domain. In some embodiments, the human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Pro230 to the C-terminus of the heavy chain (according to Kabat EU index number). Thus, the heavy chain Fc region is smaller than the heavy chain constant region, but substantially the same as it in terms of the C-terminal portion. However, the C-terminal lysine (Lys447) or C-terminal glycine-lysine dipeptide (G446-Lys447) of the heavy chain Fc region may or may not be present (according to Kabat EU index number). The term "Fc region" also refers to a dimer containing two heavy chain Fc regions that are covalently linked to each other via cysteine ​​residues in the hinge region, forming an interchain disulfide bond.

[0368] The Fc region of an antibody (and the same constant region of the antibody) is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. Although the effect of an antibody on the complement system depends on certain conditions, binding to C1q is caused by binding sites defined in the Fc region. Such binding sites are known in the prior art, and, for example, by Lukas, TJ et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE 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, for example, amino acid residues L234, L235, D270, N297, E318, K320, K322, P331, and P329 (according to Kabat EU index numbers). Antibodies against subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, C1q binding, and C3 activation, while IgG4 does not activate the complement system, does not bind C1q, and does not activate C3.

[0369] "Antibody Fc region" is a term familiar to technical personnel and is defined based on the cleavage of antibodies by papain.

[0370] "Humanized" antibodies are antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will substantially comprise at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all FRs correspond to the FRs of a human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0371] The term "light chain" refers to a polypeptide comprising at least one light chain variable domain, with or without a leader sequence. In some embodiments, the 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 domain exchange between the CL domain and the CH1 domain, or between the light chain variable domain and the heavy chain variable domain, or both.

[0372] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that individual antibodies comprising this group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, such variants typically exist in small quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to recombinant DNA methods, such methods, and other exemplary methods for preparing the monoclonal antibodies described herein.

[0373] "Monospecific antibody" refers to an antibody with a single binding specificity, meaning it binds specifically to only one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2) or combinations thereof (e.g., a full-length antibody with an additional scFv or Fab fragment). Monospecific antibodies do not need to be monovalent; that is, a monospecific antibody can contain more than one binding site that specifically binds to a single antigen. For example, natural antibodies are monospecific but bivalent.

[0374] "Multispecific antibody" refers to an antibody that has binding specificity to at least two distinct epitopes (also referred to herein as bicomplementary sites) on the same antigen or to two distinct antigens. A "bispecific" antibody is an antibody that specifically binds to two antigens, and a "trispecific" antibody is an antibody that specifically binds to three antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody) or combinations thereof (e.g., a full-length antibody with an additional scFv or Fab fragment). Multispecific antibodies are at least bivalent, meaning they contain two antigen-binding sites. Furthermore, multispecific antibodies are at least bispecific. Therefore, bivalent bispecific antibodies are the simplest form of multispecific antibodies. Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites have also been reported (see, e.g., US 2002 / 0004587).

[0375] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable heavy chain domain (VH), followed by three constant heavy chain domains (CH1, CH2, and CH3), thereby positioning the hinge region between the first and second constant heavy chain domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable light chain domain (VL), followed by a constant light chain domain (CL). The light chains of natural antibodies, based on the amino acid sequence of their constant domains, can be classified into one of two types, called kappa (κ) and lamuda (λ).

[0376] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric antibodies, humanized antibodies, and human antibodies) prepared, expressed, created, or isolated by recombinant means such as using recombinant mammalian cells according to the invention.

[0377] As used herein, the term "substitute for each other" in relation to the corresponding heavy-chain and light-chain domains refers to the aforementioned domain crossings. Therefore, when the CH1 and CL domains "substitute for each other," it refers to the domain crossing mentioned under item (i) and the resulting heavy-chain and light-chain domain sequence. Similarly, when VH and VL "substitute for each other," it refers to the domain crossing mentioned in item (ii); and when the CH1 and CL domains "substitute for each other" and the VH and VL domains "substitute for each other," it refers to the domain crossing mentioned in item (iii). For example, bispecific antibodies involving domain exchanges have been reported 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 commonly referred to as CrossMab.

[0378] As used in this application, the terms "valent" or "valency" indicate the presence of a specified number of binding sites in an antibody. Therefore, the terms "bivalent," "trivalent," and "tetravalent" indicate the presence of two, three, and four binding sites in an antibody, respectively.

[0379] The term "variable domain" refers to the portion of the antibody heavy or light chain involved in the binding of the antibody to its antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A variable domain may contain: a heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3, with or without all or a portion of FR1 and / or FR4; and a 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, as long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated from antibodies binding to that antigen using either a VH or VL domain to screen libraries of complementary VL or VH domains, respectively (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)). It must be noted that variable domains are defined herein by the amino acid sequence beginning with the first residue following the signal peptide. However, the variable domain sequences given herein include variant sequences in which the first N-terminal glutamine (Q) residue or the first N-terminal glutamate (E) residue is replaced by a pyroglutamic acid (pE) residue.

[0380] In some cases, antibodies can be bispecific or trispecific. In other cases, antibodies can bind directly to another molecule or via a linker, such as through labeling.

[0381] Multispecific antibodies

[0382] In all aspects of the subject matter and in certain embodiments of the present invention, the antibody according to the invention is at least a bivalent bispecific antibody. In some embodiments, one of the binding specificities is against TREM2, and the other is against amyloid β protein.

[0383] Multispecific antibodies can be used to target cytotoxic agents to cells expressing one or more antigens.

[0384] Multispecific antibodies can be prepared as full-length antibodies, antibody-antibody fragment fusions, or antibody fragment-antibody fragment fusions.

[0385] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and engineered “mortar and pestle structures” (see, for example, US 5,731,168). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation effects for the preparation of antibody Fc-heterodimeric molecules (see, for example, WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, US 4,676,980, and Brennan, M. et al., Science, 229 (1985) 81-83); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); using common light chain techniques to avoid light chain mismatch problems (see, for example, WO 98 / 50431); using specific techniques for the preparation of bispecific antibody fragments (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and as Tutt, A. et al., J. The preparation of trispecific antibodies is described in Immunol. 147 (1991) 60-69.

[0386] This document also includes engineered antibodies having three or more antigen-binding sites, including, for example, “octopus antibodies” or DVD-Ig (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having 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. Bispecific antibodies or their antigen-binding fragments also include “dual-acting Fab” or “DAF” (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0387] Multispecific antibodies can also be provided in an asymmetric form with domain crossover, i.e., by exchanging the VH / VL domain (see, for example, WO 2009 / 080252 and WO2015 / 150447), CH1 / CL domain (see, for example, WO 2009 / 080253), or the complete Fab arm (see, for example, WO 2009 / 080251, WO 2016 / 016299, also see Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-1020). In all aspects and certain embodiments of the invention, cells according to the invention express multispecific antibodies comprising the Cross-Fab fragment. The term "Cross-Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab fragment comprises a polypeptide chain consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), or a polypeptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide the correct pairing of the Fab heavy chain fragment and its homologous light chain. See, for example, WO 2016 / 172485.

[0388] The antibody or fragment may also be a multispecific antibody, as described in WO 2009 / 080254, WO 2010 / 112193, WO2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, or WO 2010 / 145793.

[0389] Antibodies or fragments thereof may also be multispecific antibodies as reported in WO 2012 / 163520.

[0390] Various other molecular forms of multispecific antibodies are known in the art and can be generated using the binding sites according to the invention (see, for example, Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0391] Bispecific antibodies are typically antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

[0392] In all aspects and in certain embodiments, the form of the multispecific antibody according to the invention is selected from the group consisting of the following antibody forms:

[0393] Bivalent bispecific full-length antibody with domain exchange

[0394] (That is, a bivalent, bispecific, full-length antibody containing a first Fab fragment, a second Fab fragment, and an Fc region.)

[0395] In the first Fab segment

[0396] a) (Only) the CH1 and CL domains are interchangeable (i.e., the light chain of the first Fab segment contains the VL and CH1 domains, and the heavy chain of the first Fab segment contains the VH and CL domains).

[0397] b) (only) the VH and VL domains are interchanged (i.e., the light chain of the first Fab segment contains the VH and CL domains, and the heavy chain of the first Fab segment contains the VL and CH1 domains); or c) the CH1 and CL domains are interchanged and the VH and VL domains are interchanged (i.e., the light chain of the first Fab segment contains the VH and CH1 domains, and the heavy chain of the first Fab segment contains the VL and CL domains).

[0398] The second Fab segment comprises a light-chain Fab segment and a heavy-chain Fab segment. The light-chain Fab segment contains VL and CL structural domains, and the heavy-chain Fab segment contains VH and CH1 structural domains.

[0399] The first Fab fragment specifically binds to the first antigen, and the second Fab fragment specifically binds to the second antigen; and

[0400] The Fc region comprises a first Fc region polypeptide and a second Fc region polypeptide. The first Fc region polypeptide contains a CH3 domain, and the second Fc region polypeptide contains a CH3 domain. The two CH3 domains are engineered in a complementary manner through their respective amino acid substitutions, thereby supporting the heterodimerization of a first heavy chain containing the first Fc region polypeptide and a second heavy chain containing the second Fc region polypeptide, as 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 The one reported in 2013 / 096291 (incorporated herein by reference) preferably has a pestle-and-mortar mutation;

[0401] Trivalent bispecific antibodies containing a bivalent full-length antibody and an additional monovalent heavy chain C-terminal binding site (BS).

[0402] (That is, a trivalent bispecific antibody, which contains a bivalent full-length antibody and an additional monovalent (third) binding site conjugated to the C-terminus of one of the heavy chains of the bivalent full-length antibody, wherein...)

[0403] a) A full-length antibody comprises two pairs, each consisting of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of the full-length heavy chain and the full-length light chain specifically binds to the first antigen, and

[0404] b) An additional binding site, wherein the additional binding site is conjugated to the C-terminus of a 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, which i) contains domain crossovers such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are interchanged, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are interchanged, or ii) is a single-chain Fab fragment.

[0405] A trivalent trispecific antibody, which comprises a bivalent bispecific full-length antibody with domain exchange and an additional monovalent heavy chain C-terminal binding site (BS).

[0406] (That is, a trivalent trispecific antibody, which comprises a bispecific full-length bivalent antibody with domain exchange and an additional monovalent (third) binding site conjugated to the C-terminus of one of the heavy chains of the full-length bivalent antibody.)

[0407] in

[0408] a) The full-length antibody contains a first Fab fragment, a second Fab fragment, and an Fc region.

[0409] In the first Fab segment

[0410] a) (Only) the CH1 and CL domains are interchangeable (i.e., the light chain of the first Fab segment contains the VL and CH1 domains, and the heavy chain of the first Fab segment contains the VH and CL domains).

[0411] b) (only) the VH and VL domains are interchanged (i.e., the light chain of the first Fab segment contains the VH and CL domains, and the heavy chain of the first Fab segment contains the VL and CH1 domains); or c) the CH1 and CL domains are interchanged and the VH and VL domains are interchanged (i.e., the light chain of the first Fab segment contains the VH and CH1 domains, and the heavy chain of the first Fab segment contains the VL and CL domains).

[0412] The second Fab segment comprises a light-chain Fab segment and a heavy-chain Fab segment. The light-chain Fab segment contains VL and CL structural domains, and the heavy-chain Fab segment contains VH and CH1 structural domains.

[0413] The first Fab fragment specifically binds to the first antigen, and the second Fab fragment specifically binds to the second antigen;

[0414] The Fc region comprises a first Fc region polypeptide and a second Fc region polypeptide. The first Fc region polypeptide contains a CH3 domain, and the second Fc region polypeptide contains a CH3 domain. The two CH3 domains are engineered in a complementary manner through their respective amino acid substitutions, thereby supporting the heterodimerization of a first heavy chain containing the first Fc region polypeptide and a second heavy chain containing the second Fc region polypeptide, as 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 The one reported in 2013 / 096291 (incorporated herein by reference) preferably has a pestle-and-mortar mutation;

[0415] b) An additional binding site, wherein the additional binding site is conjugated to the C-terminus of a 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 that specifically binds to a third antigen, i) comprising domain crossovers such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are interchanged, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are interchanged, or ii) is a single-chain Fab fragment.

[0416] Bivalent bispecific single-arm single-chain antibody

[0417] (That is, an antibody containing a first binding site and a second binding site, the first binding site specifically binding to a first antigen, the second binding site specifically binding to a second antigen, wherein the individual chains are as follows)

[0418] - Light chains (containing both variable and constant structural domains of the light chain in the direction from the N-terminus to the C-terminus);

[0419] -A combination of light and heavy chains (containing a light chain variable domain, a light chain constant domain, a peptide linker, a heavy chain variable domain, and a heavy chain constant region with a club-shaped mutation in the CH3 domain, in the direction from the N-terminus to the C-terminus); and

[0420] - Heavy chain (containing a heavy chain variable domain in the direction from the N-terminus to the C-terminus and a heavy chain constant region with a mortise mutation in the CH3 domain);

[0421] Bivalent bispecific two-arm single-chain antibody

[0422] (That is, an antibody containing a first binding site and a second binding site, the first binding site specifically binding to a first antigen, the second binding site specifically binding to a second antigen, wherein the individual chains are as follows)

[0423] - The first combination of light chain and heavy chain (containing a light chain variable domain, a light chain constant domain, a peptide linker, a heavy chain variable domain, and a heavy chain constant region having i) a mortar mutation or ii) a pestle mutation in the CH3 domain); and

[0424] - The second combination of light chain-heavy chain (containing a light chain variable domain, a light chain constant domain, a peptide linker, a variable heavy chain domain, and a heavy chain constant region having i) a club-shaped mutation or ii) a mortar-shaped mutation in the CH3 domain) in the direction from the N-terminus to the C-terminus.

[0425] Common light chain bispecific antibodies

[0426] (That is, an antibody containing a first binding site and a second binding site, the first binding site specifically binding to a first antigen, the second binding site specifically binding to a second antigen, wherein the individual chains are as follows)

[0427] - Light chains (containing both variable and constant structural domains of the light chain in the direction from the N-terminus to the C-terminus);

[0428] - The first heavy chain (containing a heavy chain variable domain in the direction from the N-terminus to the C-terminus and a heavy chain constant region with i) a mortar mutation or ii) a pestle mutation in the CH3 domain) and

[0429] - The second heavy chain (containing a heavy chain variable domain in the direction from the N-terminus to the C-terminus and a heavy chain constant region having i) a club-shaped mutation or ii) a mortar-shaped mutation in the CH3 domain).

[0430] T-cell bispecific antibody (TCB)

[0431] (That is, a bivalent monospecific full-length antibody having: an additional N-terminal binding site of the insert heavy chain with domain exchange, which contains)

[0432] - A first Fab fragment and a second Fab fragment, wherein each binding site of the first Fab fragment and the second Fab fragment specifically binds to the first antigen.

[0433] - A third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to the second antigen, and wherein the third Fab fragment contains domain crossovers such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are interchanged, and

[0434] - An Fc region containing a first Fc region polypeptide and a second Fc region polypeptide.

[0435] The first Fab segment and the second Fab segment each contain a heavy-chain Fab segment and a full-length light chain.

[0436] The C-terminus of the heavy chain Fab fragment of the first Fab fragment is fused to the N-terminus of the polypeptide in the first Fc region.

[0437] The C-terminus of the heavy chain Fab fragment of the second Fab fragment is fused to the N-terminus of the variable domain of the light chain of the third Fab fragment, and the C-terminus of the CH1 domain of the third Fab fragment is fused to the N-terminus of the second Fc region polypeptide.

[0438] The “mortar and pestle structure” dimer module and its use in antibody engineering are described in Carter P., Ridgway J.BB, Presta LG: Immunotechnology, Vol. 2, No. 1, February 1996, pp. 73-73(1).

[0439] The CH3 domains in the heavy chain of an antibody can be altered using a "knob-hole" technique, described in detail with several examples, such as WO96 / 027011; Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM 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 increase the heterodimerization of the polypeptide containing them. One of the two CH3 domains (of the two heavy chains) can be a "knob" and the other a "hole". The introduction of disulfide bridges further stabilizes the heterodimer (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0440] The mutation T366W in the CH3 domain of the antibody heavy chain is designated as a "jawdrop mutation" or "mutant jawdropper," while the mutations T366S, L368A, and Y407V in the CH3 domain of the antibody heavy chain are designated as "mortar mutations" or "mutant mortar" (according to Kabat EU index number). Additional interchain disulfide bridges located between the CH3 domains can also be used by introducing the S354C mutation into the CH3 domain of a heavy chain with a "jawdrop mutation" (designated as a "jawdrop-cys-mutant" or "mutant jawdrop-cys") or by introducing the Y349C mutation into the CH3 domain of a heavy chain with a "mortar mutation" (designated as a "mortar-cys-mutant" or "mutant mortar-cys") (according to Kabat EU index number). (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681).

[0441] In any of the embodiments herein, the antibody may be an antibody fragment, such as Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2. In other embodiments, the antibody may be a complete antibody (i.e., comprising a heavy chain constant region and a light chain constant region). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgG1 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 IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. If it is a mouse IgG antibody, the antibody may be an mIgG1 or mIgG2 or an mIgG2 LALAPG antibody. In some cases, antibodies may contain a full-length heavy chain and / or a full-length light chain. In some cases, antibodies may lack the C-terminal Lys or C-terminal Lys and Gly residues in the heavy chain constant region. In other cases, antibodies contain one or both of these C-terminal residues.

[0442] Anti-TREM2 antibody

[0443] In one aspect, the present invention provides an antibody that binds to human TREM2. In another aspect, an isolated antibody that binds to human TREM2 is provided. In yet another aspect, the present invention provides a dual complementary site antibody that specifically binds to human TREM2.

[0444] Antibodies that bind to or specifically bind to human TREM2 according to the present invention are called anti-TREM2 antibodies.

[0445] In some respects, anti-TREM2 antibodies

[0446] a) Inducing acute uptake of MX04-labeled amyloid and Aβ proteins in APPswePS2 transgenic mice in a form with Fc effector functional capacity, and / or

[0447] b) Preferably, macrophage migration is induced at a concentration of 0.14 to 34 nM in the migration assay as described herein, and / or

[0448] c) Increased LPC- and C5a-stimulated cell migration in THP-1 and iPSC-derived M0 cells in the absence of any Aβ protein, and / or

[0449] d) The antibody does not induce pSyk in the absence or presence of human Aβ protein, preferably an antagonist of the Syk pathway, and / or

[0450] e) No dose-dependent phosphorylation of Syk and S6 was observed in HEK cells expressing DAP12 and TREM2, and / or

[0451] f) In the absence of crosslinking, pSyk is not induced in TREM2 / DAP12-overexpressing HEK or iPSC macrophages, and / or

[0452] g) It has non-inflammatory neuroprotective properties by activating TREM2 signaling (agonist), and / or

[0453] h) Does not induce the release of TNFα, MIP-1α, or IL-8 from iPSC-derived macrophages, and / or

[0454] i) Block the shedding of sTREM2, and / or

[0455] j) Stabilize sTREM2 in the biological fluid, and / or

[0456] k) binds to the ECD of TREM2, leading to the accumulation of sTREM2 in the biological fluid.

[0457] In one aspect, the present invention provides an anti-TREM2 antibody comprising i) a first binding site for binding to TREM2, the first binding site comprising CDR-H1, CDR-H2, CDR-H3 and CDR-L1, CDR-L2, CDR-L3 containing the following amino acid sequences:

[0458] SEQ ID NO: 129-131 and 133-135, or

[0459] SEQ ID NO: 137-139 and 141-143, or

[0460] SEQ ID NO: 145-147 and 149-151, or

[0461] SEQ ID NO: 153-155 and 157-159, or

[0462] SEQ ID NO: 161-163 and 165-167, or

[0463] SEQ ID NO: 169-171 and 173-175, or

[0464] SEQ ID NO: 177-179 and 181-183, or

[0465] SEQ ID NO: 185-187 and 189-191, or

[0466] SEQ ID NO: 193-195 and 197-199, or

[0467] SEQ ID NO: 201-203 and 205-207, or

[0468] SEQ ID NO: 209-211 and 213-215, or

[0469] SEQ ID NO: 217-219 and 221-223, or

[0470] SEQ ID NO: 225-227 and 229-231, or

[0471] SEQ ID NO: 233-235 and 237-239, or

[0472] SEQ ID NO: 241-243 and 245-247, or

[0473] SEQ ID NO: 249-251 and 253-255, or

[0474] SEQ ID NO: 257-259 and 261-263, or

[0475] SEQ ID NO: 265-267 and 269-271, or

[0476] SEQ ID NO: 273-275 and 277-279, or

[0477] SEQ ID NO: 281-283 and 285-287, or

[0478] SEQ ID NO: 289-291 and 293-295, or

[0479] SEQ ID NO: 297-299 and 301-303, or

[0480] SEQ ID NO: 305-307 and 309-311, or

[0481] SEQ ID NO: 313-315 and 317-319, or

[0482] SEQ ID NO: 321-323 and 325-327, or

[0483] SEQ ID NO: 329-331 and 333-335, or

[0484] SEQ ID NO: 337-339 and 341-343, or

[0485] SEQ ID NO: 345-347 and 349-351.

[0486] In another aspect, the antibody of the present invention comprises

[0487] (a) A VH structural domain containing at least one, at least two, or all three VH CDR sequences selected from the following:

[0488] SEQ ID NO: 129-131, or

[0489] SEQ ID NO: 137-139, or

[0490] SEQ ID NO: 145-147, or

[0491] SEQ ID NO: 153-155, or

[0492] SEQ ID NO: 161-163, or

[0493] SEQ ID NO: 169-171, or

[0494] SEQ ID NO: 177-179, or

[0495] SEQ ID NO: 185-187, or

[0496] SEQ ID NO: 193-195, or

[0497] SEQ ID NO: 201-203, or

[0498] SEQ ID NO: 209-211, or

[0499] SEQ ID NO: 217-219, or

[0500] SEQ ID NO: 225-227, or

[0501] SEQ ID NO: 233-235, or

[0502] SEQ ID NO: 241-243, or

[0503] SEQ ID NO: 249-251, or

[0504] SEQ ID NO: 257-259, or

[0505] SEQ ID NO: 265-267, or

[0506] SEQ ID NO: 273-275, or

[0507] SEQ ID NO: 281-283, or

[0508] SEQ ID NO: 289-291, or

[0509] SEQ ID NO: 297-299, or

[0510] SEQ ID NO: 305-307, or

[0511] SEQ ID NO: 313-315, or

[0512] SEQ ID NO: 321-323, or

[0513] SEQ ID NO: 329-331, or

[0514] SEQ ID NO: 337-339, or

[0515] SEQ ID NO: 345-347.

[0516] Or (b) a VL domain containing at least one, at least two, or all three VL CDR sequences selected from the following:

[0517] SEQ ID NO: 133-135, or

[0518] SEQ ID NO: 141-143, or

[0519] SEQ ID NO: 149-151, or

[0520] SEQ ID NO: 157-159, or

[0521] SEQ ID NO: 165-167, or

[0522] SEQ ID NO: 173-175, or

[0523] SEQ ID NO: 181-183, or

[0524] SEQ ID NO: 189-191, or

[0525] SEQ ID NO: 197-199, or

[0526] SEQ ID NO: 205-207, or

[0527] SEQ ID NO: 213-215, or

[0528] SEQ ID NO: 221-223, or

[0529] SEQ ID NO: 229-231, or

[0530] SEQ ID NO: 237-239, or

[0531] SEQ ID NO: 245-247, or

[0532] SEQ ID NO: 253-255, or

[0533] SEQ ID NO: 261-263, or

[0534] SEQ ID NO: 269-271, or

[0535] SEQ ID NO: 277-279, or

[0536] SEQ ID NO: 285-287, or

[0537] SEQ ID NO: 293-295, or

[0538] SEQ ID NO: 301-303, or

[0539] SEQ ID NO: 309-311, or

[0540] SEQ ID NO: 317-319, or

[0541] SEQ ID NO: 325-327, or

[0542] SEQ ID NO: 333-335, or

[0543] SEQ ID NO: 341-343, or

[0544] SEQ ID NO: 349-351.

[0545] In all respects provided herein, the anti-TREM2 antibody is humanized. In one respect, the anti-TREM2 antibody further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework.

[0546] On the other hand, anti-TREM2 antibodies contain one or more of the following CDR sequences of VH:

[0547] SEQ ID NO: 132, or

[0548] SEQ ID NO: 140, or

[0549] SEQ ID NO: 148, or

[0550] SEQ ID NO: 156, or

[0551] SEQ ID NO: 164, or

[0552] SEQ ID NO: 172, or

[0553] SEQ ID NO: 180, or

[0554] SEQ ID NO: 188, or

[0555] SEQ ID NO: 196, or

[0556] SEQ ID NO: 204, or

[0557] SEQ ID NO: 212, or

[0558] SEQ ID NO: 220, or

[0559] SEQ ID NO: 228, or

[0560] SEQ ID NO: 236, or

[0561] SEQ ID NO: 244, or

[0562] SEQ ID NO: 252, or

[0563] SEQ ID NO: 260, or

[0564] SEQ ID NO: 268, or

[0565] SEQ ID NO: 276, or

[0566] SEQ ID NO: 284, or

[0567] SEQ ID NO: 292, or

[0568] SEQ ID NO: 300, or

[0569] SEQ ID NO: 308, or

[0570] SEQ ID NO: 316, or

[0571] SEQ ID NO: 324, or

[0572] SEQ ID NO: 323, or

[0573] SEQ ID NO: 340, or

[0574] SEQ ID NO: 348.

[0575] In another embodiment, the anti-TREM2 antibody comprises one or more of the following VL CDR sequences:

[0576] SEQ ID NO: 136, or

[0577] SEQ ID NO: 144, or

[0578] SEQ ID NO: 152, or

[0579] SEQ ID NO: 160, or

[0580] SEQ ID NO: 168, or

[0581] SEQ ID NO: 176, or

[0582] SEQ ID NO: 184, or

[0583] SEQ ID NO: 192, or

[0584] SEQ ID NO: 200, or

[0585] SEQ ID NO: 208, or

[0586] SEQ ID NO: 216, or

[0587] SEQ ID NO: 224, or

[0588] SEQ ID NO: 232, or

[0589] SEQ ID NO: 240, or

[0590] SEQ ID NO: 248, or

[0591] SEQ ID NO: 256, or

[0592] SEQ ID NO: 264, or

[0593] SEQ ID NO: 272, or

[0594] SEQ ID NO: 280, or

[0595] SEQ ID NO: 288, or

[0596] SEQ ID NO: 296, or

[0597] SEQ ID NO: 304, or

[0598] SEQ ID NO: 312, or

[0599] SEQ ID NO: 320, or

[0600] SEQ ID NO: 328, or

[0601] SEQ ID NO: 336, or

[0602] SEQ ID NO: 344, or

[0603] SEQ ID NO: 352.

[0604] In another embodiment, the anti-TREM2 antibody comprises one or more of the following VH CDR sequences:

[0605] SEQ ID NO: 132, or

[0606] SEQ ID NO: 140, or

[0607] SEQ ID NO: 148, or

[0608] SEQ ID NO: 156, or

[0609] SEQ ID NO: 164, or

[0610] SEQ ID NO: 172, or

[0611] SEQ ID NO: 180, or

[0612] SEQ ID NO: 188, or

[0613] SEQ ID NO: 196, or

[0614] SEQ ID NO: 204, or

[0615] SEQ ID NO: 212, or

[0616] SEQ ID NO: 220, or

[0617] SEQ ID NO: 228, or

[0618] SEQ ID NO: 236, or

[0619] SEQ ID NO: 244, or

[0620] SEQ ID NO: 252, or

[0621] SEQ ID NO: 260, or

[0622] SEQ ID NO: 268, or

[0623] SEQ ID NO: 276, or

[0624] SEQ ID NO: 284, or

[0625] SEQ ID NO: 292, or

[0626] SEQ ID NO: 300, or

[0627] SEQ ID NO: 308, or

[0628] SEQ ID NO: 316, or

[0629] SEQ ID NO: 324, or

[0630] SEQ ID NO: 323, or

[0631] SEQ ID NO: 340, or

[0632] SEQ ID NO: 348

[0633] The CDR sequence of VL for the following items:

[0634] SEQ ID NO: 136, or

[0635] SEQ ID NO: 144, or

[0636] SEQ ID NO: 152, or

[0637] SEQ ID NO: 160, or

[0638] SEQ ID NO: 168, or

[0639] SEQ ID NO: 176, or

[0640] SEQ ID NO: 184, or

[0641] SEQ ID NO: 192, or

[0642] SEQ ID NO: 200, or

[0643] SEQ ID NO: 208, or

[0644] SEQ ID NO: 216, or

[0645] SEQ ID NO: 224, or

[0646] SEQ ID NO: 232, or

[0647] SEQ ID NO: 240, or

[0648] SEQ ID NO: 248, or

[0649] SEQ ID NO: 256, or

[0650] SEQ ID NO: 264, or

[0651] SEQ ID NO: 272, or

[0652] SEQ ID NO: 280, or

[0653] SEQ ID NO: 288, or

[0654] SEQ ID NO: 296, or

[0655] SEQ ID NO: 304, or

[0656] SEQ ID NO: 312, or

[0657] SEQ ID NO: 320, or

[0658] SEQ ID NO: 328, or

[0659] SEQ ID NO: 336, or

[0660] SEQ ID NO: 344, or

[0661] SEQ ID NO: 352.

[0662] In another aspect, the anti-TREM2 antibody comprises the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the VH domain of SEQ ID NO: 308 or 332 and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the VL domain of SEQ ID NO: 312 or 336.

[0663] On the one hand, anti-TREM2 antibodies contain one or more of the following amino acid sequences in the heavy chain CDR of the VH domain:

[0664] SEQ ID NO: 132, or

[0665] SEQ ID NO: 140, or

[0666] SEQ ID NO: 148, or

[0667] SEQ ID NO: 156, or

[0668] SEQ ID NO: 164, or

[0669] SEQ ID NO: 172, or

[0670] SEQ ID NO: 180, or

[0671] SEQ ID NO: 188, or

[0672] SEQ ID NO: 196, or

[0673] SEQ ID NO: 204, or

[0674] SEQ ID NO: 212, or

[0675] SEQ ID NO: 220, or

[0676] SEQ ID NO: 228, or

[0677] SEQ ID NO: 236, or

[0678] SEQ ID NO: 244, or

[0679] SEQ ID NO: 252, or

[0680] SEQ ID NO: 260, or

[0681] SEQ ID NO: 268, or

[0682] SEQ ID NO: 276, or

[0683] SEQ ID NO: 284, or

[0684] SEQ ID NO: 292, or

[0685] SEQ ID NO: 300, or

[0686] SEQ ID NO: 308, or

[0687] SEQ ID NO: 316, or

[0688] SEQ ID NO: 324, or

[0689] SEQ ID NO: 323, or

[0690] SEQ ID NO: 340, or

[0691] SEQ ID NO: 348

[0692] And the framework, which has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the framework amino acid sequence of the VH domain of the following:

[0693] SEQ ID NO: 132, or

[0694] SEQ ID NO: 140, or

[0695] SEQ ID NO: 148, or

[0696] SEQ ID NO: 156, or

[0697] SEQ ID NO: 164, or

[0698] SEQ ID NO: 172, or

[0699] SEQ ID NO: 180, or

[0700] SEQ ID NO: 188, or

[0701] SEQ ID NO: 196, or

[0702] SEQ ID NO: 204, or

[0703] SEQ ID NO: 212, or

[0704] SEQ ID NO: 220, or

[0705] SEQ ID NO: 228, or

[0706] SEQ ID NO: 236, or

[0707] SEQ ID NO: 244, or

[0708] SEQ ID NO: 252, or

[0709] SEQ ID NO: 260, or

[0710] SEQ ID NO: 268, or

[0711] SEQ ID NO: 276, or

[0712] SEQ ID NO: 284, or

[0713] SEQ ID NO: 292, or

[0714] SEQ ID NO: 300, or

[0715] SEQ ID NO: 308, or

[0716] SEQ ID NO: 316, or

[0717] SEQ ID NO: 324, or

[0718] SEQ ID NO: 323, or

[0719] SEQ ID NO: 340, or

[0720] SEQ ID NO: 348.

[0721] On one hand, the anti-TREM2 antibody contains the following three heavy chain CDR amino acid sequences of the VH domain:

[0722] SEQ ID NO: 132, or

[0723] SEQ ID NO: 140, or

[0724] SEQ ID NO: 148, or

[0725] SEQ ID NO: 156, or

[0726] SEQ ID NO: 164, or

[0727] SEQ ID NO: 172, or

[0728] SEQ ID NO: 180, or

[0729] SEQ ID NO: 188, or

[0730] SEQ ID NO: 196, or

[0731] SEQ ID NO: 204, or

[0732] SEQ ID NO: 212, or

[0733] SEQ ID NO: 220, or

[0734] SEQ ID NO: 228, or

[0735] SEQ ID NO: 236, or

[0736] SEQ ID NO: 244, or

[0737] SEQ ID NO: 252, or

[0738] SEQ ID NO: 260, or

[0739] SEQ ID NO: 268, or

[0740] SEQ ID NO: 276, or

[0741] SEQ ID NO: 284, or

[0742] SEQ ID NO: 292, or

[0743] SEQ ID NO: 300, or

[0744] SEQ ID NO: 308, or

[0745] SEQ ID NO: 316, or

[0746] SEQ ID NO: 324, or

[0747] SEQ ID NO: 323, or

[0748] SEQ ID NO: 340, or

[0749] SEQ ID NO: 348

[0750] And the framework, which has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the framework amino acid sequence of the VH domain of the following:

[0751] SEQ ID NO: 132, or

[0752] SEQ ID NO: 140, or

[0753] SEQ ID NO: 148, or

[0754] SEQ ID NO: 156, or

[0755] SEQ ID NO: 164, or

[0756] SEQ ID NO: 172, or

[0757] SEQ ID NO: 180, or

[0758] SEQ ID NO: 188, or

[0759] SEQ ID NO: 196, or

[0760] SEQ ID NO: 204, or

[0761] SEQ ID NO: 212, or

[0762] SEQ ID NO: 220, or

[0763] SEQ ID NO: 228, or

[0764] SEQ ID NO: 236, or

[0765] SEQ ID NO: 244, or

[0766] SEQ ID NO: 252, or

[0767] SEQ ID NO: 260, or

[0768] SEQ ID NO: 268, or

[0769] SEQ ID NO: 276, or

[0770] SEQ ID NO: 284, or

[0771] SEQ ID NO: 292, or

[0772] SEQ ID NO: 300, or

[0773] SEQ ID NO: 308, or

[0774] SEQ ID NO: 316, or

[0775] SEQ ID NO: 324, or

[0776] SEQ ID NO: 323, or

[0777] SEQ ID NO: 340, or

[0778] SEQ ID NO: 348.

[0779] On one hand, the anti-TREM2 antibody contains the following three heavy chain CDR amino acid sequences of the VH domain:

[0780] SEQ ID NO: 132, or

[0781] SEQ ID NO: 140, or

[0782] SEQ ID NO: 148, or

[0783] SEQ ID NO: 156, or

[0784] SEQ ID NO: 164, or

[0785] SEQ ID NO: 172, or

[0786] SEQ ID NO: 180, or

[0787] SEQ ID NO: 188, or

[0788] SEQ ID NO: 196, or

[0789] SEQ ID NO: 204, or

[0790] SEQ ID NO: 212, or

[0791] SEQ ID NO: 220, or

[0792] SEQ ID NO: 228, or

[0793] SEQ ID NO: 236, or

[0794] SEQ ID NO: 244, or

[0795] SEQ ID NO: 252, or

[0796] SEQ ID NO: 260, or

[0797] SEQ ID NO: 268, or

[0798] SEQ ID NO: 276, or

[0799] SEQ ID NO: 284, or

[0800] SEQ ID NO: 292, or

[0801] SEQ ID NO: 300, or

[0802] SEQ ID NO: 308, or

[0803] SEQ ID NO: 316, or

[0804] SEQ ID NO: 324, or

[0805] SEQ ID NO: 323, or

[0806] SEQ ID NO: 340, or

[0807] SEQ ID NO: 348

[0808] And the framework, which has at least 95% sequence identity with the framework amino acid sequence of the VH domain of the following:

[0809] SEQ ID NO: 132, or

[0810] SEQ ID NO: 140, or

[0811] SEQ ID NO: 148, or

[0812] SEQ ID NO: 156, or

[0813] SEQ ID NO: 164, or

[0814] SEQ ID NO: 172, or

[0815] SEQ ID NO: 180, or

[0816] SEQ ID NO: 188, or

[0817] SEQ ID NO: 196, or

[0818] SEQ ID NO: 204, or

[0819] SEQ ID NO: 212, or

[0820] SEQ ID NO: 220, or

[0821] SEQ ID NO: 228, or

[0822] SEQ ID NO: 236, or

[0823] SEQ ID NO: 244, or

[0824] SEQ ID NO: 252, or

[0825] SEQ ID NO: 260, or

[0826] SEQ ID NO: 268, or

[0827] SEQ ID NO: 276, or

[0828] SEQ ID NO: 284, or

[0829] SEQ ID NO: 292, or

[0830] SEQ ID NO: 300, or

[0831] SEQ ID NO: 308, or

[0832] SEQ ID NO: 316, or

[0833] SEQ ID NO: 324, or

[0834] SEQ ID NO: 323, or

[0835] SEQ ID NO: 340, or

[0836] SEQ ID NO: 348.

[0837] On the other hand, the anti-TREM2 antibody contains the following three heavy chain CDR amino acid sequences of the VH domain:

[0838] SEQ ID NO: 132, or

[0839] SEQ ID NO: 140, or

[0840] SEQ ID NO: 148, or

[0841] SEQ ID NO: 156, or

[0842] SEQ ID NO: 164, or

[0843] SEQ ID NO: 172, or

[0844] SEQ ID NO: 180, or

[0845] SEQ ID NO: 188, or

[0846] SEQ ID NO: 196, or

[0847] SEQ ID NO: 204, or

[0848] SEQ ID NO: 212, or

[0849] SEQ ID NO: 220, or

[0850] SEQ ID NO: 228, or

[0851] SEQ ID NO: 236, or

[0852] SEQ ID NO: 244, or

[0853] SEQ ID NO: 252, or

[0854] SEQ ID NO: 260, or

[0855] SEQ ID NO: 268, or

[0856] SEQ ID NO: 276, or

[0857] SEQ ID NO: 284, or

[0858] SEQ ID NO: 292, or

[0859] SEQ ID NO: 300, or

[0860] SEQ ID NO: 308, or

[0861] SEQ ID NO: 316, or

[0862] SEQ ID NO: 324, or

[0863] SEQ ID NO: 323, or

[0864] SEQ ID NO: 340, or

[0865] SEQ ID NO: 348

[0866] And the framework, which has at least 98% sequence identity with the framework amino acid sequence of the VH domain of the following:

[0867] SEQ ID NO: 132, or

[0868] SEQ ID NO: 140, or

[0869] SEQ ID NO: 148, or

[0870] SEQ ID NO: 156, or

[0871] SEQ ID NO: 164, or

[0872] SEQ ID NO: 172, or

[0873] SEQ ID NO: 180, or

[0874] SEQ ID NO: 188, or

[0875] SEQ ID NO: 196, or

[0876] SEQ ID NO: 204, or

[0877] SEQ ID NO: 212, or

[0878] SEQ ID NO: 220, or

[0879] SEQ ID NO: 228, or

[0880] SEQ ID NO: 236, or

[0881] SEQ ID NO: 244, or

[0882] SEQ ID NO: 252, or

[0883] SEQ ID NO: 260, or

[0884] SEQ ID NO: 268, or

[0885] SEQ ID NO: 276, or

[0886] SEQ ID NO: 284, or

[0887] SEQ ID NO: 292, or

[0888] SEQ ID NO: 300, or

[0889] SEQ ID NO: 308, or

[0890] SEQ ID NO: 316, or

[0891] SEQ ID NO: 324, or

[0892] SEQ ID NO: 323, or

[0893] SEQ ID NO: 340, or

[0894] SEQ ID NO: 348.

[0895] On the one hand, anti-TREM2 antibodies contain one or more of the following amino acid sequences in the light chain CDR of the VL domain:

[0896] SEQ ID NO: 136, or

[0897] SEQ ID NO: 144, or

[0898] SEQ ID NO: 152, or

[0899] SEQ ID NO: 160, or

[0900] SEQ ID NO: 168, or

[0901] SEQ ID NO: 176, or

[0902] SEQ ID NO: 184, or

[0903] SEQ ID NO: 192, or

[0904] SEQ ID NO: 200, or

[0905] SEQ ID NO: 208, or

[0906] SEQ ID NO: 216, or

[0907] SEQ ID NO: 224, or

[0908] SEQ ID NO: 232, or

[0909] SEQ ID NO: 240, or

[0910] SEQ ID NO: 248, or

[0911] SEQ ID NO: 256, or

[0912] SEQ ID NO: 264, or

[0913] SEQ ID NO: 272, or

[0914] SEQ ID NO: 280, or

[0915] SEQ ID NO: 288, or

[0916] SEQ ID NO: 296, or

[0917] SEQ ID NO: 304, or

[0918] SEQ ID NO: 312, or

[0919] SEQ ID NO: 320, or

[0920] SEQ ID NO: 328, or

[0921] SEQ ID NO: 336, or

[0922] SEQ ID NO: 344, or

[0923] SEQ ID NO: 352,

[0924] And the framework, which has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the framework amino acid sequence of the VL domain of the following:

[0925] SEQ ID NO: 136, or

[0926] SEQ ID NO: 144, or

[0927] SEQ ID NO: 152, or

[0928] SEQ ID NO: 160, or

[0929] SEQ ID NO: 168, or

[0930] SEQ ID NO: 176, or

[0931] SEQ ID NO: 184, or

[0932] SEQ ID NO: 192, or

[0933] SEQ ID NO: 200, or

[0934] SEQ ID NO: 208, or

[0935] SEQ ID NO: 216, or

[0936] SEQ ID NO: 224, or

[0937] SEQ ID NO: 232, or

[0938] SEQ ID NO: 240, or

[0939] SEQ ID NO: 248, or

[0940] SEQ ID NO: 256, or

[0941] SEQ ID NO: 264, or

[0942] SEQ ID NO: 272, or

[0943] SEQ ID NO: 280, or

[0944] SEQ ID NO: 288, or

[0945] SEQ ID NO: 296, or

[0946] SEQ ID NO: 304, or

[0947] SEQ ID NO: 312, or

[0948] SEQ ID NO: 320, or

[0949] SEQ ID NO: 328, or

[0950] SEQ ID NO: 336, or

[0951] SEQ ID NO: 344, or

[0952] SEQ ID NO: 352.

[0953] On one hand, the anti-TREM2 antibody contains the following three light chain CDR amino acid sequences of the VL domain:

[0954] SEQ ID NO: 136, or

[0955] SEQ ID NO: 144, or

[0956] SEQ ID NO: 152, or

[0957] SEQ ID NO: 160, or

[0958] SEQ ID NO: 168, or

[0959] SEQ ID NO: 176, or

[0960] SEQ ID NO: 184, or

[0961] SEQ ID NO: 192, or

[0962] SEQ ID NO: 200, or

[0963] SEQ ID NO: 208, or

[0964] SEQ ID NO: 216, or

[0965] SEQ ID NO: 224, or

[0966] SEQ ID NO: 232, or

[0967] SEQ ID NO: 240, or

[0968] SEQ ID NO: 248, or

[0969] SEQ ID NO: 256, or

[0970] SEQ ID NO: 264, or

[0971] SEQ ID NO: 272, or

[0972] SEQ ID NO: 280, or

[0973] SEQ ID NO: 288, or

[0974] SEQ ID NO: 296, or

[0975] SEQ ID NO: 304, or

[0976] SEQ ID NO: 312, or

[0977] SEQ ID NO: 320, or

[0978] SEQ ID NO: 328, or

[0979] SEQ ID NO: 336, or

[0980] SEQ ID NO: 344, or

[0981] SEQ ID NO: 352

[0982] And the framework, which has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the framework amino acid sequence of the VL domain of the following:

[0983] SEQ ID NO: 136, or

[0984] SEQ ID NO: 144, or

[0985] SEQ ID NO: 152, or

[0986] SEQ ID NO: 160, or

[0987] SEQ ID NO: 168, or

[0988] SEQ ID NO: 176, or

[0989] SEQ ID NO: 184, or

[0990] SEQ ID NO: 192, or

[0991] SEQ ID NO: 200, or

[0992] SEQ ID NO: 208, or

[0993] SEQ ID NO: 216, or

[0994] SEQ ID NO: 224, or

[0995] SEQ ID NO: 232, or

[0996] SEQ ID NO: 240, or

[0997] SEQ ID NO: 248, or

[0998] SEQ ID NO: 256, or

[0999] SEQ ID NO: 264, or

[1000] SEQ ID NO: 272, or

[1001] SEQ ID NO: 280, or

[1002] SEQ ID NO: 288, or

[1003] SEQ ID NO: 296, or

[1004] SEQ ID NO: 304, or

[1005] SEQ ID NO: 312, or

[1006] SEQ ID NO: 320, or

[1007] SEQ ID NO: 328, or

[1008] SEQ ID NO: 336, or

[1009] SEQ ID NO: 344, or

[1010] SEQ ID NO: 352.

[1011] On one hand, the anti-TREM2 antibody contains the following three light chain CDR amino acid sequences of the VL domain:

[1012] SEQ ID NO: 136, or

[1013] SEQ ID NO: 144, or

[1014] SEQ ID NO: 152, or

[1015] SEQ ID NO: 160, or

[1016] SEQ ID NO: 168, or

[1017] SEQ ID NO: 176, or

[1018] SEQ ID NO: 184, or

[1019] SEQ ID NO: 192, or

[1020] SEQ ID NO: 200, or

[1021] SEQ ID NO: 208, or

[1022] SEQ ID NO: 216, or

[1023] SEQ ID NO: 224, or

[1024] SEQ ID NO: 232, or

[1025] SEQ ID NO: 240, or

[1026] SEQ ID NO: 248, or

[1027] SEQ ID NO: 256, or

[1028] SEQ ID NO: 264, or

[1029] SEQ ID NO: 272, or

[1030] SEQ ID NO: 280, or

[1031] SEQ ID NO: 288, or

[1032] SEQ ID NO: 296, or

[1033] SEQ ID NO: 304, or

[1034] SEQ ID NO: 312, or

[1035] SEQ ID NO: 320, or

[1036] SEQ ID NO: 328, or

[1037] SEQ ID NO: 336, or

[1038] SEQ ID NO: 344, or

[1039] SEQ ID NO: 352

[1040] And the framework, which has at least 95% sequence identity with the framework amino acid sequence of the VL domain of the following:

[1041] SEQ ID NO: 136, or

[1042] SEQ ID NO: 144, or

[1043] SEQ ID NO: 152, or

[1044] SEQ ID NO: 160, or

[1045] SEQ ID NO: 168, or

[1046] SEQ ID NO: 176, or

[1047] SEQ ID NO: 184, or

[1048] SEQ ID NO: 192, or

[1049] SEQ ID NO: 200, or

[1050] SEQ ID NO: 208, or

[1051] SEQ ID NO: 216, or

[1052] SEQ ID NO: 224, or

[1053] SEQ ID NO: 232, or

[1054] SEQ ID NO: 240, or

[1055] SEQ ID NO: 248, or

[1056] SEQ ID NO: 256, or

[1057] SEQ ID NO: 264, or

[1058] SEQ ID NO: 272, or

[1059] SEQ ID NO: 280, or

[1060] SEQ ID NO: 288, or

[1061] SEQ ID NO: 296, or

[1062] SEQ ID NO: 304, or

[1063] SEQ ID NO: 312, or

[1064] SEQ ID NO: 320, or

[1065] SEQ ID NO: 328, or

[1066] SEQ ID NO: 336, or

[1067] SEQ ID NO: 344, or

[1068] SEQ ID NO: 352.

[1069] On the other hand, the anti-TREM2 antibody contains the following three light chain CDR amino acid sequences of the VL domain:

[1070] SEQ ID NO: 136, or

[1071] SEQ ID NO: 144, or

[1072] SEQ ID NO: 152, or

[1073] SEQ ID NO: 160, or

[1074] SEQ ID NO: 168, or

[1075] SEQ ID NO: 176, or

[1076] SEQ ID NO: 184, or

[1077] SEQ ID NO: 192, or

[1078] SEQ ID NO: 200, or

[1079] SEQ ID NO: 208, or

[1080] SEQ ID NO: 216, or

[1081] SEQ ID NO: 224, or

[1082] SEQ ID NO: 232, or

[1083] SEQ ID NO: 240, or

[1084] SEQ ID NO: 248, or

[1085] SEQ ID NO: 256, or

[1086] SEQ ID NO: 264, or

[1087] SEQ ID NO: 272, or

[1088] SEQ ID NO: 280, or

[1089] SEQ ID NO: 288, or

[1090] SEQ ID NO: 296, or

[1091] SEQ ID NO: 304, or

[1092] SEQ ID NO: 312, or

[1093] SEQ ID NO: 320, or

[1094] SEQ ID NO: 328, or

[1095] SEQ ID NO: 336, or

[1096] SEQ ID NO: 344, or

[1097] SEQ ID NO: 352

[1098] And the framework, which has at least 98% sequence identity with the framework amino acid sequence of the VH domain of the following:

[1099] SEQ ID NO: 136, or

[1100] SEQ ID NO: 144, or

[1101] SEQ ID NO: 152, or

[1102] SEQ ID NO: 160, or

[1103] SEQ ID NO: 168, or

[1104] SEQ ID NO: 176, or

[1105] SEQ ID NO: 184, or

[1106] SEQ ID NO: 192, or

[1107] SEQ ID NO: 200, or

[1108] SEQ ID NO: 208, or

[1109] SEQ ID NO: 216, or

[1110] SEQ ID NO: 224, or

[1111] SEQ ID NO: 232, or

[1112] SEQ ID NO: 240, or

[1113] SEQ ID NO: 248, or

[1114] SEQ ID NO: 256, or

[1115] SEQ ID NO: 264, or

[1116] SEQ ID NO: 272, or

[1117] SEQ ID NO: 280, or

[1118] SEQ ID NO: 288, or

[1119] SEQ ID NO: 296, or

[1120] SEQ ID NO: 304, or

[1121] SEQ ID NO: 312, or

[1122] SEQ ID NO: 320, or

[1123] SEQ ID NO: 328, or

[1124] SEQ ID NO: 336, or

[1125] SEQ ID NO: 344, or

[1126] SEQ ID NO: 352.

[1127] In one aspect, the anti-TREM2 antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 305; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 306; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 307; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 309; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 310; and (f) a 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 with the amino acid sequence of SEQ ID NO: 308; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 312 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 308. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 312.

[1128] In one aspect, the anti-TREM2 antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 305; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 306; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 307; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 309; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 310; and (f) a 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 with the amino acid sequence of SEQ ID NO: 308; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 312 ... The amino acid sequence of the antibody has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; wherein the antibody specifically binds to TREM2. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 308. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 312.

[1129] In one aspect, the anti-TREM2 antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 329; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 330; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 331; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 333; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 334; and (f) a 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 with the amino acid sequence of SEQ ID NO: 332; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 336 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 332. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 336.

[1130] In one aspect, the anti-TREM2 antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 329; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 330; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 331; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 333; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 334; and (f) a 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 with the amino acid sequence of SEQ ID NO: 332; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 336 ... The amino acid sequence of the antibody has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; wherein the antibody specifically binds to TREM2. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 332. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 336.

[1131] On the other hand, the 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 with the amino acid sequence of SEQ ID NO:308. In one aspect, the anti-TREM2 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:308. In some aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-TREM2 antibody containing this sequence retains its ability to bind to TREM2. In some aspects, in SEQ ID NO:308, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). Optionally, the anti-TREM2 antibody comprises the VH sequence of SEQ ID NO: 308, including post-translational modifications of that sequence. In one specific 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 with the amino acid sequence of SEQ ID NO: 312. In one aspect, the anti-TREM2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 312. In some aspects, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-TREM2 antibody containing this sequence retains its ability to bind to TREM2. In some aspects, in SEQ ID NO: 312, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted.In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). Optionally, the anti-TREM2 antibody comprises the VL sequence of SEQ ID NO: 312, including post-translational modifications of that sequence. In one specific aspect, the VL comprises one, two, or three CDRs selected from the following: (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.

[1132] On the other hand, the 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 with the amino acid sequence of SEQ ID NO:332. In one aspect, the anti-TREM2 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:332. In some aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-TREM2 antibody containing this sequence retains its ability to bind to TREM2. In some aspects, in SEQ ID NO:332, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). Optionally, the anti-TREM2 antibody comprises the VH sequence shown in SEQ ID NO: 332, including post-translational modifications of that sequence. In one specific 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 with the amino acid sequence of SEQ ID NO: 336. In one aspect, the anti-TREM2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 336. In some aspects, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-TREM2 antibody containing this sequence retains its ability to bind to TREM2. In some aspects, in SEQ ID NO: 336, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted.In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). Optionally, the anti-TREM2 antibody comprises the VL sequence of SEQ ID NO: 336, including post-translational modifications of that sequence. In one specific aspect, the VL comprises one, two, or three CDRs selected from the following: (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.

[1133] In a preferred aspect, an anti-TREM2 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects described above and a VL sequence as provided in any of the aspects described above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 308 and SEQ ID NO: 312, respectively, which include post-translational modifications of those sequences. In one embodiment, the heavy chain variable domain further comprises a glutamic acid (E) residue or a pyroglutamic acid (pE) residue as a first N-terminal amino acid residue.

[1134] In another preferred aspect, an anti-TREM2 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects described above and a VL sequence as provided in any of the aspects described above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 332 and SEQ ID NO: 336, respectively, which include post-translational modifications of those sequences. In one embodiment, the heavy chain variable domain further comprises a glutamic acid (E) residue or a pyroglutamic acid (pE) residue as a first N-terminal amino acid residue.

[1135] In another aspect, the present invention provides an antibody that binds to the same epitope as the anti-TREM2 antibody provided herein. For example, in some aspects, an antibody is provided that binds to the same epitope as TREM2 3295 or TREM2 3306. In some aspects, an antibody is provided that binds to an epitope within the fragment of TREM2 consisting of amino acids 41 to 45 of SEQ ID NO: 416 (TREM2 3295) or amino acids 67 to 68 and 74 to 80 of SEQ ID NO: 416.

[1136] In another aspect of the invention, the anti-TREM2 antibody according to any of the foregoing aspects is a monoclonal antibody, including chimeric, humanized, or human antibodies. In another aspect, the anti-TREM2 antibody is an antibody fragment having the same complementary site as the full-length antibody, such as Fv, Fab, Fab', scFv, biantibody, or F(ab')2 fragment.

[1137] On the other hand, the antibody is a full-length antibody, such as a full-length IgG1 antibody or IgG1LALAPG antibody or other antibody class or isotype as defined herein.

[1138] The terms "anti-TREM2 antibody" and "TREM2-binding antibody" refer to antibodies that bind to human TREM2 with sufficient affinity, making them usable as diagnostic and / or therapeutic agents targeting TREM2. In one respect, as measured, for example by surface plasmon resonance (SPR), anti-TREM2 antibodies bind to less than about 10% of unrelated, non-TREM2 proteins as they bind to TREM2. In other respects, TREM2-binding antibodies have a dissociation constant (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM (e.g., 1E-8 M or less, such as 1E-8 M to 10-11 M, such as 1E-9 to 1E-11 M). When the KD of an antibody is 1 μM or less, the antibody is said to "specifically bind" to TREM2.

[1139] In some respects, this article provides an antibody having a dissociation constant (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM or ≤ 0.1 nM (e.g., 1E-8 M or less, e.g., 1E-8 M to 10-11 M, e.g., 1E-9 M to 1E-11 M).

[1140] On one hand, KD was measured using BIACORE® surface plasmon resonance measurement.

[1141] Exemplary antibody variants, fragments, and constant regions

[1142] In another aspect, the antibodies according to the invention may be combined, alone or in combination, with any of the features described in the following sections.

[1143] antibody fragments

[1144] In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, F(ab')2 and (scFv)2 fragments, as well as other fragments described below, provided they are at least bivalent. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); also see WO 93 / 16185; and US 5,571,894 and 5,587,458. For a discussion of Fab fragments and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see US 5,869,046.

[1145] In another respect, antibody fragments are bisomatic, trisomatic, or tetrasomatic antibodies. A bisomatic antibody is an antibody fragment having two antigen-binding sites, and it can 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). Trisomatic and tetrasomatic antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[1146] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a humanized single-domain antibody (Domantis, Inc., Waltham, MIT; see, for example, U.S. Patent No. 6,248,516).

[1147] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and production from recombinant host cells, as described herein.

[1148] Chimeric and humanized antibodies

[1149] In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, 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 another instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments, provided that these fragments bind to their respective targets.

[1150] In some embodiments, the chimeric antibody is a humanized antibody. Typically, nonhuman antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent nonhuman antibody. Typically, humanized antibodies comprise one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the nonhuman antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[1151] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US patents 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) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface reshaping”); Dall'Acqua Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach for FR shuffling).

[1152] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from specific subgroups of human antibody common sequences of the light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok ... Chem. 271:22611-22618 (1996)).

[1153] In some embodiments, the humanized antibody may include a human IgG1 heavy chain constant region.

[1154] Bispecific antibodies or multispecific antibodies

[1155] The antibodies described herein are multispecific antibodies, such as bispecific or trispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigens or epitopes of the same antigen. In some embodiments, one of the binding specificities is against human TREM2, and the other is against human amyloid β protein. In some embodiments, the multispecific antibody may bind to two different epitopes of TREM2. Bispecific antibodies can also be used to target drugs such as cytotoxic agents or to target detection markers to cells expressing TREM2. In some embodiments, the multispecific antibody (e.g., a bispecific antibody) comprises a first variable domain containing a CDR or variable region as described herein. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[1156] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)); and engineered “mortar and pestle structures” (see, for example, US 5,731,168). Multispecific antibodies can also be manufactured using the following techniques: engineered electrostatic manipulation effects to create antibody Fc-heterodimer molecules (WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, US 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 128(5):1547-1553 (1992)); using “dual antibody” techniques to create bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers ((scFv)2) (see, for example, Gruber et al., J. Immunol., 152:5368). (1994)); and prepare trispecific antibodies according to, for example, the description in Tutt et al. J. Immunol. 147: 60 (1991).

[1157] Engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies,” are also included in this document (see, for example, US 2006 / 0025576).

[1158] Other antibody variants

[1159] In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[1160] Substitution, insertion, and deletion variants

[1161] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVR and FR. Conservative substitutions are shown under the heading “Preferred Substitutions” in Table A. Further substantial changes are provided under the heading “Exemplary Substitutions” in Table A and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into target antibodies, and products can be screened for desired activities (e.g., maintaining / improving antigen binding, reducing immunogenicity, or increasing or decreasing ADCC or CDC activity).

[1162] Table A

[1163]

[1164] Amino acids can be grouped based on their common side-chain characteristics:

[1165] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[1166] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[1167] (3) Acidic: Asp, Glu;

[1168] (4) Alkaline: His, Lys, Arg;

[1169] (5) Residues affecting chain orientation: Gly, Pro;

[1170] (6) Fang ethnic group: Trp, Tyr, Phe.

[1171] Non-conservative substitution would require swapping members of one of these categories for members of another category.

[1172] One type of substitution variant involves substituting one or more highly variable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more resulting variants selected for further research will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[1173] Modifications (e.g., substitutions) can be made in the HVR to improve antibody affinity, for example. Such modifications can occur in HVR “hotspots,” which are residues encoded by codons that undergo frequent mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen (to detect the binding affinity of the resulting variant VH or VL). Affinity maturation achieved by constructing and reselecting from a secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation purposes by any of a variety of methods (e.g., error-prone PCR, strand 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 approach to introducing diversity involves HVR-directed methods, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutations or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[1174] In some embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to its antigen. For example, conserved changes (e.g., conserved substitutions as described herein) that do not substantially reduce binding affinity may be made in the HVR. Such changes may occur outside the antigen-contact residues of the HVR. In some embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or contains no more than one, two, or three amino acid substitutions.

[1175] A method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they possess the desired properties.

[1176] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include fusion of the N-terminus or C-terminus of an antibody with an enzyme that increases the serum half-life of the antibody (e.g., for ADEPT) or a peptide.

[1177] Glycosylation variants

[1178] Changes in Fc region glycosylation and certain Fc region mutations can affect antibody effector function by enhancing or reducing effector function, or in some cases silence antibody effector function (effect-free).

[1179] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[1180] When an antibody contains an Fc region, the carbohydrates attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically linked to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[1181] On one hand, antibodies can be modified to reduce or eliminate glycosylation at Asn297, such as by mutating that residue to glycine or another amino acid (N297G). On the other hand, other residues in the Fc region can be modified to reduce ADCC and / or CDC activity or to reduce or modify Fc-γ receptor binding.

[1182] In another embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose intrinsically present at Asn297 within the glycan chain relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297 as determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 in the Fc region (EU number of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 (EU number). These fucosylated variants may have improved ADCC function. See, for example, US 2003 / 0157108; US 2004 / 0093621. Examples of publications associated with “defucosylated” or “fucosylated” 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; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 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 defucosylation antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, especially in Example 11), and knockout cell lines, such as CHO cells with the α-1,6-fucosylation gene FUT8 knocked out (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107). In some embodiments, the antibody may have a human IgG heavy chain constant region, for example, containing a mutation (EU number) at Asn297 to reduce fucosylation or alternatively eliminate glycosylation. In some embodiments, the antibody according to the invention may have an Asn297Ala or Asn297Gly mutation.

[1183] Antibody variants also provide bipartite oligosaccharides, for example, wherein the bitendril oligosaccharide attached to the Fc region of the antibody is bipartitely divided by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; US 6,602,684; and US 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[1184] Fc region variant

[1185] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[1186] In some embodiments, the invention considers antibody variants possessing some, but not all, effector functions (i.e., antibodies producing reduced effector functions or antibodies producing effector-silencing functions), which can make them desirable candidates for applications where the antibody's half-life in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR (FcgR) binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. 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 for assessing ADCC activity of target molecules are described in US 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l 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 can be used (see, for example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc., Mountain View, CA); and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo in animal models such as those reported in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int. Immunol. 18(12):1759-1769 (2006)).

[1187] Antibodies with reduced effector function include those with substitutions at one or more Fc region residues 234, 235, 238, 265, 269, 270, 297, 327, and 329 (e.g., US 6,737,056; EU number for residues). Such Fc region mutants include Fc region mutants with substitutions at two or more of the amino acid positions 234, 235, 265, 269, 270, 297, 327, and 329, including the so-called “DANA” Fc region mutant (US 7,332,581; EU number) with residues 265 and 297 replaced by alanine, and the so-called “PGLALA” Fc region mutant (WO 2012 / 130831) with residues 329, 234, and 235 replaced by glycine, alanine, and alanine, respectively. In some embodiments, the antibody contains engineered alanine at amino acid position 265 according to EU numbering convention. In some embodiments, the antibody contains engineered alanine at amino acid position 297 according to EU numbering convention.

[1188] Some antibodies that have improved or reduced binding to FcR are described, for example, in US 6,737,056; WO2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[1189] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbers of the residues).

[1190] In some embodiments, such as those described in US 6,194,551, WO 99 / 51642 and Idusogie et al. J. Immunol. 164: 4178-4184 (2000), alterations are made in the Fc region resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC).

[1191] Antibodies with prolonged half-life and improved neonatal Fc receptor (FcRn) binding responsible for transferring maternal IgG 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. These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more of the following 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 number), such as the substitution of Fc region residue 434 (US 7,371,826). For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); US 5,648,260; US 5,624,821; and WO 94 / 29351.

[1192] In some embodiments, the antibody may have a wild-type human IgG1 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 IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, a human IgG1 N297G / M428L / N434S Fc region, or a human IgG1 LALAPG YTE (L234A / L235A / P329G / M252Y / S254T / T256E) Fc region. (All locations are assigned EU numbers.) In a preferred embodiment of all aspects and embodiments, the antibody according to the invention comprises the human IgG1 LALAPG (L234A / L235A / P329G) Fc region.

[1193] Cysteine-engineered antibody variants

[1194] In some embodiments, it is desirable to generate cysteine-engineered antibodies, such as “thioMAb”, wherein one or more residues of the antibody are substituted with cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites on the antibody. As further described herein, by substituting those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the antibody and can be used to conjugate the antibody to other parts (such as a pharmaceutical part or a linker-pharmaceutical part) to produce an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described, for example, in US 7,521,541.

[1195] KTG engineered antibody variant

[1196] In some embodiments, it may be desirable to generate KTG-engineered antibodies in which a Q tag is inserted after one or more residues of the antibody. In certain embodiments, the inserted residues are located at an accessible site on the antibody. In some embodiments, the Q tag is selected from RYGQR (SEQ ID NO: 422), RWRQR (SEQ ID NO: 423), YRQRT (SEQ ID NO: 424), IRQRQ (both Qs may be modified; SEQ ID NO: 425), FRYRQ (SEQ ID NO: 426), and YRYRQ (SEQ ID NO: 427), preferably YRYRQ (SEQ ID NO: 427). As further described herein, transglutaminase (KTG) from Kutzneria albida can be used to localize the reactive group to an accessible site on the antibody by inserting those residues, and can be used to conjugate the antibody to other parts, such as drug parts or linker-drug parts, to produce immunoconjugates. In some embodiments, the Q tag may be inserted at any one or more of positions 110 (LC110), 143 (LC143), and 214 (LC214) on the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) on the heavy chain (according to the Kabat number), preferably after HC297. In some embodiments, one or more recognition sites of transglutaminase (KTG) from *Kuznerella alba* are inserted between two flexible peptide linkers. In some embodiments, one or more recognition sites of transglutaminase (KalbTG) from *Kuznerella alba* have the amino acid sequence GGGSYRYRQGGGS (SEQ ID NO: 431).

[1197] KTG engineered antibodies can be generated, for example, as described in WO 2023 / 118398.

[1198] Fc region variant

[1199] The term "(human) Fc region polypeptide" refers to a polypeptide with the same amino acid sequence as a "natural" or "wild-type" (human) Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to a polypeptide derived from a "natural" or "wild-type" (human) Fc region polypeptide whose amino acid sequence differs by at least one "amino acid alteration." A "variant (human) Fc region" consists of two Fc region polypeptides, both of which can be variant (human) Fc region polypeptides, or one can be a human Fc region polypeptide and the other a variant (human) Fc region polypeptide.

[1200] In some embodiments, the human Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region polypeptide or a variant thereof, or the amino acid sequence of the human IgG2 Fc region polypeptide or a variant thereof, or the amino acid sequence of the human IgG3 Fc region polypeptide or a variant thereof, or the amino acid sequence of the human IgG4 Fc region polypeptide or a variant thereof. In some embodiments, the Fc region polypeptide is derived from the Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441 and has at least one amino acid mutation compared to the Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In some embodiments, the Fc region polypeptide contains / has about one to about ten amino acid mutations. In some embodiments, the Fc region polypeptide contains / has about one to about five amino acid mutations.

[1201] In some embodiments, the Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region polypeptide with LALAPG mutation and pestle-cys mutation (SEQ ID NO: 436), wherein the C-terminal lysine residue is optionally deleted. In some embodiments, the Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region with LALAPG mutation and pestle-cys or mortar-cys mutation (SEQ ID NO: 440 or SEQ ID NO: 439), wherein the C-terminal lysine residue is optionally deleted.

[1202] In some embodiments, the Fc region polypeptide shares at least about 80% sequence homology with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In some embodiments, the Fc region polypeptide shares at least about 90% sequence homology with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In some embodiments, the Fc region polypeptide shares at least about 95% sequence homology with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In a preferred embodiment, the Fc region polypeptide shares at least about 97.5% homology with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441.

[1203] In some embodiments, the Fc region polypeptide shares at least about 80% sequence identity with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In some embodiments, the Fc region polypeptide shares at least about 90% sequence identity with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In some embodiments, the Fc region polypeptide shares at least about 95% sequence identity with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In a preferred embodiment, the Fc region polypeptide shares at least about 97.5% identity with the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441. In a preferred embodiment, the C-terminal lysine residue is deleted.

[1204] A variant Fc region polypeptide derived from the parental (human) Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441 is further defined by the amino acid changes contained compared to the parental or wild-type sequence. Thus, for example, the term P329G refers to an Fc region polypeptide derived from the (human) Fc region polypeptide having a proline-to-glycine mutation at amino acid position 329 relative to the human Fc region polypeptide of SEQ ID NO: 429 or SEQ ID NO: 441 (according to Kabat numbering).

[1205] The human IgG1 Fc region polypeptide has the following amino acid sequence:

[1206] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 429), optionally with an additional lysine residue (K) added to the C-terminus.

[1207] The Fc region polypeptide derived from the human IgG1 Fc region with mutations L234A and L235A (LALA mutation) has the following amino acid sequence:

[1208] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 430), optionally with an additional lysine residue (K) added to the C-terminus.

[1209] The Fc region polypeptide derived from the human IgG1 Fc region with mutations Y349C, T366S, L368A, and Y407V (cys mutation) has the following amino acid sequence:

[1210] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 431), optionally with an additional lysine residue (K) added to the C-terminus.

[1211] The Fc region polypeptide derived from the human IgG1 Fc region with the S354C, T366W (sodium-cys mutation) has the following amino acid sequence:

[1212] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 432), optionally with an additional lysine residue (K) added to the C-terminus.

[1213] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A mutations and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[1214] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 433), optionally with an additional lysine residue (K) added to the C-terminus.

[1215] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, S354C, and T366W mutations has the following amino acid sequence:

[1216] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 434), optionally with an additional lysine residue (K) added to the C-terminus.

[1217] The Fc region polypeptide derived from the human IgG1 Fc region with the P329G mutation (PG mutation) has the following amino acid sequence:

[1218] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 435), optionally with an additional lysine residue (K) added to the C-terminus.

[1219] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A mutations and P329G mutation (LALAPG mutation) has the following amino acid sequence:

[1220] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 436), optionally with an additional lysine residue (K) added to the C-terminus.

[1221] The Fc region polypeptide derived from the human IgG1 Fc region with P329G mutation and Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

[1222] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 437), optionally with an additional lysine residue (K) added to the C-terminus.

[1223] The Fc region polypeptide derived from the human IgG1 Fc region with P329G mutation and S354C, T366W mutation has the following amino acid sequence:

[1224] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 438), optionally with an additional lysine residue (K) added to the C-terminus.

[1225] The Fc region polypeptide derived from the human IgG1 Fc region with mutations L234A, L235A, P329G and Y349C, T366S, L368A, Y407V has the following amino acid sequence:

[1226] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 439), optionally with an additional lysine residue (K) added to the C-terminus.

[1227] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:

[1228] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 440), optionally with an additional lysine residue (K) added to the C-terminus.

[1229] The human IgG4 Fc region polypeptide has the following amino acid sequence:

[1230] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:441).

[1231] The Fc region polypeptide derived from the human IgG4 Fc region with S228P and L235E mutations has the following amino acid sequence:

[1232] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:442).

[1233] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E mutations and P329G mutations has the following amino acid sequence:

[1234] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:443).

[1235] The Fc region polypeptide derived from the human IgG4 Fc region with the S354C, T366W mutation has the following amino acid sequence:

[1236] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:444).

[1237] The Fc region polypeptide derived from the human IgG4 Fc region with mutations Y349C, T366S, L368A, and Y407V has the following amino acid sequence:

[1238] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVCTLPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:445).

[1239] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E and S354C, T366W mutations has the following amino acid sequence:

[1240] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:446).

[1241] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[1242] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVCTLPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:447).

[1243] The Fc region polypeptide derived from the human IgG4 Fc region with the P329G mutation has the following amino acid sequence:

[1244] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:448).

[1245] The Fc region polypeptide derived from the human IgG4 Fc region with P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[1246] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVCTLPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:449).

[1247] The Fc region polypeptide derived from the human IgG4 Fc region with P329G, S354C, and T366W mutations has the following amino acid sequence:

[1248] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:450).

[1249] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[1250] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVCTLPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:451).

[1251] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E, P329G and S354C, T366W mutations has the following amino acid sequence:

[1252] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:452).

[1253] Antibody derivatives and conjugates

[1254] In some embodiments, the antibodies provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers linked to the antibody may vary, and if more than one polymer is linked, they may be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for a limited therapy.

[1255] In another embodiment, a conjugate of an antibody and a non-protein portion that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature at which cells near the antibody-non-protein portion are killed.

[1256] In some embodiments, the anti-TREM2 / amyloid β protein antibody according to the present invention is conjugated with a detection marker and / or a drug. As used herein, a detection marker is a portion of the antibody and / or a molecule to which the antibody binds that facilitates detection. Non-limiting exemplary detection markers include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc.

[1257] Nucleic acid encoding antibody

[1258] Also provided are nucleic acid molecules comprising polynucleotides encoding one or more anti-TREM2 antibody chains. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of the anti-TREM2 antibody. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy chain of the anti-TREM2 antibody and a polynucleotide encoding its light chain. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding the heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain.

[1259] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides by one or two separate nucleic acid molecules. In some embodiments, such as when the antibody is scFv, a single polynucleotide encodes a single polypeptide comprising a heavy and light chain linked together.

[1260] In some embodiments, the polynucleotide encoding the heavy or light chain of the anti-TREM2 antibody includes a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain during translation. As described above, the leader sequence can be a natural heavy or light chain leader sequence, or it can be another heterologous leader sequence.

[1261] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[1262] Vectors, host cells, and production methods

[1263] Vectors containing polynucleotides encoding an anti-TREM2 heavy chain and / or an anti-TREM2 light chain are provided. Vectors containing polynucleotides encoding an anti-TREM2 heavy chain and / or an anti-TREM2 light chain are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector contains a first polynucleotide sequence encoding the heavy chain and a second polynucleotide sequence encoding the light chain. In some embodiments, the heavy chain and light chain are expressed by 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 scFv.

[1264] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain, and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first and second vectors are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, the first and second vectors are transfected into host cells in a molar ratio or mass ratio between 5:1 and 1:5. In some embodiments, a mass ratio between 1:1 and 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[1265] In some embodiments, a vector optimized for expression of the peptide in CHO or CHO-derived cells or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[1266] In some embodiments, a vector is selected for in vivo expression of the anti-TREM2 heavy chain and / or the anti-TREM2 light chain in animals (including humans). In some such embodiments, peptide expression is controlled by a promoter that functions in a tissue-specific manner. For example, liver-specific promoters are described, for example, in WO 2006 / 076288.

[1267] To generate anti-TREM2 antibodies through recombinant synthesis, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibodies).

[1268] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totoa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*.) Antibodies can be separated from the bacterial cell paste in a soluble fraction after expression and can be further purified.

[1269] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. These eukaryotic microorganisms include fungal and yeast strains whose glycosylation pathways have been "humanized," thereby enabling the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[1270] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[1271] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES that produce antibodies in transgenic plants). TM technology).

[1272] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney lines (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); and TRI cells (as described, for example, in Mather et al., Annals NYAcad. Sci.). (As described in 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, which include DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, HumanaPress, Totowa, NJ), pp. 255–268 (2003).

[1273] Anti-TREM2 antibodies can 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 TREM2 ECDs and ligands that bind to the antibody constant region. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind to the constant region and purify anti-TREM2 antibodies. Hydrophobic interaction chromatography, such as butyl or phenyl columns, is also suitable for purifying some peptides. Many methods for purifying peptides are known in the art. In some embodiments, anti-TREM2 antibodies are generated in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, 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).

[1274] Medication usage instructions

[1275] This invention also reports methods for using the anti-TREM2 antibody described herein, for example, in pharmaceutical therapy. For instance, this invention reports methods for treating subjects with symptoms associated with loss of TREM2 function. The invention also includes methods for reducing the shedding of membrane-bound TREM2 in subjects that leads to low levels of sTREM2. Furthermore, the invention covers methods for increasing sTREM2 levels by binding the TREM2 antibody to the ECD of TREM2, thereby adapting the clearance of sTREM2 to the clearance of the bound antibody, thus accumulating sTREM2 in their respective biological fluids (i.e., blood, cerebrospinal fluid, or cerebrospinal fluid).

[1276] In some cases, the symptoms are neuroinflammatory diseases or neurodegenerative diseases. Examples include, for instance, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nasu-Hacola disease, Guillain-Barré syndrome (GBS), lysosomal storage diseases, sphingomyelin storage diseases (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neurobehçet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some cases, the symptoms are Alzheimer's disease. In some cases, the symptoms are MS. In some cases, the symptoms are Parkinson's disease.

[1277] For example, the pathological features of Alzheimer's disease (AD) include extracellular deposits of β-amyloid peptides that form amyloid plaques and intracellular deposits of hyperphosphorylated tau aggregates called neurofibrillary tangles. These lesions are accompanied by increased activation of immune pathways in the brain, including inflammatory activation of astrocytes and microglia, and associated synaptic loss and neurodegeneration with neuronal loss. Familial AD may be caused by mutations in the presenilin 1 / 2 and amyloid precursor protein genes. Transgenic mice expressing these human mutations, as well as mutations in the tau protein, show an age-dependent increase in Aβ pathology, hyperphosphorylated tau, and neurodegeneration, in some respects similar to what is observed in the human brain. Certain mutations leading to loss of TREM2 function result in irregular microglial compaction of plaques, increased inflammatory dystrophy of the neuroplasm surrounding the plaque, increased Aβ-induced tau lesions, and neurodegeneration. Without being bound by theory, enhancing TREM2 activity can be used to treat Alzheimer's disease by phagocytizing or compacting it into low-toxicity amyloid plaques, thereby enhancing microglial activity and promoting the removal of toxic amyloid β protein.

[1278] Multiple sclerosis (MS) is a disease characterized by autoimmune-related demyelination in the central nervous system. In patients, the disease manifests, among other neurological effects, particularly symptoms such as ataxia, limb weakness, and optic neuritis. Current treatments involve immunosuppressive agents and may have limited effectiveness, with no therapy effectively preventing or reversing the disease. Myelin regeneration therapy has been proposed as a treatment and potential reversal approach for MS. Compounds and therapies that promote the differentiation of oligodendrocytes from progenitor cells (OPCs) into mature myelinated oligodendrocytes are considered potential avenues for myelin regeneration therapy and glial cell modification. Specifically, microglia are thought to play a role in clearing myelin debris and promoting new myelin formation. Loss of TREM2 function leads to myelin regeneration-reducing leukodystrophy, and TREM2 knockout mice exhibit severe impairment in myelin regeneration and recovery in animal models of MS. Without being bound by theoretical constraints, activation of the TREM2 pathway could accelerate myelin regeneration, thereby potentially treating MS.

[1279] In various embodiments, anti-TREM2 antibodies can be administered in vivo via various routes, including but not limited to oral, intravenous, subcutaneous, parenteral, intranasal, intramuscular, intradermal, topical, percutaneous, and intrathecal administration, or by implantation into antibody-expressing cells, inhalation, or gene therapy. The subject composition can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. Nucleic acid molecules encoding anti-TREM2 antibodies can be administered directly or in a vector such as a viral vector. Appropriate formulations and routes of administration can be selected according to the intended application.

[1280] In various embodiments, compositions comprising anti-TREM2 antibodies are provided in formulations having a variety of pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacology). y With Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, including mediators, adjuvants, and diluents. In addition, a variety of pharmaceutically acceptable excipients are available, such as pH adjusters and buffers, tonicants, stabilizers, wetting agents, etc. Non-limiting exemplary carriers include saline, buffered saline, dextran, water, glycerol, ethanol, and combinations thereof.

[1281] In various embodiments, compositions containing anti-TREM2 antibodies can be formulated for injection or infusion by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable oils or other oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In various embodiments, compositions can be formulated for inhalation, for example, using pressurizable propellants such as dichlorodifluoromethane, propane, nitrogen, etc. In various embodiments, compositions can also be formulated into sustained-release microcapsules, such as those formulated with biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic acid-glycolic acid polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. For example, some methods for preparing such formulations are described in EP 1 125 584 A1.

[1282] Also provided are pharmaceutical packages and kits comprising one or more containers, each container containing one or more doses of anti-TREM2 antibody. In some embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of composition comprising anti-TREM2 antibody, with or without one or more additional agents. In some embodiments, such unit doses are provided in disposable pre-filled syringes for injection. In various embodiments, the composition contained in the unit dose may comprise saline, sucrose, etc.; buffers, such as phosphates, etc.; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, such as 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, the compositions of the present invention comprise heparin and / or proteoglycans.

[1283] The following sequences, figures, and examples are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention.

[1284] Sequence Description

[1285] Numbers 01 to 415 in the table below refer to the corresponding SEQ ID NO.

[1286]

[1287] Attached Figure Description

[1288]

[1289] Description of exemplary experimental work

[1290] Example 1

[1291] Antibody production

[1292] antibody form

[1293] Monospecific and bispecific antibodies were generated via transient transfection in HEK293 cells. Two expression plasmids were used for the normal monospecific bivalent antibody (normal IgG), three expression plasmids were used for the bispecific tetravalent antibody (2+2 bispecific form), and four expression plasmids were used for the domain-exchange bispecific bivalent antibody (cross-mAb) to provide the coding sequences for the light and heavy chains. Normal IgG is formed from two identical pairs, each pair containing one light chain (VL-CL) and one heavy chain (VH-CH1-hinge-CH2-CH3). The cross mAb is formed from four different polypeptides, one of which is a domain-exchangeable heavy chain with “cys-jaw” mutations T366W and S354C (HC(k), VH(1)-CL-hinge-CH2-CH3(k)), one of which is a heavy chain with “cys-jaw” mutations T366S, Y407V and L368A and Y349C (VH(2)-CH1-hinge-CH2-CH3(h)), one of which is a light chain (VL-CL) and one of which is a domain-exchangeable light chain (VL-CH1) (see, for example, Schaefer, W. et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192). The 2+2 bispecific antibody is formed from two identical pairs, each containing a heavy chain with a light chain variable domain at its C-terminus, followed by a heavy chain constant domain 1 genetically fused via a (GGSGG)2 linker (SEQ ID NO: 428) or a (GGSGG)4 linker (SEQ ID NO: 453) (VH(1)-CH1-hinge-CH2-CH3-linker-VL(2)-CH1), a light chain (VL(1)-CL), and a domain-exchange light chain (VH(2)-CL).

[1294] Transient antibody production

[1295] According to the manufacturer's instructions (Invitrogen, USA), use FreeStyle TM Antibodies were produced by transient transfection of human embryonic kidney 293-F cells with the 293 expression system. In short, the suspension of FreeStyle... TM 293-F cells were grown at 37°C / 8% CO2 in FreeStyle TM 293 cells were cultured in expression medium and seeded in fresh medium at a density of 1 to 2 x 1E6 viable cells / mL on the day of transfection. 325 µL of 293fectin was used in Opti-MEM I medium (Invitrogen, USA). TM (Invitrogen, Germany) and a total of 250 µg of the corresponding plasmid DNA were used to prepare DNA293fectin at a final transfection volume of 250 mL in a molar ratio of 1:1, 1:1:1, or 1:1:1:1. TM Complex. Cell culture supernatant containing the antibody was harvested 7 days after transfection.

[1296] Purification of recombinant antibodies

[1297] Cell culture supernatant containing antibodies was filtered and purified using a two-step chromatographic process. HiTrapMabSelect was used for purification, equilibrated with PBS (equilibration buffer, 1 mM KH₂PO₄, 10 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4). TM Sure TM (Cytiva) Antibodies are captured by affinity chromatography. Unbound proteins are removed by washing the chromatographic material with equilibration buffer. Antibodies are recovered by elution with a solution containing 100 mM sodium acetate (pH 3.0). Immediately after elution, the eluent is adjusted to pH 6.0 with 2 M Tris (pH 9.0). Superdex 200 TM Size exclusion chromatography on (Cytiva) was used as the second purification step. Size exclusion chromatography was performed using 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody-containing solution was stored at -80°C.

[1298] Example 2

[1299] THP-1 Aβ bead binding and uptake assay (also known as "Aβ bead phagocytosis assay")

[1300] To evaluate the potential of antibody constructs to mimic plaque decoration and target cell binding to Aβ plaques, microbeads were coated with Aβ protein to represent artificial plaques. These Aβ-coated beads were then co-incubated with THP-1 cells, showing expression of TREM2 and a migration response upon stimulation with certain TREM2 monoclonal antibodies (mAbs). Since the size of the microbeads also allows for their uptake by THP-1 cells, the Aβ-coated microbeads were labeled with a pH-sensitive dye that fluoresces in the acidic pH of lysosomes. Therefore, the degree of bead uptake could be quantified and used as an indicator of THP-1 binding mediated by the respective tested antibodies.

[1301] Preparation of pHRodo-labeled Aβ-coated beads

[1302] Interamidinium latex beads (100 µL, 1.0 µm; ThermoFisher) were diluted in 1 mL PBS, centrifuged (5 sec, 16,000 xg), and resuspended in 50 µL PBS after discarding the fluid. 1 mg / mL of Aβ42 peptide solution (100 µL) (Anaspec; dissolved in PBS according to manufacturer's instructions) was added, and the suspension was incubated overnight at 37°C without shaking. After washing once with PBS, the beads were resuspended in 100 µL PBS (pH 8.3), and 20 µL of 1 mg / mL pHRodo red succinimide ester (ThermoFisher) solution was added. The mixture was then incubated in the dark at room temperature for 1 hour, followed by further washing with PBS and resuspending in 1 mL PBS.

[1303] Aβ-beta binding and uptake assay

[1304] THP-1 cells were cultured in RPMI / 10% FBS / 50 µM 2-mercaptoethanol at a cell density of 0.5 x 1E6 cells / mL. On the day of the experiment, cells were harvested and resuspended in fresh medium at 0.5 x 1E6 cells / mL. Aliquots of this suspension (70 µL) were added to each well of a flat-bottomed 96-well plate (Greiner), followed by 25 µL of the appropriate concentration of test antibody solution and 5 µL of bead suspension. The plate was then shaken on a plate shaker (900 to 1200 rpm) for 20 seconds, rotated at 120 g for 5 minutes, and incubated for 16 hours in an IncuCyte S3 analyzer. Phase and red fluorescence channel images were acquired every 4 hours at 20x magnification. The relative uptake of microbeads was quantified using the IncuCyte cell-by-cell algorithm. A fluorescence intensity gate was set on untreated cells, resulting in 90% of cells being negative and 10% being positive.

[1305] Example 3

[1306] Phosphorylation assays using HEK / DAP12 / TREM2 for Syk and S6 phosphorylation

[1307] HeK cells were transiently transfected with human TREM2 / DAP12.

[1308] HEK293A cells expressing DAP12 and TREM2 were generated by transfection with an insert vector containing the human DAP12-2A-TREM2 sequence and a vector containing the piggybac transposase sequence. Following electroporation, cells were cultured in DMEM supplemented with 10% FCS (Gibco, 10082), 1 mM sodium pyruvate (Gibco, 11360), 100 U / ml penicillin / streptomycin (Gibco, 14140), and GlutaMax-I (Gibco, 31966). Selection was initiated 48 hours post-transfection by adding 1 µg / ml puromycin. Five days after selection, cells were isolated with trypsin, and single-cell clones were generated by limiting dilution seeding at 0.5 cells / well in 384-well plates. Clonal growth was monitored using Incucyte whole-well scanning, and wells with only one colony were picked for further characterization.

[1309] Cell preparation

[1310] For each experiment, 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 µg / ml puromycin (Gibco, 10131).

[1311] The conditioning effects of amidine latex beads with Aβ1-42

[1312] Prepare the Aβ1-42 stock solution according to the manufacturer's instructions as follows: Reconstitute 1 mg of lyophilized Aβ1-42 (Anaspec, AS-20276) in 1% ammonium hydroxide (Anaspec, AS-61322) and immediately dilute in PBS to a stock concentration of 1.25 mg / ml. Aliquot the Aβ1-42 and store at -80°C.

[1313] Amidine latex beads (4 mg; ThermoFisher, A37322) were washed with PBS and incubated overnight at 37°C with 125 µg Aβ1-42. The Aβ-coated beads were washed once with PBS and resuspended in 1 ml PBS pH 7.2 (Gibco, 20012). The Aβ beads were stored at 4°C.

[1314] Aβ bead bonding assay

[1315] To maintain low endogenous pSyk levels and avoid interference from phenol red in the AlphaLisa assay, all dilutions were performed in FCS-deficient DMEM medium without phenol red (Gibco, 31053) and supplemented with 1% GlutaMax (Gibco, 35050), 1 mM sodium pyruvate, 100 U / ml penicillin / streptomycin, and 1 µg / ml puromycin. This medium will be referred to below as the assay medium.

[1316] Eight times concentrated antibody / isotype control stock solution was mixed with Aβ-beads at a 1:1 ratio (v:v), diluted 1 to 8 in assay medium in a round-bottom 96-well plate, and incubated with shaking at room temperature for two hours to produce antibody concentrations of 250 nM and / or 50 nM in the assay. Three technical replicates were performed.

[1317] Forty-five minutes before the end of the two-hour antibody-bead mixture incubation period, harvest the cells and resuspend them in assay medium for counting. Centrifuge the cells and resuspend them in assay medium at 3.33 million cells / ml. Transfer 10 µl of antibody-Aβ bead mixture to the wells of a flat-bottomed 96-well plate and add 1E5 cells / well. Briefly vortex the plate and then centrifuge. Incubate the cells with the antibody-bead mixture at 37°C for 30 minutes. Then lyse the cells at 4°C with shaking for 30 minutes and analyze immediately.

[1318] Aβ-coated plate stimulation assay

[1319] Collagen I-coated 96-well plates (Greiner, 655956 or Invitrogen, A11428-03) were coated with 5 and 15 µg / ml Aβ42 at 37°C for 20 h ± 4 h.

[1320] Three-fold serial dilutions of the antibody were prepared in the assay medium as follows: for the TREM2 antibody, 2000 nM was reduced to 0.3 nM, and for the isotype control, 200 nM was reduced to 0.03 nM. After removing the Aβ solution, the antibody was added to the plate and incubated at room temperature for two hours with shaking. Three technical replicates were performed.

[1321] Forty-five minutes before the end of the two-hour antibody-bead incubation period, harvest the cells and resuspend the cell pellet in assay medium for counting. Centrifuge the cells and resuspend them in assay medium at a concentration of 3.33 million cells / ml. Wash the plate twice with assay medium and add 1E5 cells / well. Briefly vortex the plate, centrifuge, and then incubate at 37°C for 30 minutes. Lyse the cells at 4°C with shaking for 30 minutes and analyze immediately.

[1322] AlphaLisa

[1323] Phosphorylated (Tyr525 / 526) Syk and total Syk levels were measured using the Alpha SureFire® Ultra™ Multiple Phosphorylation / Total SYK Assay Kit (Perkin Elmer, MPSU-PTSYK_K10K) on an EnVision plate reader. In this assay, the following two dual emission wavelengths were measured sequentially: a 615 nm signal (Europium) corresponding to phosphorylated (Tyr525 / 526) Syk, and a 545 nm signal (Tellerium) corresponding to total Syk. S6 and pS6 were measured using the Alpha SureFire Ultra Multiple Phosphorylation (Ser235 / 236) / Total Ribosomal Protein S6 Assay Kit (Perkin Elmer, MPSU-PTRPS6-B10K).

[1324] Perform the measurements according to the manufacturer's instructions.

[1325] In short, 10 µl of undiluted cell lysate was transferred to the wells of a white, opaque 384 Optiplate, and 5 µl of recipient bead mixture was added to each well. The plate was sealed with aluminum foil and incubated at room temperature with shaking for one hour. Subsequently, donor bead mixture (5 µl) was added to each well under reduced light exposure, the plate was sealed, and incubated at room temperature with shaking for one hour.

[1326] Syk or S6 activation in HEK293A cells expressing TREM2 and DAP12 was determined by the ratio of phosphorylated Syk or S6 to total Syk or S6 (in percentage).

[1327] Based on treatment of HEK cells transiently transfected with TREM2 / DAP12, different baseline pSyk levels were observed. This allowed for testing of the antibody's effect on the induction of the inactive TREM2 / pSyk pathway at the top of the tetanic TREM2-mediated pSyk signaling (i.e., elevated baseline pSyk, possibly due to putative activation of TREM2 ligands under culture conditions) or near the detection limit of pSyk.

[1328]

Claims

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

2. The anti-TREM2 antibody according to claim 1, wherein the antibody has one or more of the following characteristics: a) The antibody, as a dual complementary anti-TREM2 antibody, induces macrophage phagocytosis of amyloid plaques, and / or b) The antibody, as a dual-complementary anti-TREM2 antibody, induces amyloid uptake in microglia in the presence of FcgRII and FcgRIII receptor binding, and / or c) The antibody, in a form with Fc effector function, induces acute uptake of MX04-labeled amyloid and Aβ proteins in APPswePS2 transgenic mice, and / or d) The antibody preferably induces macrophage migration in a migration assay as described herein at a concentration of 0.14 to 34 nM, and / or e) The antibody does not induce pSyk in the absence or presence of human Aβ protein, preferably the antibody is an antagonist of the Syk pathway, and / or f) The antibody does not induce the release of TNFα, MIP-1α, or IL-8 from iPSC-derived macrophages.

3. The anti-TREM2 antibody according to any one of claims 1 to 2, wherein the antibody comprises a) The heavy-chain variable structural domains of SEQ ID NO: 308 (VQLVESGGGLVQPGRSLRLSCAASGFTFGDYAMSWFRQAPGKGLEWVSIIGDSGDNTYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCMNYDIDVWGQGTTVTVSS) and the light-chain variable structural domains of SEQ ID NO: 312 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKRLIYAASDLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK), or b) The heavy chain variable domain of SEQ ID NO: 332 (VQLLESGGGLVQPGGSLRLSCVASGFIFNSYAMNWVRQAPGKGLEWVSTMSGSGGDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAKEGGTVFDNWGQGTLVTVSS) and the light chain variable domain of SEQ ID NO: 336 (AIQMTQSPSSLSTSVGDRVTITCRASQDISNDLGWYQQKPGKAPKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNLPFTFGPGTKVDFK).

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

5. The anti-TREM2 antibody according to any one of claims 1 to 4, wherein the antibody is induced to migrate in vitro at a concentration of 5 to 15 nM.

6. The anti-TREM2 antibody according to any one of claims 1 to 5, wherein the antibody is a multispecific antibody.

7. The anti-TREM2 antibody according to claim 6, wherein the antibody comprises... a) A first binding site for binding to human TREM2, the first binding site comprising The heavy chain variable domains comprising HVR-H1 (SEQ ID NO: 305), HVR-H2 (SEQ ID NO: 306), and HVR-H3 (SEQ ID NO: 307), and the light chain variable domains comprising HVR-L1 (SEQ ID NO: 309), HVR-L2 (SEQ ID NO: 310), and HVR-L3 (SEQ ID NO: 311) (TREM2 3295), as well as b) A second binding site for binding to human TREM2, the second binding site comprising Heavy chain variable domains comprising HVR-H1 (SEQ ID NO: 329), HVR-H2 (SEQ ID NO: 330), and HVR-H3 (SEQ ID NO: 331), and light chain variable domains comprising HVR-L1 (SEQ ID NO: 333), HVR-L2 (SEQ ID NO: 334), and HVR-L3 (SEQ ID NO: 335) (TREM2 3306).

8. The anti-TREM2 antibody according to any one of claims 6 to 7, wherein the antibody comprises a) A first binding site for binding to human TREM2, the first binding site comprising the heavy chain variable domain of SEQ ID NO: 308 and the light chain variable domain of SEQ ID NO: 312 (TREM2 3295), as well as b) A second binding site that binds to human TREM2, the second binding site comprising the heavy chain variable domain of SEQ ID NO: 332 and the light chain variable domain of SEQ ID NO: 336 (TREM2 3306).

9. The anti-TREM2 antibody according to any one of claims 1 to 8, wherein the antibody is a) Full-length antibody against the human IgG1 subclass. b) Full-length antibody against the human IgG4 subclass. c) Full-length antibodies against human IgG1 subclasses with mutations L234A, L235A, and P329G. d) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, and P329G in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain. e) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, and P329G in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain. f) Full-length antibodies against the human IgG4 subclass containing mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain. g) Full-length antibodies against the human IgG4 subclass containing mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain. h) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, I253A, H310A, and H435A in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain. i) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, I253A, H310A, and H435A in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain. j) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain. k) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain, or l) Full-length antibodies against human IgG1 subclasses containing mutations L234A, L235A, P329G, H310A, H433A, and Y436A in both heavy chains, and mutations i) T366W and ii) S354C or Y349C in one heavy chain and mutations i) T366S, L368A, and Y407V and ii) Y349C or S354C in the corresponding other heavy chain, or One of m)a) to l) that does not contain a C-terminal lysine residue.

10. A pharmaceutical composition comprising: an antibody according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

11. The anti-TREM2 antibody according to any one of claims 1 to 9, which is used as a drug.

12. The antibody according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10, for treating symptoms associated with loss of TREM2 function in a subject with this need.

13. The antibody according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10, for reducing the level of amyloid plaques in a subject with this need.

14. The anti-TREM2 antibody or use according to any one of claims 11 to 13, wherein the drug is used to treat Alzheimer's disease or the condition is Alzheimer's disease.

15. The anti-TREM2 antibody or use according to any one of claims 11 to 13, wherein the drug is used to treat multiple sclerosis or the condition is multiple sclerosis.

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