Anti-TREM2 antibodies and methods of use thereof

Abstract

To provide therapeutic agents that modulate TREM2 activity or levels of sTREM2.SOLUTION: In one aspect, antibodies that specifically bind to a human triggering receptor expressed on myeloid cells 2 (TREM2) protein are provided. In some embodiments, the antibody decreases levels of soluble TREM2 (sTREM2). In some embodiments, the antibody enhances TREM2 activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 808,141, filed February 20, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] background Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor expressed in microglia that is thought to function in regulating phagocytosis, cell survival, and the production of inflammatory cytokines. Mutations in TREM2 have been identified in neurodegenerative diseases, including Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia. In addition, altered levels of soluble TREM2 (sTREM2) have been reported in the cerebrospinal fluid of patients with Alzheimer's disease or frontotemporal dementia who have mutations in TREM2.

[0003] There remains a need for therapeutic agents that modulate TREM2 activity or levels of sTREM2. Summary of the Invention

[0004] overview In one aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to human myeloid cell expressed triggering receptor 2 (TREM2) is provided. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TREM2 is (a) a CDR-H1 sequence comprising the sequence of GFSIEDFYIH (SEQ ID NO: 29); (b) a CDR-H2 sequence comprising the sequence WIDPE-β6-G-β8-SKYAPKFQG (SEQ ID NO: 47), wherein β6 is N or Q and β8 is D or E; (c) a CDR-H3 sequence comprising the sequence HADHGNYGSTMDY (SEQ ID NO: 31); (d) a CDR-L1 sequence comprising the sequence HASQHINVWLS (SEQ ID NO: 32); (e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID NO: 33); and (f) CDR-L3 sequence comprising the sequence QQGQTYPRT (SEQ ID NO: 34) Includes.

[0005] In some embodiments, the CDR-H2 sequence is selected from SEQ ID NOs: 30, 39, 41, and 43.

[0006] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34 Includes.

[0007] In some embodiments, the antibody or antigen-binding fragment has a V that has at least 85% sequence identity to any one of SEQ ID NOs: 27, 35, 37, 38, 40, 42, 44, 45, and 46. H In some embodiments, the V H The sequence has at least 90% sequence identity to SEQ ID NO: 27. H The sequence has at least 95% sequence identity to SEQ ID NO: 27. H The sequence comprises SEQ ID NO: 27. In some embodiments, V H The sequence has at least 90% sequence identity to SEQ ID NO: 42. H The sequence has at least 95% sequence identity to SEQ ID NO: 42. H The sequence comprises SEQ ID NO: 42. In some embodiments, V H The sequence has at least 90% sequence identity to SEQ ID NO: 45. H The sequence has at least 95% sequence identity to SEQ ID NO: 45. H The sequence comprises SEQ ID NO:45.

[0008] In some embodiments, the antibody or antigen-binding fragment has a V that has at least 85% sequence identity to SEQ ID NO: 28 or 36. L In some embodiments, the V L The sequence has at least 90% sequence identity to SEQ ID NO: 28. L The sequence has at least 95% sequence identity to SEQ ID NO: 28. L The sequence comprises SEQ ID NO: 28. In some embodiments, V L The sequence has at least 90% sequence identity to SEQ ID NO: 36. L The sequence has at least 95% sequence identity to SEQ ID NO: 36. LThe sequence comprises SEQ ID NO:36.

[0009] In some embodiments, the antibody or antigen-binding fragment comprises: (a) V comprising SEQ ID NO: 27 H V comprising the sequence SEQ ID NO:28 L Array, or (b) V comprising SEQ ID NO: 35 H V comprising the sequence SEQ ID NO:36 L Array, or (c) V comprising SEQ ID NO: 37 H V comprising the sequence SEQ ID NO:36 L Array, or (d) V comprising SEQ ID NO: 38 H V comprising the sequence SEQ ID NO:36 L Array, or (e) V comprising SEQ ID NO: 40 H V comprising the sequence SEQ ID NO:36 L Array, or (f) V comprising SEQ ID NO: 42 H V comprising the sequence SEQ ID NO:36 L Array, or (g) V comprising SEQ ID NO: 44 H V comprising the sequence SEQ ID NO:36 L Array, or (h) V comprising SEQ ID NO: 45 H V comprising the sequence SEQ ID NO:36 L Array, or (i) V comprising SEQ ID NO: 46 H V comprising the sequence SEQ ID NO:36 L array Includes.

[0010] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 is (a) a CDR-H1 sequence comprising the sequence of GFTFT-α6-FYMS (SEQ ID NO: 48), wherein α6 is D or N; (b) VIRN-β5-β6-N-β8-YT-β 11 -β 12- A CDR-H2 sequence comprising the sequence of YNPSVKG (SEQ ID NO: 49), wherein β5 is K or R, β6 is A or P, β8 is G or A, and 11 is A or T, and β 12 is G or D; (c) a CDR-H3 sequence comprising the sequence γ1-RL-γ4-YGFDY (SEQ ID NO: 50), wherein γ1 is A or T and γ4 is T or S; (d) QSSKSLLHS-δ 10 - a CDR-L1 sequence comprising the sequence GKTYLN (SEQ ID NO: 51), 10 is N or T, (e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); and (f) a CDR-L3 sequence comprising the sequence QQFLE-φ6-PFT (SEQ ID NO: 52), wherein φ6 is Y or F; Includes.

[0011] In some embodiments, the CDR-H1 sequence is selected from any one of SEQ ID NOs: 4 and 12. In some embodiments, the CDR-H2 sequence is selected from any one of SEQ ID NOs: 5, 13, and 25. In some embodiments, the CDR-H3 sequence is selected from any one of SEQ ID NOs: 6, 14, and 17. In some embodiments, the CDR-L1 sequence is selected from any one of SEQ ID NOs: 7 and 23. In some embodiments, the CDR-L3 sequence is selected from any one of SEQ ID NOs: 9 and 18.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9 Includes.

[0013] In some embodiments, the antibody or antigen-binding fragment has a V sequence having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, and 79. H In some embodiments, the V H The sequence has at least 90% sequence identity to SEQ ID NO: 15. H The sequence has at least 95% sequence identity to SEQ ID NO: 15. H The sequence comprises SEQ ID NO: 15. In some embodiments, V H The sequence has at least 90% sequence identity to SEQ ID NO: 24. H The sequence has at least 95% sequence identity to SEQ ID NO: 24. H The sequence comprises SEQ ID NO: 24. In some embodiments, V H The sequence has at least 90% sequence identity to SEQ ID NO: 79. H The sequence has at least 95% sequence identity to SEQ ID NO: 79. H The sequence comprises SEQ ID NO:79.

[0014] In some embodiments, the antibody or antigen-binding fragment has a V sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 68. L In some embodiments, the V L The sequence has at least 90% sequence identity to SEQ ID NO: 16. L The sequence has at least 95% sequence identity to SEQ ID NO: 16. L The sequence comprises SEQ ID NO: 16. In some embodiments, V L The sequence has at least 90% sequence identity to SEQ ID NO: 22. L The sequence has at least 95% sequence identity to SEQ ID NO: 22.L The sequence comprises SEQ ID NO: 22. In some embodiments, V L The sequence has at least 90% sequence identity to SEQ ID NO: 68. L The sequence has at least 95% sequence identity to SEQ ID NO: 68. L The sequence comprises SEQ ID NO:68.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises: (a) V comprising SEQ ID NO: 15 H V comprising the sequence SEQ ID NO:16 L Array, or (b) V comprising SEQ ID NO: 19 H V comprising sequence SEQ ID NO: 20 L Array, or (c) V comprising SEQ ID NO: 21 H V comprising sequence SEQ ID NO: 20 L Array, or (d) V comprising SEQ ID NO: 19 H V comprising sequence SEQ ID NO:22 L Array, or (e) V comprising SEQ ID NO: 79 H V comprising sequence SEQ ID NO:22 L Array, or (f) V comprising SEQ ID NO: 24 H V comprising sequence SEQ ID NO: 20 L Array, or (g) V comprising SEQ ID NO: 26 H V comprising sequence SEQ ID NO: 20 L Array, or (h) V comprising SEQ ID NO: 24 H V comprising sequence SEQ ID NO:22 L Array, or (i) V comprising SEQ ID NO: 26 H V comprising sequence SEQ ID NO:22 L Array, or (j) V containing SEQ ID NO: 2 H V comprising the sequence SEQ ID NO:3 L Array, or (k) V containing SEQ ID NO: 10H V comprising the sequence SEQ ID NO:11 L Array, or (l) V comprising SEQ ID NO: 24 H V comprising the sequence SEQ ID NO:68 L array Includes.

[0016] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 is (a) a CDR-H1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 4, 12, and 29; (b) a CDR-H2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 5, 13, 25, 30, 39, 41, and 43; (c) a CDR-H3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 6, 14, 17, and 31; (d) a CDR-L1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 7, 23, and 32; (e) a CDR-L2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 8 and 33; and (f) a CDR-L3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 9, 18, and 34; Includes.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (h) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (j) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (k) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9 Includes.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, 28, 36, and 68.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a V having at least 85% sequence identity to SEQ ID NO:2 H and V having at least 85% sequence identity to SEQ ID NO:3. L Array, or (b) a V having at least 85% sequence identity to SEQ ID NO: 10 H and V having at least 85% sequence identity to SEQ ID NO:11. LArray, or (c) a V having at least 85% sequence identity to SEQ ID NO: 15 H and V having at least 85% sequence identity to SEQ ID NO:16. L Array, or (d) a V having at least 85% sequence identity to SEQ ID NO: 19 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (e) a V having at least 85% sequence identity to SEQ ID NO: 21 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (f) a V having at least 85% sequence identity to SEQ ID NO: 19 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (g) a V having at least 85% sequence identity to SEQ ID NO: 79 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (h) a V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (i) a V having at least 85% sequence identity to SEQ ID NO: 26 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (j) V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (k) V having at least 85% sequence identity to SEQ ID NO: 26 H and V having at least 85% sequence identity to SEQ ID NO:22. LArray, or (l) a V having at least 85% sequence identity to SEQ ID NO: 27 H and V having at least 85% sequence identity to SEQ ID NO:28. L Array, or (m) a V having at least 85% sequence identity to SEQ ID NO: 35 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (n) a V having at least 85% sequence identity to SEQ ID NO: 37 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (o) a V having at least 85% sequence identity to SEQ ID NO: 38 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (p) a V having at least 85% sequence identity to SEQ ID NO: 40 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (q) a V having at least 85% sequence identity to SEQ ID NO: 42 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (r) V having at least 85% sequence identity to SEQ ID NO: 44 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (s) a V having at least 85% sequence identity to SEQ ID NO: 45 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (t) V having at least 85% sequence identity to SEQ ID NO: 46 H and V having at least 85% sequence identity to SEQ ID NO:36. LArray, or (u) V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:68. L array Includes.

[0020] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 is selected from the group consisting of clone CL0020306, clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL00 The antibody clone recognizes the same or substantially the same epitope as that recognized by an antibody clone selected from the group consisting of 20307, ​​clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7, and clone CL0020123-8.

[0021] In some embodiments, the antibody or antigen-binding fragment recognizes an epitope that is the same as or substantially the same as an epitope recognized by an antibody clone selected from the group consisting of clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7, and clone CL0020123-8. In certain embodiments, the antibody or antigen-binding fragment recognizes one or more of the following epitopes in SEQ ID NO:1: (i) amino acid residues 55-63 (GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO:72)). In another aspect, the disclosure features an isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, where the antibody or antigen-binding fragment thereof recognizes an epitope including or consisting of one or more of the following epitopes in SEQ ID NO:1: (i) amino acid residues 55-63 (GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO:72)). In some embodiments, the antibody or antigen-binding fragment recognizes an epitope that is the same or substantially the same as an epitope recognized by an antibody clone selected from the group consisting of clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307, ​​and clone CL0020306. In certain embodiments, the antibody or antigen-binding fragment recognizes amino acid residues 143-149 in SEQ ID NO:1 (FPGESES (SEQ ID NO:69)).In another aspect, the disclosure features an isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope including or consisting of amino acid residues 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO:69)).

[0022] In some embodiments, the antibodies or antigen-binding fragments disclosed herein reduce the level of soluble TREM2 protein (sTREM2). In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to soluble TREM2 protein (sTREM2) in healthy human CSF or cynomolgus monkey CSF with better potency compared to the reference antibody. In some embodiments, the reference antibody is represented by a combination of sequences selected from the group consisting of SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, and SEQ ID NOs: 77 and 78. In some embodiments, the potency assay is performed substantially as described in Example 11. In some embodiments, the antibodies or antigen-binding fragments disclosed herein enhance TREM2 activity. In some embodiments, the antibodies or antigen-binding fragments thereof enhance phagocytosis or enhance migration, differentiation, function, or survival of bone marrow cells, microglia, or macrophages. In some embodiments, the antibodies or antigen-binding fragments thereof enhance microglial function without increasing neuroinflammation. In some embodiments, the antibodies or antigen-binding fragments thereof enhance Syk phosphorylation. In some embodiments, the antibody or antigen-binding fragment thereof enhances Syk phosphorylation in the presence of a TREM2 ligand, hi some embodiments, the antibody or antigen-binding fragment thereof exhibits cross-reactivity with cynomolgus TREM2 protein.

[0023] In some embodiments, the antibody or antigen-binding fragment disclosed herein is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment disclosed herein is a chimeric antibody. In some embodiments, the antibody or antigen-binding fragment disclosed herein is a humanized antibody. In some embodiments, the antibody or antigen-binding fragment disclosed herein is a fully human antibody. In some embodiments, the antibody or antigen-binding fragment disclosed herein is a Fab, F(ab')2, scFv, or bivalent scFv.

[0024] In another aspect, the disclosure provides an antibody or antigen-binding fragment thereof that competes with an isolated anti-TREM2 antibody disclosed herein for binding to human TREM2 protein.

[0025] In another aspect, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment disclosed herein that specifically binds to TREM2 and a pharma- ceutically acceptable carrier.

[0026] In yet another aspect, the disclosure provides a kit comprising a pharmaceutical composition comprising an antibody or antigen-binding fragment disclosed herein that specifically binds to TREM2, or an anti-TREM2 antibody or antigen-binding fragment, and instructions for use thereof.

[0027] In yet another aspect, the disclosure provides a method for treating a neurodegenerative disease in a subject. In some embodiments, the method comprises administering to the subject an anti-TREM2 antibody or antigen-binding fragment disclosed herein, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment disclosed herein.

[0028] In some embodiments, the neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica ... pugilistica, diffuse neurofibrillary tangle disease with calcifications, Down's syndrome, familial British dementia, familial Danish dementia, Gerstmann-Sträussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, PSP of the Guadeloupean type, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle predominant dementia, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Parkinson's disease, Pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle only dementia.

[0029] In yet another aspect, the disclosure provides a method for reducing the level of sTREM2 in a subject having a neurodegenerative disease. In some embodiments, the method comprises administering to the subject an anti-TREM2 antibody or antigen-binding fragment disclosed herein, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment disclosed herein.

[0030] In yet another aspect, the disclosure provides a method for enhancing TREM2 activity in a subject with a neurodegenerative disease. In some embodiments, the method comprises administering to the subject an anti-TREM2 antibody or antigen-binding fragment disclosed herein, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment disclosed herein. [The present invention 1001] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, (a) a CDR-H1 sequence comprising the sequence of GFTFT-α6-FYMS (SEQ ID NO: 48), wherein α6 is D or N; (b) VIRN-β5-β6-N-β8-YT-β 11 -β 12 - A CDR-H2 sequence comprising the sequence of YNPSVKG (SEQ ID NO: 49), wherein β5 is K or R, β6 is A or P, β8 is G or A, and 11 is A or T, and β 12 is G or D, (c) a CDR-H3 sequence comprising the sequence γ1-RL-γ4-YGFDY (SEQ ID NO: 50), wherein γ1 is A or T and γ4 is T or S; (d) QSSKSLLHS-δ 10 - a CDR-L1 sequence comprising the sequence GKTYLN (SEQ ID NO: 51), 10 is N or T, (e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); and (f) a CDR-L3 sequence comprising the sequence QQFLE-φ6-PFT (SEQ ID NO: 52), wherein φ6 is Y or F; The antibody or antigen-binding fragment thereof. [The present invention 1002] 1001. The isolated antibody or antigen-binding fragment of the present invention, wherein said CDR-H1 sequence is selected from any one of SEQ ID NOs: 4 and 12. [The present invention 1003] 1003. The isolated antibody or antigen-binding fragment of claim 1001 or 1002, wherein said CDR-H2 sequence is selected from any one of SEQ ID NOs: 5, 13, and 25. [The present invention 1004] The isolated antibody or antigen-binding fragment of any of claims 1001 to 1003, wherein the CDR-H3 sequence is selected from any one of SEQ ID NOs: 6, 14, and 17. [The present invention 1005] The isolated antibody or antigen-binding fragment of any of claims 1001 to 1004, wherein the CDR-L1 sequence is selected from any one of SEQ ID NOs: 7 and 23. [The present invention 1006] The isolated antibody or antigen-binding fragment of any of claims 1001 to 1005, wherein the CDR-L3 sequence is selected from any one of SEQ ID NOs: 9 and 18. [The present invention 1007] (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9 The isolated antibody or antigen-binding fragment of any of claims 1001 to 1006. [The present invention 1008] V having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, and 79 H 8. The isolated antibody or antigen-binding fragment of any of claims 1001 to 1007, comprising the sequence: [The present invention 1009] The above V H 1008. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:15. [The present invention 1010] The above V H 1009. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 95% sequence identity to SEQ ID NO:15. [The present invention 1011] The above V H 10. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence comprises SEQ ID NO:15. [The present invention 1012] The above VH 1008. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:24. [The present invention 1013] The above V H 1012. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 95% sequence identity to SEQ ID NO:24. [The present invention 1014] The above V H 1013. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence comprises SEQ ID NO:24. [The present invention 1015] The above V H 1008. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:79. [The present invention 1016] The above V H 1015. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 95% sequence identity to SEQ ID NO:79. [The present invention 1017] The above V H 1016. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence comprises SEQ ID NO:79. [The present invention 1018] V having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 68 L 8. The isolated antibody or antigen-binding fragment of any of claims 1001 to 1017, comprising the sequence: [The present invention 1019] The above V L 1018. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:16. [The present invention 1020] The above V L 1020. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 95% sequence identity to SEQ ID NO:16. [The present invention 1021] The above V L1020. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence comprises SEQ ID NO:16. [The present invention 1022] The above V L 1018. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:22. [The present invention 1023] The above V L An isolated antibody or antigen-binding fragment of the present invention 1022, whose sequence has at least 95% sequence identity to SEQ ID NO:22. [The present invention 1024] The above V L 1023. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence comprises SEQ ID NO:22. [The present invention 1025] The above V L 1018. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:68. [The present invention 1026] The above V L An isolated antibody or antigen-binding fragment of the present invention 1025, wherein the sequence has at least 95% sequence identity to SEQ ID NO:68. [The present invention 1027] The above V L 1026. The isolated antibody or antigen-binding fragment of the present invention, wherein the sequence comprises SEQ ID NO:68. [The present invention 1028] (a) V comprising SEQ ID NO: 15 H V comprising the sequence SEQ ID NO:16 L Array, or (b) V comprising SEQ ID NO: 19 H V comprising sequence SEQ ID NO: 20 L Array, or (c) V comprising SEQ ID NO: 21 H V comprising sequence SEQ ID NO: 20 L Array, or (d) V comprising SEQ ID NO: 19 H V comprising sequence SEQ ID NO:22 L Array, or (e) V comprising SEQ ID NO: 79H V comprising sequence SEQ ID NO:22 L Array, or (f) V comprising SEQ ID NO: 24 H V comprising sequence SEQ ID NO: 20 L Array, or (g) V comprising SEQ ID NO: 26 H V comprising sequence SEQ ID NO: 20 L Array, or (h) V comprising SEQ ID NO: 24 H V comprising sequence SEQ ID NO:22 L Array, or (i) V comprising SEQ ID NO: 26 H V comprising sequence SEQ ID NO:22 L Array, or (j) V containing SEQ ID NO: 2 H V comprising the sequence SEQ ID NO:3 L Array, or (k) V containing SEQ ID NO: 10 H V comprising the sequence SEQ ID NO:11 L Array, or (l) V comprising SEQ ID NO: 24 H V comprising the sequence SEQ ID NO:68 L array The isolated antibody or antigen-binding fragment of any of claims 1001 to 1027. [The present invention 1029] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, (a) a CDR-H1 sequence comprising the sequence of GFSIEDFYIH (SEQ ID NO: 29); (b) a CDR-H2 sequence comprising the sequence WIDPE-β6-G-β8-SKYAPKFQG (SEQ ID NO: 47), wherein β6 is N or Q and β8 is D or E; (c) a CDR-H3 sequence comprising the sequence HADHGNYGSTMDY (SEQ ID NO: 31); (d) a CDR-L1 sequence comprising the sequence HASQHINVWLS (SEQ ID NO: 32); (e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID NO: 33); and (f) CDR-L3 sequence comprising the sequence QQGQTYPRT (SEQ ID NO: 34) The antibody or antigen-binding fragment thereof. [The present invention 1030] The isolated antibody or antigen-binding fragment of the present invention 1029, wherein the CDR-H2 sequence is selected from SEQ ID NOs: 30, 39, 41, and 43. [The present invention 1031] (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34 1029 or 1030. An isolated antibody or antigen-binding fragment of the present invention comprising: [The present invention 1032] V having at least 85% sequence identity to any one of SEQ ID NOs: 27, 35, 37, 38, 40, 42, 44, 45, and 46 H2. An isolated antibody or antigen-binding fragment of any of claims 1029 to 1031, comprising the sequence: [The present invention 1033] The above V H 1032. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:27. [The present invention 1034] The above V H An isolated antibody or antigen-binding fragment of the present invention 1033, whose sequence has at least 95% sequence identity to SEQ ID NO:27. [The present invention 1035] The above V H The isolated antibody or antigen-binding fragment of the present invention 1034, wherein the sequence comprises SEQ ID NO:27. [The present invention 1036] V having at least 85% sequence identity to SEQ ID NO: 28 or 36 L 6. An isolated antibody or antigen-binding fragment of any of claims 1029 to 1035, comprising the sequence: [The present invention 1037] The above V L 1036. An isolated antibody or antigen-binding fragment of the present invention, wherein the sequence has at least 90% sequence identity to SEQ ID NO:28. [The present invention 1038] The above V L The isolated antibody or antigen-binding fragment of the present invention 1037, wherein the sequence has at least 95% sequence identity to SEQ ID NO:28. [The present invention 1039] The above V L The isolated antibody or antigen-binding fragment of the present invention 1038, wherein the sequence comprises SEQ ID NO:28. [The present invention 1040] (a) V comprising SEQ ID NO: 27 H V comprising the sequence SEQ ID NO:28 L Array, or (b) V comprising SEQ ID NO: 35 H V comprising the sequence SEQ ID NO:36 L Array, or (c) V comprising SEQ ID NO: 37 H V comprising the sequence SEQ ID NO:36L Array, or (d) V comprising SEQ ID NO: 38 H V comprising the sequence SEQ ID NO:36 L Array, or (e) V comprising SEQ ID NO: 40 H V comprising the sequence SEQ ID NO:36 L Array, or (f) V comprising SEQ ID NO: 42 H V comprising the sequence SEQ ID NO:36 L Array, or (g) V comprising SEQ ID NO: 44 H V comprising the sequence SEQ ID NO:36 L Array, or (h) V comprising SEQ ID NO: 45 H V comprising the sequence SEQ ID NO:36 L Array, or (i) V comprising SEQ ID NO: 46 H V comprising the sequence SEQ ID NO:36 L array 1036. The isolated antibody or antigen-binding fragment of the present invention comprising: [The present invention 1041] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, (a) a CDR-H1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 4, 12, and 29; (b) a CDR-H2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 5, 13, 25, 30, 39, 41, and 43; (c) a CDR-H3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 6, 14, 17, and 31; (d) a CDR-L1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 7, 23, and 32; (e) a CDR-L2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 8 and 33; and (f) a CDR-L3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 9, 18, and 34; The antibody or antigen-binding fragment thereof. [The present invention 1042] (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (h) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (j) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (k) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9 1041. An isolated antibody or antigen-binding fragment of the present invention comprising: [The present invention 1043] 1041 or 1042, an isolated antibody or antigen-binding fragment of the invention, comprising a heavy chain variable region having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79. [The present invention 1044] The isolated antibody or antigen-binding fragment of any of claims 1041 to 1043, comprising a light chain variable region having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, 28, and 36. [The present invention 1045] (a) a V having at least 85% sequence identity to SEQ ID NO:2 H and V having at least 85% sequence identity to SEQ ID NO:3. L Array, or (b) a V having at least 85% sequence identity to SEQ ID NO: 10 H and V having at least 85% sequence identity to SEQ ID NO:11. L Array, or (c) a V having at least 85% sequence identity to SEQ ID NO: 15 H and V having at least 85% sequence identity to SEQ ID NO:16. L Array, or (d) a V having at least 85% sequence identity to SEQ ID NO: 19 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (e) a V having at least 85% sequence identity to SEQ ID NO: 21 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (f) a V having at least 85% sequence identity to SEQ ID NO: 19 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (g) a V having at least 85% sequence identity to SEQ ID NO: 79 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (h) a V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:20.L Array, or (i) a V having at least 85% sequence identity to SEQ ID NO: 26 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (j) V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (k) V having at least 85% sequence identity to SEQ ID NO: 26 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (l) a V having at least 85% sequence identity to SEQ ID NO: 27 H and V having at least 85% sequence identity to SEQ ID NO:28. L Array, or (m) a V having at least 85% sequence identity to SEQ ID NO: 35 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (n) a V having at least 85% sequence identity to SEQ ID NO: 37 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (o) a V having at least 85% sequence identity to SEQ ID NO: 38 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (p) a V having at least 85% sequence identity to SEQ ID NO: 40 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (q) a V having at least 85% sequence identity to SEQ ID NO: 42 H and V having at least 85% sequence identity to SEQ ID NO:36.L Array, or (r) V having at least 85% sequence identity to SEQ ID NO: 44 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (s) a V having at least 85% sequence identity to SEQ ID NO: 45 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (t) V having at least 85% sequence identity to SEQ ID NO: 46 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (u) V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:68. L array The isolated antibody or antigen-binding fragment of any of claims 1041 to 1044. [The present invention 1046] An isolated antibody or antigen-binding fragment thereof that specifically binds to human myeloid cell expressed triggering receptor 2 (TREM2), comprising: clone CL0020306, clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020188-9, clone CL0020188-10, clone CL0020188-11, clone CL0020188-12, clone CL0020188-13, clone CL0020188-14, clone CL0020188-15, clone CL0020188-16, clone CL0020188-17, clone CL0020188-18, clone CL0020188-19, clone CL0020188-20, clone CL0020188-21, clone CL0020188-22, clone CL0020188-23, clone CL0020188-24, clone CL0020188-25, clone CL0020188-26, clone CL0020188-27, clone CL0020188-28, clone CL0020188-29, clone CL0020188-30, clone CL0020188-31, clone CL0020188-32, clone CL0020188-33, clone CL0 The antibody or antigen-binding fragment thereof recognizes the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7, and clone CL0020123-8. [The present invention 1047] An isolated antibody or antigen-binding fragment of the present invention 1046, which recognizes the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7, and clone CL0020123-8. [The present invention 1048] In SEQ ID NO:1 (i) amino acid residues 55 to 63 (GEKGPCQRV (SEQ ID NO: 70)); (ii) amino acids 96 to 107 (TLRNLQPHDAGL (SEQ ID NO: 71)), and (iii) amino acid residues 126 to 129 (VEVL (SEQ ID NO: 72)) The isolated antibody or antigen-binding fragment of the present invention 1047, which recognizes one or more of: [The present invention 1049] An isolated antibody or antigen-binding fragment of the present invention 1046, which recognizes the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307, ​​and clone CL0020306. [The present invention 1050] 1049. An isolated antibody or antigen-binding fragment of the present invention which recognizes amino acid residues 143 to 149 in SEQ ID NO:1 (FPGESES (SEQ ID NO:69)). [The present invention 1051] An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, comprising the antibody of SEQ ID NO:1 (i) amino acid residues 55 to 63 (GEKGPCQRV (SEQ ID NO: 70)); (ii) amino acids 96 to 107 (TLRNLQPHDAGL (SEQ ID NO: 71)), and (iii) amino acid residues 126 to 129 (VEVL (SEQ ID NO: 72)) The antibody or antigen-binding fragment thereof, which recognizes an epitope comprising or consisting of one or more of the following: [The present invention 1052] An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope comprising or consisting of amino acid residues 143 to 149 in SEQ ID NO:1 (FPGESES (SEQ ID NO:69)). [The present invention 1053] An isolated antibody or antigen-binding fragment thereof according to any of claims 1001 to 1052, which reduces the level of soluble TREM2 protein (sTREM2). [The present invention 1054] An isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1053, which enhances TREM2 activity. [The present invention 1055] The isolated antibody or antigen-binding fragment thereof of the present invention 1054 which enhances phagocytosis or enhances the migration, differentiation, function, or survival of bone marrow cells, microglia, or macrophages. [The present invention 1056] An isolated antibody or antigen-binding fragment thereof of the present invention 1055 that enhances microglial function without increasing neuroinflammation. [The present invention 1057] The isolated antibody or antigen-binding fragment thereof of the present invention 1054, which enhances Syk phosphorylation. [The present invention 1058] The isolated antibody or antigen-binding fragment thereof of the present invention 1057, which enhances Syk phosphorylation in the presence of a TREM2 ligand. [The present invention 1059] An isolated antibody or an antigen-binding fragment thereof of any one of claims 1001 to 1058 of the present invention, which shows cross-reactivity with cynomolgus monkey TREM2 protein. [The present invention 1060] The isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1059, wherein the antibody is a monoclonal antibody. [The present invention 1061] The isolated antibody or antigen-binding fragment thereof of any of claims 1001 to 1059, wherein the antibody is a chimeric antibody. [The present invention 1062] The isolated antibody or antigen-binding fragment thereof of any of claims 1001 to 1059, wherein the antibody is a humanized antibody. [The present invention 1063] The isolated antibody or antigen-binding fragment thereof of any of claims 1001 to 1059 of the present invention, wherein the antibody is a fully human antibody. [The present invention 1064] The isolated antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1059 of the present invention, wherein the antigen-binding fragment is Fab, F(ab')2, scFv, or bivalent scFv. [The present invention 1065] An isolated antibody or antigen-binding fragment thereof according to any one of 1001 to 1064 of the present invention, A pharma- ceutically acceptable carrier and 13. A pharmaceutical composition comprising: [The present invention 1066] An antibody or antigen-binding fragment thereof that competes with any of the isolated antibodies of the present inventions 1001 to 1064 for binding to human TREM2 protein. [The present invention 1067] An isolated antibody or antigen-binding fragment thereof according to any one of the present inventions 1001 to 1064, or a pharmaceutical composition according to the present invention 1065; Its instruction manual and Including the kit. [The present invention 1068] A method for treating a neurodegenerative disease in a subject, the method comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any of claims 1001 to 1064 or a pharmaceutical composition of claim 1065. [The present invention 1069] The neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica (dementia pugilistica), and others. pugilistica, diffuse neurofibrillary tangle disease with calcifications, Down's syndrome, familial British dementia, familial Danish dementia, Gerstmann-Sträussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, PSP of the Guadeloupean type, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Parkinson's disease, Pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle only dementia. The method of the present invention 1068, wherein the patient is selected from the group consisting of dementia. [The present invention 1070] A method for reducing the level of sTREM2 in a subject having a neurodegenerative disease, the method comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1001 to 1064 or a pharmaceutical composition of claim 1065. [The present invention 1071] A method for enhancing TREM2 activity in a subject having a neurodegenerative disease, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1001 to 1064 of the present invention or a pharmaceutical composition of claim 1065 of the present invention. [Brief description of the drawings]

[0031] [Figure 1] Included are representative flow cytometry histograms depicting binding of exemplary anti-TREM2 antibodies to surface TREM2 on TREM2-expressing HEK cells. [Diagram 2] 1 includes representative dose-response curves of pSyk signaling activation by exemplary anti-TREM2 antibodies in primary human macrophage cells. Filled circles (●) represent anti-TREM2 antibodies and open circles (○) represent isotype controls. [Figure 3A] 1 includes a representative dose-response curve of pSyk signaling activation in human iPSC-microglial cells after 5 min pretreatment with an exemplary anti-TREM2 antibody followed by administration of lipid vesicles to assess liposome response in the cells. [Figure 3B] 1 includes a representative dose-response curve of pSyk signaling activation in human iPSC-microglial cells after 24-hour pretreatment with an exemplary anti-TREM2 antibody followed by administration of lipid vesicles to assess liposome response in the cells. [Figure 4] 1 includes representative dose-response curves of NFAT-luciferase reporter activity in Jurkat NFAT cells expressing human TREM2 / DAP12 in response to stimulation with exemplary anti-TREM2 antibodies. Filled circles (●) represent anti-TREM2 antibodies and open circles (○) represent isotype controls. [Diagram 5] 1 shows a representative dose-response curve of cell survival in human macrophage cells in response to treatment with an exemplary anti-TREM2 antibody. [Figure 6] 1 shows representative soluble TREM2 levels (sTREM2) as a function of anti-TREM2 antibody concentration for exemplary anti-TREM2 antibodies. [Figure 7]1 is a bar graph showing the mean pHrodo fluorescence intensity per cell in human macrophages treated with exemplary anti-TREM2 antibodies. [Figure 8A] Representative microscopic images of lipid accumulation in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody or isotype control. [Figure 8B] Representative bar graph of Nile Red staining (indicating lipid accumulation) of iPSC-microglia imaged in FIG. 8A. [Figure 8C] 1 includes a bar graph showing quantified levels of cholesteryl ester species in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody. [Figure 8D] 1 includes a bar graph showing quantified levels of triacylglyceride lipid species in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody. [Figure 8E] 1 includes a bar graph showing quantified levels of cholesteryl ester species in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody. Data is presented for iPSC-microglia in which a myelin washout step was included prior to incubation with an exemplary anti-TREM2 antibody. [Figure 8F] 1 includes a bar graph showing quantification levels of triacylglyceride lipid species in iPSC-microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody. Data is presented for iPSC-microglia in which a myelin washout step was included prior to incubation with an exemplary anti-TREM2 antibody. [Figure 9] 1 includes exemplary mouse plasma pharmacokinetic profiles of exemplary anti-TREM2 antibodies. [Figure 10A] 1 includes a bar graph showing the change in total soluble TREM2 (sTREM2) in mouse plasma for an exemplary anti-TREM2 antibody injected into TREM2 cDNA KI (huTrem2KI / KI) mice. [Figure 10B] 1 includes a bar graph showing the change in antibody-bound TREM2 in mouse plasma for an exemplary anti-TREM2 antibody injected into TREM2 cDNA KI (huTrem2KI / KI) mice. [Figure 11A] 1 includes dose-response binding curves of exemplary humanized, sequence-optimized anti-TREM2 antibodies to human TREM2 in HEK cells. [Figure 11B] 1 includes dose-response binding curves of exemplary humanized, sequence-optimized anti-TREM2 antibodies to human TREM2 in HEK cells. [Figure 12A] 1 includes dose-response curves of pSyk signaling activation in HEK293-H6 cells by exemplary humanized and sequence-optimized anti-TREM2 antibodies. [Figure 12B] 1 includes dose-response curves of pSyk signaling activation in HEK293-H6 cells by exemplary humanized and sequence-optimized anti-TREM2 antibodies. [Figure 13] 1 shows dose-response curves of cell survival in human macrophage cells in response to treatment with an exemplary humanized, sequence-optimized anti-TREM2 antibody. [Figure 14A] 1 includes a dose-response curve of lipid clearance in iPSC-microglia in response to treatment with an exemplary humanized, sequence-optimized anti-TREM2 antibody. [Figure 14B] 1 includes a dose-response curve of lipid clearance in iPSC-microglia in response to treatment with an exemplary humanized, sequence-optimized anti-TREM2 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description I. Introduction TREM2 is a transmembrane receptor expressed on the cell surface of microglia, dendritic cells, macrophages, and osteoclasts. Without wishing to be bound by a particular theory, it is believed that after ligand binding, TREM2 forms a signaling complex with the transmembrane adaptor protein DNAX-activating protein 12 (DAP12), which is then tyrosine 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 activities such as phagocytosis, cell proliferation and survival, inflammatory cytokine secretion, and migration of cells such as microglia and macrophages. TREM2 undergoes regulated intramembrane proteolysis, in which membrane-bound full-length TREM2 is cleaved by the metalloprotease ADAM10 into a sTREM2 portion that is shed from the cell and a membrane-retained C-terminal fragment that is further degraded by gamma secretase. Altered levels of sTREM2 have been reported in patients with Alzheimer's disease or frontotemporal dementia and with mutations in TREM2. Furthermore, mutations in TREM2 are associated with altered function, such as impaired phagocytosis and reduced microglial function.

[0033] As detailed in the Examples section below, antibodies have been generated that specifically bind to human TREM2 and modulate one or more downstream functions of the TREM2 / DAP12 signaling complex. Thus, in one aspect, the disclosure provides anti-TREM2 antibodies and antigen-binding fragments thereof. Thus, in one aspect, the disclosure provides anti-TREM2 antibodies and antigen-binding portions thereof.

[0034] In some embodiments, the anti-TREM2 antibody enhances TREM2 activity (e.g., enhances phagocytosis or enhances differentiation, function, migration, or survival of myeloid cells, microglia, or macrophages). Thus, in another aspect, a method is provided for enhancing TREM2 activity, for example in a subject with a neurodegenerative disease.

[0035] In some embodiments, the anti-TREM2 antibodies reduce sTREM2 shedding. Thus, in another aspect, methods are provided for decreasing levels of sTREM2, for example in a subject with a neurodegenerative disease.

[0036] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to an "antibody" optionally includes a combination of two or more such molecules, and the like.

[0037] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate reasonable deviations from that numerical value and values ​​known to one of ordinary skill in the art, for example, ±20%, ±10%, or ±5%, within the intended meaning of the stated value.

[0038] As used herein, the term "TREM2 protein" refers to myeloid cell-expressed triggering receptor 2 protein encoded by the gene TREM2. As used herein, "TREM2 protein" refers to native (i.e., wild-type) TREM2 protein of any vertebrate, including, but not limited to, human, non-human primate (e.g., cynomolgus monkey), rodent (e.g., mouse, rat), and other mammals. In some embodiments, the TREM2 protein is a human TREM2 protein having a sequence identified in UniprotKB Accession No. Q9NZC2 (SEQ ID NO: 1).

[0039] As used herein, the term "anti-TREM2 antibody" refers to an antibody that specifically binds to a TREM2 protein (eg, human TREM2).

[0040] As used herein, the term "antibody" refers to a protein having an immunoglobulin fold and specifically binds to an antigen through its variable region. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The term "antibody" as used herein also includes antibody fragments, including but not limited to Fab, F(ab')2, Fv, scFv, and bivalent scFv, that retain binding specificity through their variable regions. Antibodies may contain light chains classified as either kappa or lambda. Antibodies may contain heavy chains classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0041] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. A "variable light chain" (VL) and a "variable heavy chain" (VH) are H ) refer to each of these light and heavy chains.

[0042] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and confers to the antibody its antigen-binding specificity. Antibody variable regions usually contain four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

[0043] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that interrupt the four framework regions formed by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also usually identified by the chain in which a particular CDR is located. Thus, V H CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain in which it is found, whereas V L CDR1 or CDR-L1 is the CDR1 from the variable region of the antibody light chain in which it is found.

[0044] The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, function to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database for human and mouse sequences.

[0045] The amino acid sequences of the CDRs and framework regions can be determined using a variety of definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), AbM, and observed antigen contact ("Contact"). In some embodiments, the CDRs are determined according to the Contact definition. See MacCallum et al., J. Mol. Biol., 262:732-745 (1996). In some embodiments, the CDRs are determined by a combination of the Kabat, Chothia, and / or Contact CDR definitions.

[0046] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a TREM2 protein) via its variable regions. Examples of antigen-binding fragments include Fab fragments (VL, V H F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region), single-chain Fv (scFv), disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), V L (light chain variable region), and V H (heavy chain variable region), but are not limited to these.

[0047] The term "epitope" refers to the area or region of an antigen to which the CDR of an antibody specifically binds, and may include a few amino acids or a portion of a few amino acids, for example, 5 or 6 or more, for example, 20 or more amino acids, or a portion thereof. For example, when the target is a protein, the epitope may be composed of consecutive amino acids (e.g., linear epitope), or amino acids from different parts of the protein that are in close proximity due to protein folding (e.g., discontinuous or conformational epitope). In some embodiments, the epitope is phosphorylated at one amino acid (e.g., at a serine or threonine residue).

[0048] As used herein, the phrase "recognizes an epitope," when used in reference to an anti-TREM2 antibody, means that the antibody CDR interacts with or specifically binds to an antigen (i.e., a TREM2 protein) at that epitope or at a portion of the antigen that contains that epitope.

[0049] As used herein, the term "multispecific antibody" refers to an antibody that comprises two or more different antigen-binding portions, each of which comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two or more different antigens through its variable regions. As used herein, the term "bispecific antibody" refers to an antibody that comprises two different antigen-binding portions, each of which comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two different antigens through its variable regions.

[0050] "Monoclonal antibody" refers to an antibody produced by a single clone of cells or a single cell line and consisting of or consisting essentially of antibody molecules that are identical in their primary amino acid sequence.

[0051] A "polyclonal antibody" refers to an antibody obtained from a heterogeneous population of antibodies, where different antibodies in the population bind to different epitopes of an antigen.

[0052] "Chimeric antibody" refers to an antibody molecule in which the constant region or a portion thereof has been altered, replaced, or exchanged, such that the antigen binding site (i.e., variable region, CDR, or portion thereof) is linked to a constant region of a different or altered class, effector function, and / or species, or the variable region or a portion thereof has been altered, replaced, or exchanged by a variable region having a different or altered antigen specificity (e.g., CDR and framework regions from a different species). In some embodiments, a chimeric antibody is a monoclonal antibody that comprises a variable region from one source or species (e.g., mouse) and a constant region from another source or species (e.g., human). Methods for making chimeric antibodies have been described in the art.

[0053] A "humanized antibody" is a chimeric immunoglobulin derived from a non-human source (e.g., mouse) that contains minimal sequence derived from the non-human immunoglobulin outside the CDRs. Generally, a humanized antibody contains at least one (e.g., two) antigen-binding variable domain(s), in which the CDR regions substantially correspond to those of a non-human immunoglobulin and the framework regions substantially correspond to those of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin sequence. Methods of antibody humanization are known in the art.

[0054] A "human antibody" or "fully human antibody" is an antibody that has human heavy and light chain sequences, typically derived from human germline genes. In some embodiments, the antibody is produced by human cells, by non-human animals that utilize the human antibody repertoire (e.g., transgenic mice genetically engineered to express human antibody sequences), or by phage display platforms.

[0055] The term "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding portion thereof) that binds to an epitope or target in a sample with higher affinity, higher avidity, and / or longer duration than it binds to another epitope or non-target compound (e.g., a structurally distinct antigen). In some embodiments, an antibody (or antigen-binding portion thereof) that specifically binds to an epitope or target is an antibody (or antigen-binding portion thereof) that binds to an epitope or target with at least 5-fold higher affinity than other epitopes or non-target compounds, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, or 10,000-fold or more affinity. As used herein, the terms "specific binding to," "specifically binds to," or "is specific for" a particular epitope or target refer to, for example, binding to an epitope or target that is more specific than, for example, 10-fold higher affinity ... -4 M or less, e.g., 10 -5M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Equilibrium dissociation constant K of M D It will be appreciated by those of skill in the art that an antibody that specifically binds to a target (e.g., a TREM2 protein) from one species may also specifically bind to an ortholog of that target (e.g., a TREM2 protein).

[0056] The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules, e.g., an antibody (or antigen-binding portion thereof) and an antigen. Thus, for example, unless otherwise indicated or clear from the context, the term may refer to a 1:1 interaction between an antibody (or antigen-binding portion thereof) and an antigen. Binding affinity is measured by the equilibrium dissociation constant (K D ), which can be quantified by measuring the binding rate constant (k a ,time -1 M -1 ) divided by the dissociation rate constant (k d ,time -1 ) refers to K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) techniques (e.g., a Biacore™ system), equilibrium exclusion binding techniques such as KinExA®, and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, "binding affinity" refers not only to formal binding affinity, which reflects a 1:1 interaction between an antibody (or antigen-binding portion thereof) and an antigen, but also to K, which may reflect strong binding. D It also includes the apparent affinity from which values ​​are calculated.

[0057] The term "cross-react," as used herein, refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody was raised. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from a species other than the antigen against which the antibody was raised. As a non-limiting example, an anti-TREM2 antibody described herein raised against a human TREM2 peptide may exhibit cross-reactivity with a TREM2 peptide or protein from a different species (e.g., monkey or mouse).

[0058] The term "isolated," when used with respect to a nucleic acid or protein (e.g., an antibody), indicates that the nucleic acid or protein is essentially free from other cellular components with which it is associated in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated nucleic acid or protein (e.g., an antibody) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0059] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of natural α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. "Amino acid analog" refers to a chemical compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0060] The terms "polypeptide" and "peptide" are used interchangeably herein and refer to a polymer of amino acid residues in a single chain. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to natural and non-natural amino acid polymers. The amino acid polymers may contain all L-amino acids, all D-amino acids, or a mixture of L- and D-amino acids.

[0061] As used herein, the term "protein" refers to either one polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of a single chain polypeptide. The single chain polypeptides of a protein can be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

[0062] The terms "polynucleotide" and "nucleic acid" refer interchangeably to a chain of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0063] The terms "conservative substitution" and "conservative mutation" refer to a change that results in the replacement of one amino acid with another amino acid that can be classified as having similar characteristics. Examples of classifications of conservative amino acid groups defined in this way include the "charged / polar group" which includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), the "aromatic group" which includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), and the "aliphatic group" which includes Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His, a "negatively charged subgroup" consisting of Glu and Asp, and a "polar subgroup" consisting of Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp, and a "phenyl subgroup" consisting of Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups such as an "aliphatic non-polar subgroup" consisting of Val, Leu, Gly, and Ala, and an "aliphatic slightly polar subgroup" consisting of Met, Ser, Thr, and Cys.Examples of conservative mutation classes include amino acid substitutions of amino acids within the aforementioned subgroups, such as, but not limited to, Lys for Arg or vice versa, which can maintain a positive charge; Glu for Asp or vice versa, which can maintain a negative charge; Ser for Thr or vice versa, which can maintain a free -OH; and Gln for Asn or vice versa, which can maintain a free -NH2. In some embodiments, a hydrophobic amino acid is substituted for a natural hydrophobic amino acid, for example in the active site, to maintain hydrophobicity.

[0064] The term "identical" or percent "identity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that have the same or a certain percentage of amino acid residues that are identical over a particular region, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0065] In the sequence comparison of polypeptides, one amino acid sequence usually serves as a reference sequence and is compared with the candidate sequence. To obtain maximum alignment, alignment can be performed using various methods available to those skilled in the art, such as visual alignment or publicly available software using known algorithms. Such programs include BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for alignment to obtain maximum alignment can be determined by those skilled in the art. For the purpose of this application, the sequence comparison of polypeptide sequences uses the standard protein BLAST of the BLASTP algorithm to align two protein sequences using default parameters.

[0066] The terms "subject," "individual," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the subject, individual, or patient is a human.

[0067] The terms "treat", "treatment" and the like as used herein generally mean to obtain a desired pharmacological and / or physiological effect. "Treat" or "treatment" may refer to any indication of success in treating or ameliorating a neurodegenerative disease (e.g., Alzheimer's disease or another neurodegenerative disease described herein), including any objective and subjective parameter, such as palliative, remission, improvement in the survival of the patient, increase in the duration or rate of survival, reducing symptoms or making the disease more tolerable to the patient, slowing the rate of degeneration or decline, or improving the physical or mental well-being of the patient. The treatment or amelioration of symptoms may be based on objective or subjective parameters. The effect of treatment may be compared to an individual or pool of individuals not receiving treatment, or to the same patient at different time points before or during treatment.

[0068] The term "pharmaceutical acceptable excipient" refers to non-active pharmaceutical ingredients that are biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.

[0069] As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent (e.g., an antibody described herein) is an amount of an agent that treats, relieves, alleviates, or reduces the severity of a disease symptom in a subject. A "therapeutic amount" of an agent (e.g., an antibody described herein) can improve a patient's survival, increase the length or rate of survival, reduce symptoms, make an injury, disease, or condition (e.g., a neurodegenerative disease) more tolerable, slow the rate of degeneration or decline, or improve the physical or mental well-being of a patient.

[0070] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired site of biological action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the antibodies described herein are administered intravenously.

[0071] The term "control" or "control value" refers to a reference value or baseline value. A suitable control can be determined by one skilled in the art. In some cases, the control value can be determined against a baseline within the same subject or experiment, for example, a measurement of sTREM2 taken before treatment with an anti-TREM2 antibody can be a control value for a post-treatment measurement of sTREM2 levels in the same subject. In other cases, the control value can be determined against a control subject (e.g., a healthy control or a disease control) or against a mean value in a population of control subjects (e.g., a population of healthy controls or disease controls, for example, 10, 20, 50, 100, 200, 500, 1000 or more control subjects), for example, a measurement of sTREM2 levels of either subject at baseline or after treatment can be compared to a value of a healthy control.

[0072] III. Anti-TREM2 antibody In one aspect, antibodies and antigen-binding fragments thereof that specifically bind to TREM2 protein are provided. In some embodiments, the antibodies specifically bind to human TREM2 protein. In some embodiments, the anti-TREM2 antibodies are selective for TREM2 over other TREM-like receptors (e.g., TREM1).

[0073] In some embodiments, the anti-TREM2 antibody is an antibody that comprises one or more complementarity determining region (CDR) sequences, heavy chain variable region sequences, and / or light chain variable region sequences disclosed herein. In some embodiments, the anti-TREM2 antibody comprises one or more CDR sequences, heavy chain variable region sequences, and / or light chain variable region sequences disclosed herein and further comprises one or more functional properties disclosed herein, such as an antibody that enhances TREM2 activity (e.g., enhances phagocytosis, or enhances migration, differentiation, function, or survival of cells such as myeloid cells, microglia, or macrophages), or an antibody that decreases levels of sTREM2.

[0074] Anti-TREM2 antibody sequence In some embodiments, the anti-TREM2 or antigen-binding fragment thereof comprises a heavy chain sequence or a portion thereof, and / or a light chain sequence or a portion thereof, from any of the following anti-TREM2 antibodies described herein: clone CL0020306, clone CL0020188, clone CL0020307, ​​and clone CL0020123. The amino acid sequences of the CDRs, heavy chain variable regions, and light chain variable regions of these clones are set forth in the Abbreviated Sequence Listing. In some embodiments, the anti-TREM2 antibody is a chimeric antibody. In some embodiments, the anti-TREM2 antibody is a humanized and / or affinity matured antibody.

[0075] In some embodiments, the anti-TREM2 antibody (a) a heavy chain CDR1 (CDR-H1) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 4, 12, and 29, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs: 4, 12, and 29; (b) a heavy chain CDR2 (CDR-H2) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 5, 13, 25, 30, 39, 41, and 43, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs: 5, 13, 25, 30, 39, 41, and 43; (c) a heavy chain CDR3 (CDR-H3) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 6, 14, 17, and 31, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs: 6, 14, 17, and 31; (d) a light chain CDR1 (CDR-L1) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 7, 23, and 32, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs: 7, 23, and 32; (e) a light chain CDR2 (CDR-L2) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8 and 33, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs: 8 and 33; and (f) a light chain CDR3 (CDR-L3) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 9, 18, and 34, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs: 9, 18, and 34. The nucleic acid sequence comprises one or more CDRs selected from the group consisting of:

[0076] In some embodiments, the anti-TREM2 antibody comprises two, three, four, five or all six of (a)-(f). In some embodiments, the anti-TREM2 antibody comprises CDR-H1 of (a), CDR-H2 of (b), and CDR-H3 of (c). In some embodiments, the anti-TREM2 antibody comprises CDR-L1 of (d), CDR-L2 of (e), and CDR-L3 of (f). In some embodiments, the CDR with up to two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, the CDR with up to two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

[0077] In some embodiments, the anti-TREM2 antibody (a) a CDR-H1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 4, 12, and 29; (b) a CDR-H2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 5, 13, 25, 30, 39, 41, and 43; (c) a CDR-H3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 6, 14, 17, and 31; (d) a CDR-L1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 7, 23, and 32; (e) a CDR-L2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 8 and 33; and (f) a CDR-L3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 9, 18, and 34; The nucleic acid sequence comprises one or more CDRs selected from the group consisting of:

[0078] In some embodiments, the anti-TREM2 antibody comprises two, three, four, five or all six of (a)-(f). In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 of (a), a CDR-H2 of (b), and a CDR-H3 of (c). In some embodiments, the anti-TREM2 antibody comprises a CDR-L1 of (d), a CDR-L2 of (e), and a CDR-L3 of (f).

[0079] In some embodiments, the anti-TREM2 antibody (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (h) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34, or (j) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34 Includes.

[0080] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79. In some embodiments, the anti-TREM2 comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79.

[0081] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, 28, 36, and 68. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 16, 20, 22, 28, 36, and 68.

[0082] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79, and a light chain variable region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, 28, 36, and 68. In some embodiments, the anti-TREM2 comprises a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79, and a light chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 3, 11, 16, 20, 22, 28, 36, and 68.

[0083] In some embodiments, the anti-TREM2 antibody (a) a V having at least 85% sequence identity to SEQ ID NO:2 H and V having at least 85% sequence identity to SEQ ID NO:3. L Array, or (b) a V having at least 85% sequence identity to SEQ ID NO: 10 H and V having at least 85% sequence identity to SEQ ID NO:11. L Array, or (c) a V having at least 85% sequence identity to SEQ ID NO: 15 H and V having at least 85% sequence identity to SEQ ID NO:16. L Array, or (d) a V having at least 85% sequence identity to SEQ ID NO: 19 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (e) a V having at least 85% sequence identity to SEQ ID NO: 21H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (f) a V having at least 85% sequence identity to SEQ ID NO: 19 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (g) a V having at least 85% sequence identity to SEQ ID NO: 79 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (h) a V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (i) a V having at least 85% sequence identity to SEQ ID NO: 26 H and V having at least 85% sequence identity to SEQ ID NO:20. L Array, or (j) V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (k) V having at least 85% sequence identity to SEQ ID NO: 26 H and V having at least 85% sequence identity to SEQ ID NO:22. L Array, or (l) a V having at least 85% sequence identity to SEQ ID NO: 27 H and V having at least 85% sequence identity to SEQ ID NO:28. L Array, or (m) a V having at least 85% sequence identity to SEQ ID NO: 35 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (n) a V having at least 85% sequence identity to SEQ ID NO: 37H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (o) a V having at least 85% sequence identity to SEQ ID NO: 38 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (p) a V having at least 85% sequence identity to SEQ ID NO: 40 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (q) a V having at least 85% sequence identity to SEQ ID NO: 42 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (r) V having at least 85% sequence identity to SEQ ID NO: 44 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (s) a V having at least 85% sequence identity to SEQ ID NO: 45 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (t) V having at least 85% sequence identity to SEQ ID NO: 46 H and V having at least 85% sequence identity to SEQ ID NO:36. L Array, or (u) V having at least 85% sequence identity to SEQ ID NO: 24 H and V having at least 85% sequence identity to SEQ ID NO:68. L array Includes.

[0084] In some embodiments, the anti-TREM2 antibody comprises one or more sequences encompassed by the consensus sequences disclosed herein. As a non-limiting example, a consensus sequence can be identified by aligning the heavy or light chain sequences (e.g., CDRs) to antibodies derived from the same (or similar) germline. In some embodiments, a consensus sequence can be generated from antibodies containing sequences of the same (or similar) length and / or sequences with at least one very similar CDR (e.g., a very similar CDR3). In some embodiments, such sequences in the antibodies can be aligned and compared to identify conserved amino acids or motifs (i.e., where changes in the sequence may alter protein function) and / or regions where mutations exist in the sequence (i.e., where mutations in the sequence are unlikely to significantly affect protein function). Alternatively, consensus sequences may be identified by aligning the heavy or light chain sequences (e.g., CDRs) against antibodies that bind the same or similar (e.g., overlapping) epitopes to determine conserved amino acids or motifs (i.e., where changes in the sequence may alter protein function) and regions where mutations are present in the sequence alignment (i.e., where mutations in the sequence are unlikely to significantly affect protein function). In some embodiments, one or more consensus sequences may be identified for antibodies that recognize the same or similar epitopes as the anti-TREM2 antibodies disclosed herein. Exemplary consensus sequences include SEQ ID NOs: 47-52. In the consensus sequences of SEQ ID NOs: 47-52, capital letters represent amino acid residues that are completely conserved between the aligned sequences (e.g., aligned CDR sequences), while "X" or Greek letters (e.g., "α", "β", "γ", "δ", "ε", or "φ") represent amino acid residues that are not completely conserved between the aligned sequences. It will be understood that when selecting an amino acid for insertion at a position marked by an "X" or Greek letter, in some embodiments the amino acid will be selected from those found at the corresponding position in the aligned sequences.

[0085] Clone CL0020123 and variants of CL0020123 In some embodiments, the anti-TREM2 antibody or antigen-binding fragment thereof (a) a CDR-H1 sequence comprising the sequence of GFSIEDFYIH (SEQ ID NO: 29); (b) a CDR-H2 sequence comprising the sequence WIDPE-β6-G-β8-SKYAPKFQG (SEQ ID NO: 47), wherein β6 is N or Q and β8 is D or E; (c) a CDR-H3 sequence comprising the sequence HADHGNYGSTMDY (SEQ ID NO: 31); (d) a CDR-L1 sequence comprising the sequence HASQHINVWLS (SEQ ID NO: 32); (e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID NO: 33); and (f) CDR-L3 sequence comprising the sequence QQGQTYPRT (SEQ ID NO: 34) Includes.

[0086] In some embodiments, the anti-TREM2 antibody comprises a CDR-H2 sequence selected from SEQ ID NOs: 30, 39, 41, and 43.

[0087] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs: 27, 35, 37, 38, 40, 42, 44, 45, and 46. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 27, 35, 37, 38, 40, 42, 44, 45, and 46.

[0088] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs: 28 and 36. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 28 and 36.

[0089] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:30, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:34.

[0090] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 27. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27.

[0091] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 28. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:28.

[0092] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 27, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 28. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:28.

[0093] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:29, 30, and 31, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:27. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:32, 33, and 34, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:28.

[0094] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28).

[0095] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:43, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:34.

[0096] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 42. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42.

[0097] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:36.

[0098] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:36.

[0099] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 29, 43, and 31, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 42. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36.

[0100] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 29, 43, 31, 32, 33, and 34, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36).

[0101] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:41, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:34.

[0102] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 45. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45.

[0103] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:36.

[0104] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 45, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:36.

[0105] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 29, 41, and 31, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 45. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 36.

[0106] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 29, 41, 31, 32, 33, and 34, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36).

[0107] Clones CL0020188, CL0020306, CL0020307, ​​and variants of CL0020188 In some embodiments, the anti-TREM2 antibody or antigen-binding fragment thereof (a) a CDR-H1 sequence comprising the sequence of GFTFT-α6-FYMS (SEQ ID NO: 48), wherein α6 is D or N; (b) VIRN-β5-β6-N-β8-YT-β 11 -β 12 - A CDR-H2 sequence comprising the sequence of YNPSVKG (SEQ ID NO: 49), wherein β5 is K or R, β6 is A or P, β8 is G or A, and 11 is A or T, and β 12 is G or D; (c) a CDR-H3 sequence comprising the sequence γ1-RL-γ4-YGFDY (SEQ ID NO: 50), wherein γ1 is A or T and γ4 is T or S; (d) QSSKSLLHS-δ 10 - a CDR-L1 sequence comprising the sequence GKTYLN (SEQ ID NO: 51), 10 is N or T, (e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); and (f) a CDR-L3 sequence comprising the sequence QQFLE-φ6-PFT (SEQ ID NO: 52), wherein φ6 is Y or F; Includes.

[0108] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence selected from SEQ ID NOs: 4 and 12. In some embodiments, the anti-TREM2 antibody comprises a CDR-H2 sequence selected from SEQ ID NOs: 5, 13, and 25. In some embodiments, the anti-TREM2 antibody comprises a CDR-H3 sequence selected from SEQ ID NOs: 6, 14, and 17. In some embodiments, the anti-TREM2 antibody comprises a CDR-L1 sequence selected from SEQ ID NOs: 7 and 23. In some embodiments, the anti-TREM2 antibody comprises a CDR-L3 sequence selected from SEQ ID NOs: 9 and 18.

[0109] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, and 79. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, and 79.

[0110] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 68. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 68.

[0111] Clone CL0020188 and variants of CL0020188 In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:18.

[0112] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15.

[0113] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0114] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0115] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 18, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16.

[0116] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 5, 17, 7, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16).

[0117] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:18.

[0118] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 79. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79.

[0119] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.

[0120] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 79, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.

[0121] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 79. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 23, 8, and 18, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22.

[0122] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, 17, 23, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22).

[0123] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:18.

[0124] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24.

[0125] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.

[0126] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.

[0127] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 25, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 23, 8, and 18, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 22.

[0128] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 25, 17, 23, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22).

[0129] In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:9.

[0130] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24.

[0131] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 68. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 68.

[0132] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 68. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 68.

[0133] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 25, and 17, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 24. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO: 68.

[0134] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 25, 17, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 68).

[0135] Clone CL0020306 In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:6, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:9.

[0136] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 2. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0137] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 3. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3.

[0138] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:3.

[0139] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 9, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.

[0140] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3).

[0141] Clone CL0020307 In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 9.

[0142] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10.

[0143] In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:11.

[0144] In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11. In some embodiments, an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

[0145] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and having at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11.

[0146] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 12, 13, 14, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11).

[0147] Binding characteristics of anti-TREM2 antibodies In some embodiments, antibodies described herein that specifically bind to a TREM2 protein bind to TREM2 expressed on cells (e.g., primary cells or cell lines that endogenously express TREM2, such as human macrophages, or primary cells or cell lines that have been engineered to express TREM2, e.g., as described in the Examples section below). In some embodiments, antibodies that specifically bind to a TREM2 protein described herein bind to purified or recombinant TREM2 protein or a portion thereof, or bind to a chimeric protein comprising TREM2 or a portion thereof (e.g., an Fc fusion protein comprising TREM2 or an Fc fusion protein comprising the ectodomain of TREM2).

[0148] In some embodiments, an antibody that specifically binds to the human TREM2 protein exhibits cross-reactivity with one or more other TREM2 proteins from other species. In some embodiments, an antibody that specifically binds to the human TREM2 protein exhibits cross-reactivity with cynomolgus monkey ("cyno") TREM2 protein. In some embodiments, an antibody that specifically binds to the human TREM2 protein exhibits cross-reactivity with mouse TREM2 protein. In some embodiments, an anti-TREM2 antibody exhibits cross-reactivity with human TREM2, cyno TREM2, and mouse TREM2.

[0149] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), equilibrium exclusion binding (e.g., KinExA®), flow cytometry, fluorescence activated cell sorting (FACS), BioLayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, equilibrium exclusion binding is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0150] Epitopes recognized by anti-TREM2 antibodies In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that is the same or substantially the same as the epitope recognized by the antibody clone described herein. As used herein, the term "substantially the same," when used in reference to an epitope recognized by an antibody clone described herein, means that the anti-TREM2 antibody recognizes an epitope that is identical to, within, or nearly identical to (e.g., has at least 90% sequence identity to, or has one, two or three amino acid substitutions, e.g., conservative substitutions to) the epitope recognized by the antibody clone described herein, or that substantially overlaps (e.g., overlaps at least 50%, 60%, 70%, 80%, 90%, or 95% with) the epitope.

[0151] In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that is the same or substantially the same as an epitope recognized by an antibody clone selected from the group consisting of clone CL0020306, clone CL0020188, clone CL0020307, ​​and clone CL0020123.

[0152] In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope within the stalk region of TREM2. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that includes, is within, or consists of residues 129-172 or residues 131-169 of SEQ ID NO:1. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that includes, is within, or consists of residues 129-148 of SEQ ID NO:1. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that includes, is within, or consists of amino acid residues 143-149 of SEQ ID NO:1. In some embodiments, the anti-TREM2 antibody is an agonist that activates TREM2 / DAP12 signaling (e.g., by inducing phosphorylation of a kinase such as Syk) and binds to human TREM2 at an epitope within the stalk region of TREM2. In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope within the stalk region of TREM2 and inhibits cleavage of TREM2 by proteases (eg, ADAM17).

[0153] In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope within the Ig variable (IgV) domain of TREM2. In some embodiments, the anti-TREM2 antibody is an agonist that activates TREM2 / DAP12 signaling (e.g., by inducing phosphorylation of a kinase such as Syk) and binds to human TREM2 at an epitope within the IgV domain of TREM2. In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope that includes or consists of one or more of the following: (i) amino acid residues 55-63 of SEQ ID NO:1 (GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107 of SEQ ID NO:1 (TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residues 126-129 of SEQ ID NO:1 (VEVL (SEQ ID NO:72)).

[0154] Functional characterization of anti-TREM2 antibodies In some embodiments, an anti-TREM2 antibody (e.g., an antibody having one or more of the disclosed CDR sequences, heavy chain variable region sequences, and / or light chain variable region sequences) functions in one or more TREM2 activities disclosed herein. For example, in some embodiments, an anti-TREM2 antibody is an antibody that modulates the level of sTREM2 protein (e.g., the level of sTREM2 shed from the cell surface to an extracellular sample), modulates the recruitment or phosphorylation of a kinase (e.g., Syk kinase) that interacts with the TREM2 / DAP12 signaling complex, and / or modulates one or more activities downstream of the signaling complex, such as phagocytosis, cell proliferation, cell survival, cell differentiation, cytokine secretion, or cell migration. In some embodiments, an anti-TREM2 antibody disclosed herein binds to soluble TREM2 protein (sTREM2) in healthy human CSF or cynomolgus monkey CSF with better potency than a reference antibody. In some embodiments, the reference antibody is represented by a combination of sequences selected from the group consisting of SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, and SEQ ID NOs: 77 and 78. In some embodiments, the potency assay is performed substantially as described in Example 11.

[0155] In some embodiments, the anti-TREM2 antibody enhances one or more TREM2 activities (e.g., those described herein) induced by a ligand. In some embodiments, the ligand is a lipid ligand. Examples of TREM2 lipid ligands include 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), 2-arachidonoylglycerol (2-AG), 7-ketocholesterol (7-KC), 24(S)hydroxycholesterol (24OHC), 25(S)hydroxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), acylcarnitine (AC), alkylacylglycerophosphocholine (PAF), α-galactosyltransferase (TAT), α-galactosyltransferase (AL ... Ceramide (KRN7000), bis(monoacylglycero)phosphate (BMP), cardiolipin (CL), ceramide, ceramide-1-phosphate (C1P), cholesteryl ester (CE), cholesterol phosphate (CP), diacylglycerol 34:1 (DG34:1), diacylglycerol 38:4 (DG38:4), diacylglycerol pyrophosphate (DGPP), dihydroceramide (DhCer), dihydrosphingomyelin (DhSM), ether phosphatidylcholine (PCe), and phospholipids. Free cholesterol (FC), galactosylceramide (GalCer), galactosylsphingosine (GalSo), ganglioside GM1, ganglioside GM3, glucosylsphingosine (GlcSo), Hanks' balanced salt solution (HBSS), Kdo2-lipid A (KLA), lactosylceramide (LacCer), lysoalkylacylglycerophosphocholine (LPAF), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPAF) E), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysosphingomyelin (LSM), lysophosphatidylserine (LPS), N-acyl-phosphatidylethanolamine (NAPE), N-acyl-serine (NSer), oxidized phosphatidylcholine (oxPC), palmitic acid-9-hydroxy-stearic acid (PAHSA), phosphatidylethanolamine (PE), phosphatidylethanol (PEtOH), phosphatidic acid (PA),These include, but are not limited to, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphinganine, sphinganine-1-phosphate (Sa1P), sphingomyelin (SM), sphingosine, sphingosine-1-phosphate (So1P), and sulfatide.

[0156] Regulation of sTREM2 shedding In some embodiments, the anti-TREM2 antibody alters the level of sTREM2 protein in a sample, e.g., the level of sTREM2 shed from the cell surface to an extracellular sample, hi some embodiments, the anti-TREM2 antibody reduces the level of sTREM2.

[0157] In some embodiments, the anti-TREM2 antibody reduces the level of sTREM2 if the amount of sTREM2 in the treated sample is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more compared to the control value. In some embodiments, the anti-TREM2 antibody reduces the level of sTREM2 if the amount of sTREM2 in the treated sample is reduced by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more compared to the control value. In some embodiments, the control value is the amount of sTREM2 in an untreated sample (e.g., supernatant from TREM2-expressing cells not treated with an anti-TREM2 antibody, or a sample from a subject not treated with an anti-TREM2 antibody) or a sample treated with a suitable non-TREM2 binding antibody.

[0158] In some embodiments, sTREM2 shedding is measured using a sample comprising a bodily fluid, e.g., blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises cerebrospinal fluid. In some embodiments, the sample comprises a supernatant from a cell culture (e.g., from a primary cell or cell line that endogenously expresses TREM2, such as human macrophages, or from a primary cell or cell line that has been engineered to express TREM2, e.g., as described in the Examples section below).

[0159] In some embodiments, the level of sTREM2 in the sample is measured using an immunoassay. Immunoassays are known in the art and include, but are not limited to, enzyme immunoassays (EIAs), such as enzyme multiplexed immunoassays (EMIAs), enzyme-linked immunosorbent assays (ELISAs), microparticle enzyme immunoassays (MEIAs), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoretic immunoassays (CEIAs), radioimmunoassays (RIAs), immunofluorescence, chemiluminescence immunoassays (CLs), and electrochemiluminescence immunoassays (ECLs). In some embodiments, the level of sTREM2 is measured using an ELISA assay. In some embodiments, the level of sTREM2 is measured using an ELISA assay, as described in the Examples section below.

[0160] Regulation of kinase recruitment or phosphorylation In some embodiments, the anti-TREM2 antibody induces phosphorylation of a kinase that interacts with the TREM2 / DAP12 signaling complex (such as, but not limited to, Syk, ZAP70, PI3K, Erk, AKT, or GSK3b). In some embodiments, the anti-TREM2 antibody induces phosphorylation of a kinase that interacts with the TREM2 / DAP12 signaling complex without blocking binding of a natural TREM2 ligand. In some embodiments, the anti-TREM2 antibody enhances phosphorylation of a kinase that interacts with the TREM2 / DAP12 signaling complex induced by a TREM2 ligand (e.g., a lipid ligand). In some embodiments, the anti-TREM2 antibody induces or enhances phosphorylation of Syk. In some embodiments, an anti-TREM2 antibody induces or enhances phosphorylation of Syk if the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more compared to a control value. In some embodiments, an anti-TREM2 antibody induces phosphorylation of Syk if the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more compared to a control value. In some embodiments, the control value is the level of Syk phosphorylation in an untreated sample (e.g., a sample comprising TREM2-expressing cells that have not been treated with an anti-TREM2 antibody, or a sample from a subject that has not been treated with an anti-TREM2 antibody), or a sample that has been treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample that has been treated with a suitable non-TREM2 binding antibody.

[0161] In some embodiments, an immunoassay is used to detect and / or quantify phosphorylation (e.g., Syk phosphorylation) in a sample. In some embodiments, the immunoassay is an enzyme immunoassay (EIA), an enzyme-linked immunosorbent assay (EMIA), an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA), an immunohistochemistry (IHC), an immunocytochemistry, a capillary electrophoretic immunoassay (CEIA), a radioimmunoassay (RIA), an immunofluorescence assay, a chemiluminescent immunoassay (CL), or an electrochemiluminescent immunoassay (ECL). In some embodiments, phosphorylation is detected and / or quantified using an immunoassay that utilizes an amplified luminescent proximity homogenous assay (AlphaLISA®, PerkinElmer Inc.).

[0162] In some embodiments, phosphorylation is measured using a sample comprising one or more cells, e.g., one or more TREM2-expressing cells (e.g., primary cells or cell lines that endogenously express TREM2, such as human macrophages or iPSC-derived microglia, or primary cells or cell lines that have been engineered to express TREM2, e.g., as described in the Examples section below). In some embodiments, the sample comprises a bodily fluid, e.g., blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises a tissue (e.g., lung, brain, kidney, spleen, neural tissue, or skeletal muscle) or cells from such tissue. In some embodiments, the sample comprises an endogenous bodily fluid, tissue, or cell (e.g., from a human subject or a non-human subject).

[0163] Regulation of phagocytosis In some embodiments, the anti-TREM2 antibody enhances phagocytosis of dead cell debris, tissue debris, amyloid beta particles, or foreign material. In some embodiments, the anti-TREM2 antibody enhances phagocytosis without blocking the binding of the natural TREM2 ligand. In some embodiments, the anti-TREM2 antibody enhances phagocytosis induced by a TREM2 ligand (e.g., a lipid ligand). In some embodiments, the anti-TREM2 antibody enhances phagocytosis if the level of phagocytosis in a sample treated with the anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more compared to a control value. In some embodiments, the anti-TREM2 antibody enhances phagocytosis if the level of phagocytosis in a sample treated with the anti-TREM2 antibody is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more compared to a control value. In some embodiments, the control value is the level of phagocytosis in an untreated sample, a sample treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with a suitable non-TREM2 binding antibody.

[0164] In some embodiments, phagocytosis is measured using a phagocytosis assay with a labeled substrate. Phagocytosis assays are known in the art. In some embodiments, phagocytosis assays are performed on samples that contain cells that endogenously express TREM2, such as human macrophages or microglia. In some embodiments, phagocytosis assays are performed on samples that contain cells that are engineered to express TREM2. In some embodiments, phagocytosis is measured using a human macrophage phagocytosis assay, as described in the Examples section below.

[0165] Regulation of cell differentiation, function, migration, and survival In some embodiments, the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation (e.g., of myeloid cells, macrophages, and microglia, including iPSC-derived microglia and disease-associated microglia). Disease-associated microglia and methods for detecting disease-associated microglia are described in Keren-Shaul et al., Cell, 2017, 169:1276-1290. In some embodiments, the anti-TREM2 antibody enhances cell migration of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, the anti-TREM2 antibody enhances cell survival of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, the anti-TREM2 antibody enhances cell function of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, the anti-TREM2 antibody enhances cell differentiation of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, the anti-TREM2 antibody enhances myeloid cell migration, survival, function, and / or differentiation. In some embodiments, the anti-TREM2 antibody enhances macrophage migration, survival, function, and / or differentiation. In some embodiments, the anti-TREM2 antibody enhances microglial migration, survival, function, and / or differentiation. In some embodiments, the anti-TREM2 antibody enhances microglial activation. In some embodiments, the anti-TREM2 antibody enhances disease-associated microglial migration, survival, function, and / or differentiation. In some embodiments, the anti-TREM2 antibody enhances cell migration, survival, function, or differentiation without blocking binding of the natural TREM2 ligand. In some embodiments, the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation induced by a TREM2 ligand (eg, a lipid ligand).

[0166] In some embodiments, an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of an activity (e.g., migration, survival, function, or differentiation) in a sample treated with the anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more compared to a control value. In some embodiments, an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of an activity (e.g., migration, survival, function, or differentiation) in a sample treated with the anti-TREM2 antibody is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more compared to a control value. In some embodiments, the control value is the level of activity (e.g., migration, survival, function, or differentiation) in an untreated sample (e.g., a sample not treated with an anti-TREM2 antibody), a sample treated with a TREM2 ligand but not with an anti-TREM2 antibody, or a sample treated with a suitable non-TREM2 binding antibody.

[0167] In some embodiments, cell migration is measured using a chemotaxis assay. Chemotaxis assays are known in the art. In some embodiments, cell migration assays (e.g., chemotaxis assays) are performed on samples that contain cells that endogenously express TREM2, such as human macrophages. In some embodiments, cell migration assays (e.g., chemotaxis assays) are performed on samples that contain cells that are engineered to express TREM2. In some embodiments, cell migration is measured using a human macrophage chemotaxis assay, as described in the Examples section below.

[0168] In some embodiments, cell survival is measured using a cell viability assay. Cell viability assays are known in the art. In some embodiments, the cell survival assay (e.g., cell viability assay) is performed on a sample containing cells that endogenously express TREM2, such as human macrophages. In some embodiments, the cell survival assay (e.g., cell viability assay) is performed on a sample containing cells that are engineered to express TREM2. In some embodiments, cell survival is measured using a human macrophage viability assay, as described in the Examples section below.

[0169] In some embodiments, cell function is measured using a functional assay appropriate for the cell, for example, in some embodiments, macrophage cell function is assessed using a phagocytosis assay, for example, as described in the Examples section below.

[0170] In some embodiments, cell differentiation is measured by assessing the differentiation potential of cells that endogenously express TREM2, e.g., in some embodiments, cell differentiation is measured by assessing the ability of macrophages to differentiate from monocytes, e.g., as described in the Examples section.

[0171] In some embodiments, microglial activation is measured in vivo. In some embodiments, microglial activation is measured using TSPO-PET imaging. TSPO-PET imaging methods are known in the art.

[0172] In some embodiments, anti-TREM2 antibodies enhance microglial function without increasing neuroinflammation. The level of neuroinflammation can be determined by measuring the level of cytokines (e.g., inflammatory cytokines), such as, but not limited to, TNF-α, IL-1β, IL-6, IL-1ra, TGFβ, IL-15, or IFN-γ. In some embodiments, cytokine levels are measured using immunoassays, such as enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (EMIA), enzyme-linked microbead immunoassays (ELISA), microparticle enzyme immunoassays (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoretic immunoassays (CEIA), radioimmunoassays (RIA), immunofluorescence, chemiluminescence immunoassays (CL), or electrochemiluminescence immunoassays (ECL).

[0173] IV. Preparation of Antibodies In some embodiments, the antibodies are prepared by immunizing an animal(s) (e.g., a mouse, rabbit, or rat) with an antigen or mixture of antigens to induce an antibody response. In some embodiments, the antigen or mixture of antigens is administered in conjunction with an adjuvant (e.g., Freund's adjuvant). After the initial immunization, one or more subsequent booster injections of antigen(s) may be administered to improve antibody production. After immunization, antigen-specific B cells are harvested, for example, from the spleen and / or lymphoid tissues. To generate monoclonal antibodies, the B cells are fused with myeloma cells and subsequently screened for antigen specificity. Methods for preparing antibodies are also described in the Examples section below.

[0174] Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Alternatively, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to a selected antigen. Antibodies can also be made bispecific, i.e., capable of recognizing two different antigens. Antibodies can also be heteroconjugates, for example, two covalently linked antibodies or immunotoxins.

[0175] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression system, such as a hybridoma or CHO cell expression system. Many such systems are widely available from commercial sources. H and V L In embodiments that include both regions, V H and V L The regions may be expressed using a single vector, for example, in a dicistronic expression unit or under the control of different promoters. H and V L The regions can be expressed using separate vectors. H or V L The region may optionally include a methionine at the N-terminus.

[0176] In some embodiments, the antibody is a chimeric antibody. Methods for producing chimeric antibodies are known in the art. For example, chimeric antibodies can be produced in which the antigen-binding region (heavy and light chain variable regions) of one species, such as mouse, is fused to the effector region (constant domain) of another species, such as human. As another example, "class-switched" chimeric antibodies can be produced in which the effector region of an antibody is replaced with the effector region of a different immunoglobulin class or subclass.

[0177] In some embodiments, the antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity. A humanized antibody usually comprises one or more variable regions (e.g., CDRs) or portions thereof that are non-human (e.g., derived from a mouse variable region sequence), and optionally some framework regions or portions thereof that are non-human, and further comprises one or more constant regions derived from a human antibody sequence. Methods for humanizing non-human antibodies are known in the art. Transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies. Other methods for humanizing antibodies include, for example, variable domain resurfacing, CDR grafting, specificity determining residue (SDR) grafting, guided selection, and framework shuffling.

[0178] As an alternative to humanization, fully human antibodies can be produced. As a non-limiting example, transgenic animals (e.g., mice) can be produced that, upon immunization, can produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Introduction of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen exposure. As another example, human antibodies can be produced by hybridoma-based methods, such as using primary human B cells to generate cell lines that produce human monoclonal antibodies.

[0179] Human antibodies can also be produced using phage display or yeast display technology. In phage display, a repertoire of variable heavy and variable light chain genes is amplified and expressed in a phage display vector. In some embodiments, the antibody library is a natural repertoire amplified from a human source. In some embodiments, the antibody library is a synthetic library made by cloning and recombining heavy and light chain sequences to generate a large pool of antibodies with different antigen specificities. Phages usually display antibody fragments (e.g., Fab or scFv fragments), which are then screened for binding to the antigen of interest.

[0180] In some embodiments, antibody fragments (Fab, Fab', F(ab')2, scFv, V H , or V HH(e.g., F(ab')2 fragments) are produced. Various techniques have been developed for producing antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab')2 fragments. According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0181] In some embodiments, the antibody or antibody fragment is conjugated to another molecule, for example, polyethylene glycol (PEGylated) or conjugated to serum albumin to provide increased half-life in vivo.

[0182] In some embodiments, a multispecific antibody, e.g., a bispecific antibody, is provided that comprises an anti-TREM2 antibody (or an antigen-binding fragment thereof) described herein. A multispecific antibody is an antibody that has binding specificity for at least two different sites. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) has binding specificity for TREM2 and has binding specificity for at least one other antigen. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) binds to two different TREM2 epitopes. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) can induce TREM2 clustering at the cell surface. An exemplary method for measuring receptor clustering using confocal FRET microscopy is described in Wallrabe et al., Biophys. J., 2003, 85:559-571. Methods for producing multispecific antibodies (e.g., bispecific antibodies) include, but are not limited to, recombinant co-expression of two pairs of heavy and light chains in a host cell, "knobs-into-holes" engineering, intramolecular trimerization, and fusion of an antibody fragment to the N- or C-terminus of another antibody, e.g., tandem variable domains.

[0183] V. Nucleic Acids, Vectors and Host Cells In some embodiments, the anti-TREM2 antibodies disclosed herein are prepared using recombinant methods. Thus, in some aspects, the disclosure provides isolated nucleic acids comprising a nucleic acid sequence encoding any of the anti-TREM2 antibodies described herein (e.g., any one or more of the CDRs, heavy chain variable regions, and light chain variable regions described herein); vectors comprising such nucleic acids; and host cells into which nucleic acids have been introduced that are used to replicate the nucleic acids encoding the antibodies and / or to express the antibodies.

[0184] In some embodiments, the polynucleotide (e.g., the isolated polynucleotide) comprises a nucleotide sequence encoding an antibody or antigen-binding portion thereof described herein (e.g., those described in the section above entitled "Anti-TREM2 Antibody Sequences"). In some embodiments, the polynucleotide comprises a nucleotide sequence encoding one or more amino acid sequences (e.g., CDR sequences, heavy chain sequences, or light chain sequences) disclosed in the abbreviated sequence listing below. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to a sequence (e.g., CDR sequence, heavy chain sequence, or light chain sequence) disclosed in the abbreviated sequence listing below. In some embodiments, the polynucleotide described herein is operably linked to a heterologous nucleic acid, e.g., a heterologous promoter.

[0185] Suitable vectors containing a polynucleotide encoding an antibody of the present disclosure, or a fragment thereof, include cloning vectors and expression vectors. Although the cloning vector selected may vary depending on the host cell to be used, useful cloning vectors generally have the ability to replicate autonomously, may have a single target for a specific restriction endonuclease, and / or may carry a marker gene that can be used to select clones containing the vector. Examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0186] Expression vector is generally a replicable polynucleotide construct that contains the nucleic acid of the present disclosure.Expression vector can replicate in host cell either as episome or as an integral part of chromosomal DNA.Suitable expression vector includes, but is not limited to, plasmid, virus vector (including adenovirus, adeno-associated virus, retrovirus) and any other vector.

[0187] Suitable host cells for cloning or expressing the polynucleotide or vector described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell. In some embodiments, the host cell is a human cell, such as a human embryonic kidney (HEK) cell.

[0188] In another aspect, a method of making an anti-TREM2 antibody described herein is provided. In some embodiments, the method comprises culturing a host cell described herein (e.g., a host cell expressing a polynucleotide or vector described herein) under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

[0189] VI. Therapeutic Methods Using Anti-TREM2 Antibodies In another aspect, therapeutic methods are provided using the anti-TREM2 antibodies disclosed herein (e.g., the anti-TREM2 antibodies described in Section III, supra). In some embodiments, methods are provided for treating neurodegenerative diseases. In some embodiments, methods are provided for modulating one or more TREM2 activities (e.g., in a subject with a neurodegenerative disease).

[0190] In some embodiments, a method of treating a neurodegenerative disease is provided. In some embodiments, the neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle disease with calcifications, Down's syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker syndrome, or a combination of these. In some embodiments, the neurodegenerative disease is selected from the group consisting of globuloglial tauopathy, Guadeloupe-type parkinsonism with dementia, PSP of the Guadeloupe type, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallidopontinigral degeneration, Parkinson's disease, Pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and neurofibrillary senile dementia. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neurodegenerative disease is Nasu-Hakola disease. In some embodiments, the neurodegenerative disease is frontotemporal dementia. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the method includes administering to the subject an isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2 protein (e.g., an anti-TREM2 antibody described herein), or a pharmaceutical composition comprising an anti-TREM2 antibody described herein.

[0191] In some embodiments, the anti-TREM2 antibodies (or antigen-binding portions or pharmaceutical compositions thereof) described herein are used to treat a neurodegenerative disease characterized by a mutation in TREM2. In some embodiments, the neurodegenerative disease characterized by a mutation in TREM2 is Alzheimer's disease, e.g., Alzheimer's disease characterized by a R47H mutation in TREM2.

[0192] In some embodiments, a method is provided for modulating one or more TREM2 activities in a subject (e.g., a subject with a neurodegenerative disease). In some embodiments, the method includes modulating the level of sTREM2, modulating the recruitment or phosphorylation of a kinase (e.g., Syk kinase) that interacts with the TREM2 / DAP12 signaling complex, modulating phagocytosis (e.g., phagocytosis of cell debris, amyloid beta particles, etc.), modulating cell migration (e.g., migration of bone marrow cells, macrophages, microglia, and disease-associated microglia), and / or modulating cell differentiation (e.g., of bone marrow cells, macrophages, microglia, and disease-associated microglia). In some embodiments, a method is provided for enhancing one or more TREM2 activities in a subject with a neurodegenerative disease. In some embodiments, a method is provided for decreasing the level of sTREM2 in a subject with a neurodegenerative disease. In some embodiments, a method for modulating one or more TREM2 activities in a subject includes administering to the subject an isolated antibody or antigen-binding portion thereof that specifically binds to human TREM2 protein (e.g., an anti-TREM2 antibody described herein), or a pharmaceutical composition comprising an anti-TREM2 antibody described herein.

[0193] In some embodiments, the subject being treated is a human, such as an adult human or a pediatric human.

[0194] In some embodiments, a method of reducing plaque accumulation in a subject having a neurodegenerative disease is provided. In some embodiments, the method comprises administering an antibody or pharmaceutical composition described herein to a subject. In some embodiments, the subject has Alzheimer's disease. In some embodiments, the subject is an animal model of a neurodegenerative disease (e.g., a 5XFAD or APP / PS1 mouse model). In some embodiments, plaque accumulation is measured by amyloid plaque imaging and / or tau imaging, for example, using positron emission tomography (PET) scanning. In some embodiments, administration of an anti-TREM2 antibody reduces plaque accumulation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a baseline value (e.g., the level of plaque accumulation in the subject before administration of the anti-TREM2 antibody).

[0195] In some embodiments, the anti-TREM2 antibody is administered to the subject in a therapeutically effective amount or dose. A daily dose range of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg may be used. However, the dosage may vary depending on several factors, including the selected route of administration, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight, and the judgment of the prescribing physician. The dosage may be increased or decreased over time as needed by the individual patient. In certain cases, the patient is given a low dose initially, which is then increased to an effective dosage that the patient can tolerate. The determination of an effective amount is well within the capabilities of one of ordinary skill in the art.

[0196] The route of administration of the anti-TREM2 antibodies described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or aural delivery, or any other method known in the art. In some embodiments, the antibodies are administered orally, intravenously, or intraperitoneally.

[0197] In some embodiments, the anti-TREM2 antibody (and optionally another therapeutic agent) is administered to the subject for an extended period of time, for example, at least 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 150 days, 200 days, 250 days, 300 days, or 350 days or more.

[0198] VII. Pharmaceutical Compositions and Kits In another aspect, pharmaceutical compositions and kits are provided that include an antibody that specifically binds to human TREM2 protein. In some embodiments, the pharmaceutical compositions and kits are for use in treating neurodegenerative diseases. In some embodiments, the pharmaceutical compositions and kits are for use in modulating (e.g., enhancing or inhibiting) one or more TREM2 activities, such as Syk phosphorylation. In some embodiments, the pharmaceutical compositions and kits are for use in modulating (e.g., decreasing) sTREM2 levels.

[0199] Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided comprising an anti-TREM2 antibody or antigen-binding fragment thereof, hi some embodiments, the anti-TREM2 antibody is an antibody or antigen-binding fragment thereof described in Section III, supra.

[0200] In some embodiments, a pharmaceutical composition comprises an anti-TREM2 antibody as described herein and further comprises one or more pharma- ceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating that is physiologically compatible and does not interfere with or otherwise inhibit the activity of the active agent. A variety of pharma-ceutically acceptable excipients are well known in the art.

[0201] In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical or subcutaneous administration. Pharmaceutically acceptable carriers may include one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agent, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are well known in the art.

[0202] The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The following methods and excipients are merely illustrative and in no way limiting.

[0203] For oral administration, anti-TREM2 antibodies can be formulated by combining with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with solid excipients, optionally grinding the resulting mixture, processing the mixture of granules, and obtaining tablets or dragee cores, after adding suitable auxiliaries, if desired. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0204] Anti-TREM2 antibodies can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the compound(s) can be formulated into a preparation by dissolving, suspending or emulsifying in an aqueous or non-aqueous solvent, for example, vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, and optionally with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, the compound can be formulated in an aqueous solution, for example, in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline buffer. The preparation for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of a preservative. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0205] In some embodiments, the anti-TREM2 antibody is prepared for delivery in a sustained, controlled, sustained, timed or delayed release formulation, for example in a semipermeable matrix of solid hydrophobic polymers containing the active agent. Various types of sustained release materials have been established and are well known to those skilled in the art. Current sustained release formulations include film-coated tablets, multiparticulate or pellet systems, matrix technology using hydrophilic or lipophilic materials, and wax-based tablets containing pore-forming excipients. Depending on its design, a sustained release delivery system can release the compound over a period of hours or days, for example, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours or more. In general, sustained release formulations can be prepared using natural or synthetic polymers, for example, polymeric vinylpyrrolidone such as polyvinylpyrrolidone (PVP); carboxyvinyl hydrophilic polymers; hydrophobic and / or hydrophilic hydrocolloids such as methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose; and carboxypolymethylene.

[0206] Typically, pharmaceutical compositions for use in in vivo administration are sterile. Sterilization can be achieved by methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0207] The dosage and desired drug concentration of the pharmaceutical composition of the present disclosure may vary depending on the specific application envisaged. The determination of the appropriate dosage or route of administration is well within the skill of the art. Suitable dosages are also described in Section VI above.

[0208] kit In some embodiments, a kit is provided that includes an anti-TREM2 antibody or antigen-binding fragment thereof, hi some embodiments, the anti-TREM2 antibody is an antibody or antigen-binding fragment thereof described in Section III, supra.

[0209] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises an anti-TREM2 antibody described herein and further comprises one or more additional therapeutic agents for use in treating a neurodegenerative disease, such as Alzheimer's disease. In some embodiments, the therapeutic agent is an agent for use in treating cognitive or behavioral symptoms of a neurodegenerative disease (e.g., an antidepressant, a dopamine agonist, or an antipsychotic). In some embodiments, the therapeutic agent is a neuroprotective agent (e.g., carbidopa / levodopa, an anticholinergic agent, a dopamine agonist, a monoamine oxidase B (MAO-B) inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, a glutamatergic agent, a histone deacetylase (HDAC) inhibitor, a cannabinoid, a caspase inhibitor, melatonin, an anti-inflammatory agent, a hormone (e.g., estrogen or progesterone), or a vitamin).

[0210] In some embodiments, the kit comprises an anti-TREM antibody described herein and further comprises one or more reagents for measuring sTREM2 levels. In some embodiments, the kit comprises an anti-TREM antibody described herein and further comprises one or more reagents for measuring TREM2 activity (e.g., for measuring Syk phosphorylation).

[0211] In some embodiments, the kit further comprises instructional materials (e.g., instructions for using the kit for the therapeutic methods described in Section VI, supra) containing directions (i.e., protocols) for the practice of the methods described herein. The instructional materials typically comprise written or printed material, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES

[0212] VIII. Examples The present invention will now be described in more detail by way of specific examples. The following examples are presented for illustrative purposes only and are not intended to limit the present invention in any way.

[0213] Example 1. Generation and initial characterization of anti-TREM2 antibodies Recombinant expression and purification of human TREM2 ECD fused to mouse Fc The ectodomain (residues 19-172) of human TREM2 (UniProtKB ID-Q9NZC2) was subcloned into the pRK vector, including a secretion signal derived from mouse IgG kappa chain V-III, amino acids 1-20 (UniProtKB ID-P01661) at the N-terminal region, and a mouse Fc tag (with GGGGS (SEQ ID NO: 64) between the TREM2 ECD and Fc) at the C-terminal region.

[0214] Purified plasmids were transfected into Expi293F™ cells (Thermo Fisher) using the Expi293F™ Expression System Kit according to the manufacturer's instructions. To inhibit N-linked glycan maturation and reduce glycosylation heterogeneity, kifunensine (Sigma), an inhibitor of hypermannosidase I, was added to the cultures at a concentration of 1 μg / mL immediately after transfection. Transfected cells were incubated at 37°C and 125 rpm in a humidified atmosphere of 6% CO2 in an orbital shaker (Infors HT Multitron). 16 hours after transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and 2 were added to the cells and medium supernatants were harvested 96 hours after transfection. Clarified supernatants were supplemented with EDTA-free protease inhibitors (Roche) and stored at -80°C.

[0215] For isolation of rhTREM2-Fc, the clarified culture supernatant was loaded onto a HiTrap MabSelect SuRe Protein A affinity column (GE Healthcare Life Sciences) and washed with 200 mM arginine and 137 mM succinate buffer (pH 5.0). The fusion protein was eluted in 100 mM QB citrate buffer, pH 3.0, and 50 mM NaCl. Immediately after elution, 1 M Tris-HCl buffer, pH 8.0, was added to the protein solution to neutralize the pH. Protein aggregates were separated by size-exclusion chromatography (SEC) on a Superdex 200 increase 10 / 300 GL column (GE Healthcare Life Sciences). The buffer of the SEC mobile phase was maintained at 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 50 mM arginine, which was also the protein storage buffer. All chromatography steps were performed on an AKTA pure system or an AKTA Avant system (GE Healthcare Life Sciences).

[0216] Recombinant expression and purification of His-tagged TREM2 ECD The ectodomain of TREM2 (UniProtKB-Q9NZC2) (residues 19-172) was subcloned into the pRK vector, including a secretion signal derived from mouse Ig kappa chain V-III, amino acids 1-20 (UniProtKB ID-P01661) at the N-terminal region, and a 6X-His tag (SEQ ID NO: 65) at the C-terminal region. The insert was confirmed by sequencing and maxiprep plasmid purification was performed.

[0217] Purified plasmids were transfected into Expi293F™ cells (Thermo Fisher) using the Expi293F™ Expression System Kit according to the manufacturer's instructions. Transfected cells were incubated at 37° C., 125 rpm in a humidified atmosphere of 6% CO2 in an orbital shaker (Infors HT Multitron). 16 hours after transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and 2 were added to the cells and culture medium supernatants were harvested 96 hours after transfection.

[0218] The harvested medium was supplemented with 1M imidazole, pH 8.0, to a final concentration of 10 mM and filtered using a Nalgene™ Rapid-Flow™ disposable filter unit with a pore size of 0.4 microns (Thermo Fisher). HisPur™ Ni-NTA Resin (Thermo Fisher) was washed with MQ water and equilibrated with loading buffer (20 mM Tris, pH 8.0, 150 mM NaCl, and 10 mM imidazole). Affinity purification was performed using the gravity flow method. The harvested medium was loaded onto the resin and nonspecifically bound proteins were washed with loading buffer supplemented with 50 mM and 100 mM imidazole. The bound His-tagged TREM2 ectodomain was eluted with 20 mM Tris, pH 8.0, 150 mM NaCl, and 200 mM imidazole. The eluted protein was concentrated using an Amicon 10 kDa concentrator, and the concentrated protein was further purified by gel filtration chromatography using an AKTA Avant system (GE Healthcare Life Sciences). The protein was loaded onto a HiLoad Superdex 200 16 / 600 (GE Healthcare Life Sciences) column equilibrated with 1×PBS, and eluted and fractionated using 1×PBS as the running buffer. The eluted fractions were analyzed by electrophoresis on polyacrylamide (PAGE) gels under denaturing and native conditions. The eluted fractions were further characterized by analytical size exclusion chromatography and intact protein mass measurement. Using the results from PAGE and analytical characterization, highly glycosylated protein fractions were pooled, which were aliquoted and stored at −80° C.

[0219] Antibody production Rodents (mice and rats) were immunized using standard protocols with rhTREM2-Fc immunogen or BWZ cells expressing full-length Trem2 receptor. Sera collected at various time points were used to measure titers throughout immunization. Detection of antigen-specific immune responses was performed using flow cytometry with rhTREM2-Fc immunogen and live BWZ cells expressing full-length TREM2. Selection criteria for candidate antibodies included rodent antibody production and specificity of binding to TREM2 as detected by flow cytometry. Antibody-secreting cells were isolated from immune tissues of the animals, including spleen, lymph nodes, and bone marrow.

[0220] Single cell suspensions were analyzed to determine the binding properties of secreted antibodies. Antibody-secreting cells were loaded into a microfluidic device and isolated in nanoliter volume reaction chambers, allowing detection of secreted antibodies using fluorescence and bright field image-based microscopic assays (see, e.g., U.S. Pat. No. 9,188,593). Binding assays were performed, including detection of antibodies binding to antigen-coated microbeads, detection of soluble fluorescently labeled antigens binding to antibodies immobilized on beads, and detection of antibodies binding to cell surface expressed antigens. Cell surface expressed antigens included both recombinant and native antigens displayed on the cell surface.

[0221] Image analysis was used to identify chambers exhibiting positive fluorescent signals, indicating the presence of single cells producing antibodies with the desired properties, and the chamber contents were collected and lysed in 384-well plates (see, e.g., U.S. Patent No. 10,087,408). The single cell lysates were then subjected to RT-PCR to amplify the heavy and light chain variable region sequences. The resulting amplicons were then sequenced to determine the cDNA sequences of the paired heavy and light chain variable regions from the selected single cells. The resulting sequences were manually inspected and analyzed to determine sequence diversity and somatic hypermutation. Based on the screening data and sequence diversity, sequences were selected for expression. The expressed antibodies were tested to confirm antigen binding specificity.

[0222] Primary screening of anti-TREM2 antibodies Primary antibody screening was performed in TREM2-expressing HEK293 cells, wild-type iPSCs, and TREM2 knockout iPSCs as follows.

[0223] 1. Screening for TREM2 binding in TREM2-expressing HEK cells HEK293 cell lines stably expressing human TREM2 / DAP12 were generated by transfecting cells with vectors expressing wild-type human TREM2 and DAP12, and vectors expressing DAP12 alone, respectively. Stably expressing clones were selected and cell surface TREM2 expression was assessed by flow cytometry. Surface TREM2 expression was detected using an APC-conjugated rat anti-human / mouse-TREM2 monoclonal antibody (R&D, catalog no. MAB17291). The clone showing the highest expression level of wild-type TREM2 was selected and named "HEK293-H6". Clones stably expressing DAP12 were analyzed by Western blot, and the selected clone was named "HEK293-DAP12#1".

[0224] HEK293 (HEK293-H6) highly expressing human TREM2 and HEK293 (B5) highly expressing GFP were harvested with 0.05% trypsin and incubated at 37°C for 2 hours. After incubation, the cells were centrifuged and washed twice with FACS buffer (PBS + 0.5% BSA). The mixed cells were diluted with 10 per cell line in FACS buffer containing human Trustai FcX solution (Biolegend, Cat. No. 422302). 6The cells were resuspended at a density of 100,000 cells / mL. The mixed cell lines were seeded at 200,000 cells per well in a 96-well round-bottom plate and incubated at room temperature for 20 minutes. After incubation, the cells were centrifuged and incubated with anti-TREM2 antibody at a titration of approximately 0–200 nM for 45 minutes on ice. After incubation, the cells were centrifuged and washed three times with FACS buffer. The cells were then incubated with secondary antibody (Alexa Fluor 647 AffiniPure F(ab')2 Fragment Goat Anti-human IgG(H+L), Jackson ImmunoResearch Laboratories, Cat. No. 109-606-088, 1:800 dilution) for 30 minutes on ice. After incubation, cells were washed three times with FACS buffer, resuspended in 100 μL of FACS buffer, and analyzed by flow cytometry (BD FACSCanto II, San Jose, CA) with 30,000 events acquired for each sample. The mean fluorescence intensity per cell was calculated with FlowJo software and used to generate dose-response binding curves.

[0225] FIG. 1 shows representative results of an exemplary antibody binding to the cell surface receptor TREM2 on HEK293-H6 cells.

[0226] Assessment of activation of TREM2-dependent pSyk signaling Activation of TREM2-dependent pSyk signaling was measured in human macrophage cells or HEK293-H6 cells using the commercially available AlphaLisa assay from Perkin-Elmer.

[0227] For all experiments involving the use of lipid vesicles containing 70% DOPC and 30% POPS, lipid vesicles were prepared within 2 weeks of the experiment as follows: 7 mg DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and 3 mg POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) were combined in chloroform in a glass vial and dried under a stream of N2 gas for 1-2 h or until completely dry. The lipid mixture was resuspended in 1 mL HBSS (to a final lipid concentration of approximately 10 mg / mL) and vortexed for 2-3 min. The lipid suspension was subsequently extruded using an Avanti mini extruder configured with one 100 nm pore size membrane to form small unilamellar vesicles at 10 mg / mL.

[0228] 1. Administration of antibodies to cells The day before the assay, human macrophage cells or HEK293-H6 cells were seeded at 100,000 cells / well or 40,000 cells / well, respectively, in poly-D-lysine-coated 96-well plates. Antibodies were titrated in 10-step serial dilutions starting at 300 nM in PBS with 3-fold dilutions between steps. For antagonist dose-response curves, lipid vesicles containing 70% DOPC and 30% POPS at a final concentration of 1 mg / mL were also included in the antibody / PBS mixture. Cells were washed three times with HBSS using a Biotek405 / 406 plate washer, after which 50 μL of antibody / PBS (with or without vesicles) solution was added per well using a Hamilton Nimbus liquid handler. The cell plates were then transferred to a 37°C incubator for 5 min. The plate was flicked to remove the liposome / antibody solution and 40 μL of lysis buffer containing 1 μM PMSF (Cell Signaling Technologies, CST) was added using a liquid handler. Lysates were then either frozen at -80°C or assayed immediately in the AlphaLisa assay.

[0229] Human macrophage cells were prepared for the assay as follows: Human monocytes were isolated from fresh blood following the RosetteSep Human Monocyte Enrichment Cocktail protocol (Stemcell Technologies, ref. #15068). Isolated monocytes were washed with wash buffer (PBS+2% FBS) and resuspended in 10 mL of ACK lysis buffer (ThermoFisher Scientific, Cat. No. A10492) to lyse red blood cells. 20 mL of wash buffer was added to stop cell lysis, and samples were centrifuged and washed once more with medium (RPMI, 10% Hyclone FBS, 1% Sodium Pyruvate, 1% Glutamax, 1% Non-essential Amino Acids, and 1% Penicillin-Streptomycin). Human monocytes were then differentiated into macrophage cells in 250 mL flasks in medium in the presence of 50 ng / mL human recombinant M-CSF (Gibco, Cat. No. PHC9501). Fresh human M-CSF was added on day 3, and human macrophages were subsequently harvested on day 5 and used in the assay.

[0230] 2.AlphaLisa Assay Cell lysates were assayed for pSyk using the standard protocol of the Perkin Elmer pSyk AlphaLisa kit. Briefly, 10 μL of lysate / well was transferred to an opaque white 384-well Optiplate (Perkin Elmer). Next, 5 μL of Acceptor Mix (containing working solution of acceptor beads) was added per well, followed by sealing the plate with a foil seal and incubating at room temperature for 1 hour. Then, 5 μL of Donor Mix (containing working solution of donor beads) was added to each well under reduced light conditions. The plate was resealed and incubated at room temperature for 1 hour. Finally, the plate was read using the AlphaLisa settings on a Perkin Elmer EnVision plate reader.

[0231] Figure 2 shows the dose-response curves of representative anti-TREM2 antibodies on pSyk signaling activation in primary human macrophage cells. Filled circles (●) represent anti-TREM2 antibodies and open circles (○) represent isotype controls. Each curve represents the average of three independent experiments, and the EC 50 The values ​​are shown below in Table 1. The results show that anti-TREM2 antibodies are able to activate TREM2-DAP12 ITAM signaling in primary human macrophages.

[0232] Liposome response assay in iPSC-microglia TREM2 agonist antibodies and phosphotidylserine-containing liposomes activate pSyk via TREM2. To understand the effect of anti-TREM2 antibodies on Syk signaling in the presence of liposomes, we pretreated iPSC-microglia with anti-TREM2 antibodies and then assessed the intracellular liposome response.

[0233] Prior to the assay, iPSCs were first differentiated into hematopoietic progenitor cells (HPCs) using a commercially available kit (STEMdiff Hematopoietic Kit from StemCell Technologies). HPCs were transferred to plates containing primary human astrocytes and co-cultured for 14–21 days. After identifying that the floating cells in the co-cultures were primarily (>80%) mature microglia, microglia were used in the assay.

[0234] Two days prior to the assay, human iPSC-microglia were seeded at 30,000 cells / well on poly-D-lysine-coated 96-well plates. Antibodies were diluted to 100 nM in medium containing IMDM, 10% Hyclone FBS, and 1% Pen-strep, and the antibody solution was administered to the cells for 24 hours or 5 minutes at 37°C. Subsequently, the cells were washed once with HBSS and then treated with lipid vesicles containing 70% DOPC and 30% POPS at 1 mg / mL for 5 minutes at 37°C. The liposome solution was removed by flicking the plate, and 30 μL of lysis buffer (Cell Signaling Technologies, CST) containing 1 μM PMSF was added. The lysates were then frozen at -80°C or immediately assayed in the AlphaLisa assay. Cell lysates were assayed for pSyk as previously described using the standard protocol of the Perkin Elmer pSyk AlphaLisa kit.

[0235] Figures 3A and 3B show activation of pSyk signaling in human iPSC-microglia incubated with anti-TREM2 antibody followed by administration of lipid vesicles to assess liposome response in cells. White bars indicate incubation with PBS instead of lipid vesicles as a control. Data represent the mean and standard error of 2-7 independent experiments. Figure 3A shows data from iPSC-microglia pretreated with antibody for 5 min, and Figure 3B shows data from iPSC-microglia pretreated with antibody for 24 h. The results show that pretreatment of human iPSC-microglia with anti-TREM2 antibody increases phospho-Syk signaling induced by liposomes compared to isotype control, indicating that anti-TREM2 antibody does not prevent lipid activation of pSyk signaling in cells, but instead enhances it.

[0236] Human TREM2 NFAT reporter assay Jurkat NFAT cell lines expressing human TREM2 / DAP12 were generated as follows: Jurkat NFAT reporter cells were infected with lentiviral vector expression of human TREM2 and DAP12 and cultured in RPMI containing 10% HyClone FBS and 1% penicillin / streptomycin. Stable expression clones were selected in the presence of puromycin and zeocin. Cell surface TREM2 expression was assessed by flow cytometer using biotinylated anti-TREM2 antibody (SEQ ID NO: 66 and 67). The clone that showed the highest expression level of wild-type TREM2 was selected and named hTrem2 / NFAT Jurkat reporter cells for the assay described below.

[0237] The day before the assay, 96-well plates were pre-coated with anti-TREM2 antibodies or isotype controls at dose levels ranging from 0 to 500 nM (45 μL / well, 12 levels in total) and incubated overnight at 4°C. After overnight incubation, the pre-coated plates were washed twice with PBS and then inoculated with hTrem2 / NFAT Jurkat reporter cells (10 6 Cells / well) were loaded onto the plate. Plates were incubated at 37°C for 24 hours, after which 50 μL / well of quantlucia solution was added to each well and mixed thoroughly. For analysis, 20 μL of solution was removed from each well and transferred to a 384-well white plate for measuring the signal with a luminometer (Perkin Elmer Envision).

[0238] Figure 4 contains dose-response curves of representative anti-TREM2 antibodies for NFAT activation as measured by detection of the reporter gene luciferase, and the EC 50 The values ​​are shown below in Table 1. The results in Figure 4 show that, compared to the isotype control, the candidate anti-TREM2 antibodies were able to induce NFAT activation and downstream signaling sufficient to activate a transcriptional response.

[0239] Viability assay in human macrophage cells Human monocytes were isolated according to the RosetteSep Human Monocyte Enrichment Cocktail protocol (Stemcell Technologies, Cat. No. 15068). Isolated monocytes were washed with wash buffer (PBS+2% FBS) and resuspended in 10 mL of ACK lysis solution (ThermoFisher Scientific, Cat. No. A10492) to lyse red blood cells. Lysis was stopped by adding 20 mL of wash buffer. The cell suspension was centrifuged and washed once with medium (RPMI1640+10% FBS+penicillin / streptomycin). Cells were collected at 10 6 The cells were resuspended in medium at a density of 1 μL cells / mL and used in the viability assay described below.

[0240] The day before the assay, 96-well plates were pre-coated with anti-TREM2 antibodies or isotype controls at dose levels ranging from 0 to 200 nM (45 μL / well, 12 levels in total) and incubated overnight at 4 °C. After overnight incubation, the pre-coated plates were washed twice with PBS and then incubated with human monocytes (10 5 Cells / well) were loaded onto the plate. After 5 days at 37°C, the medium was aspirated and 100 μL PBS + 100 μL Celltiter-glo medium (Promega, Cat# G7571) was added to each well. After 10 minutes of incubation, the cell medium was transferred to a multi-well plate adapted for use with a luminometer and luminescence for cell viability was recorded.

[0241] Figure 5 shows the dose-response curves of representative anti-TREM2 antibodies for cell survival in human macrophage cells under low M-CSF conditions, and the EC 50 The values ​​are shown below in Table 1. The results indicate that the TREM2 agonist antibodies have sufficient receptor activation capacity to induce transcriptional responses to regulate cell function and promote survival of human macrophage cells under low M-CSF conditions.

[0242] Biacore kinetic measurements of antibodies Surface plasmon resonance (Biacore™ 8K instrument) was used to measure the affinity of anti-TREM2 antibodies for human and cynomolgus TREM2 ECD. Anti-TREM2 antibodies were captured on a Biacore Series S CM5 sensor chip (GE Healthcare Life Sciences, Cat. No. 29149604) using a Human Fab Capture Kit (GE Healthcare Life Sciences, Cat. No. 28958325). Three-fold serial dilutions of recombinant human TREM2 or recombinant cynomolgus TREM2 were injected at a flow rate of 30 μL / min. Antibody binding was monitored for 300 s, followed by antibody dissociation over 600 s in HBS-EP+ running buffer (GE Healthcare Life Sciences, Cat. No. BR100669). Binding responses were corrected by subtracting RU values ​​from an empty flow cell. Kinetic analysis included k on and k off A 1:1 Languir model of simultaneous fitting of D The binding values ​​are shown in Table 1 below.

[0243] (Table 1) In vitro characterization of antibodies TIFF2024164116000001.tif48161NB: No bonds detected ND: Not measured

[0244] Example 2. Regulation of soluble TREM2 levels and phagocytic behavior in human macrophage cells Soluble TREM2 dose-response assay in human macrophages Human macrophage cells were generated as described above. The day before the assay, human macrophage cells were seeded at 100,000 cells / well in poly-D-lysine coated 96-well plates. Antibodies were titrated in human macrophage medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% penicillin-streptomycin) starting at 300 nM with 10-fold dilutions between steps. Cells were dosed with antibodies and incubated for 24 hours. After antibody incubation, plates were spun down to remove debris and supernatants were collected for measurement of soluble TREM2.

[0245] Soluble TREM2 was measured as follows. Briefly, MSD small spot streptavidin plates (Meso Scale Discovery) were coated with biotinylated anti-hTREM2 polyclonal antibody (R&D Systems) overnight at 4°C. Plates were then blocked with 3% BSA / TBST for 1 h at room temperature. Samples and standards were prepared by heating to 95°C for 5 min in SDS-containing buffer. Prepared samples and standards were diluted 1:10 in 3% BSA / TBST in the assay plate after blocking. TREM2-His protein diluted in 3% BSA / TBST was used as a standard for absolute quantification. After 2 h incubation at room temperature, plates were washed with TBST. Primary detection antibody, sulfo-tagged goat anti-human TREM2 (R&D Systems), was diluted in 3% BSA / TBST and added to the plate and incubated at room temperature for 1 h. After washing with TBST, MSD plates were developed using 2x MSD reading buffer T and subsequently detected using an MSD Sector plate reader. MSD values ​​were converted to absolute quantification of sTREM2 by fitting a standard curve using Prism 7.0 software (Graphpad). Modulation of TREM2 shedding was expressed as the ratio of soluble TREM2 from cells incubated with anti-TREM2 test antibodies normalized to soluble TREM2 from cells cultured without specific anti-TREM2 antibodies in the medium.

[0246] Figure 6 shows representative soluble TREM2 levels (sTREM2) as a function of anti-TREM2 antibody concentration. The results show that anti-TREM2 antibodies can dose-dependently reduce sTREM2 levels in human macrophage cells after overnight treatment.

[0247] Phagocytosis assay in human macrophages Human macrophage cells were generated as described above. Two days before the assay, human macrophage cells were seeded at 80,000 cells / well in poly-D-lysine-coated 96-well plates. Antibodies were diluted at 100 nM in medium containing RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% penicillin-streptomycin. Cells were then dosed with antibody solution for 24 hours at 37°C. Next, cell nuclei and cell membranes were stained for 10 minutes, after which pHrodo-myelin was added at 5 μg / mL. Cells were then incubated for 4 hours at 37°C. pHrodo fluorescence was measured per cell with a high-content confocal microscope (Opera Phoenix), and fluorescence intensity was quantified with the instrument software.

[0248] pHrodo-myelin was prepared by purifying myelin from wild-type C57Bl / 6 mouse brains (Jackson Laboratories) using the method described in Safaiyan et al. (2016, Nature Neuroscience 19(8):995-998). After purification, myelin was resuspended in PBS and adjusted to a protein concentration of 1 mg / mL using the DC Protein Assay Kit2 (BioRad, catalog no. 5000112). Myelin was tagged with pHrodo-red using a microscale labeling kit (ThermoFisher, catalog no. P35363) according to the manufacturer's instructions. Excess labeling was removed by pelleting myelin at 10,000 g for 5 min to remove the supernatant and repeating these steps 3–5 times.

[0249] Figure 7 shows representative results of a phagocytosis assay in human macrophage cells. Phagocytosis of myelin was measured by detecting and quantifying pHrodo fluorescence in microscopic images of TREM2-treated macrophage cells and comparing the measurements to those of an isotype control. The results show that human macrophages treated with an exemplary TREM2 agonist antibody increase pHrodo-myelin phagocytosis compared to the isotype control, indicating that anti-TREM2 antibodies can enable beneficial clearance of myelin debris in cells.

[0250] Example 3. Regulation of lipid accumulation in iPSC-derived microglia Lipid storage assay Prior to the assay, iPSCs were first differentiated into hematopoietic progenitor cells (HPCs) using a commercially available kit (STEMdiff Hematopoietic Kit from StemCell Technologies). HPCs were transferred to plates containing primary human astrocytes and co-cultured for 14–21 days. After identifying that the floating cells in the co-cultures were primarily (>80%) mature microglia, microglia were used in the assay.

[0251] Cells (iPSC-microglia, 30,000 cells / well) were seeded in PDL-coated 96-well plates in full serum medium. After 24 h at 37 °C, purified unlabeled myelin (final concentration 50 μg / mL, purified from wild-type C57Bl / 6 mouse brain (Jackson Laboratories) using the method described in Safaiyan et al. (2016, Nature Neuroscience 19(8):995-998)) was added to the wells. After 24 h of lipid treatment at 37 °C, anti-TREM2 antibody or RSV control was added to the wells to a final concentration of 100 nM. Cells were incubated at 37 °C for an additional 48–72 h before cells were harvested or imaged. For myelin washout experiments, myelin was removed after the 24-h incubation period and replaced with antibody-containing medium for the subsequent 24–48 h of incubation.

[0252] For Nile Red imaging, the supernatant was removed and cells were incubated for 30 minutes at 37°C in live cell imaging buffer (Life Technologies, Catalog No. A14291DJ) containing 1 μM Nile Red (ThermoFisher, Catalog No. N1142) and 1 drop / mL Nucblue (ThermoFisher, Catalog No. R37605). After the incubation period, the staining solution was removed and cells were fixed with 4% paraformaldehyde. Cells were then imaged using Alexa568 and DAPI illumination settings on an Opera Phoenix high content confocal imager. Lipid spots were analyzed using the spot detection algorithm in the Harmony software provided with the instrument. Figure 8A includes representative microscopy images of iPSC-microglia treated with either vehicle or myelin (final concentration 50 μg / mL) for 24 hours and then incubated with isotype control or an exemplary anti-TREM2 antibody (CL0020123) for 72 hours. Figure 8B is a representative bar graph of the same anti-TREM2 antibody used in the microscopy images in Figure 8 A. Quantification of Nile Red staining was performed by total spot intensity per cell and data are presented as the mean and standard deviation of three technical replicates in different fields of the same microscopy sample.

[0253] For lipidomic analysis, cells were washed once with PBS while kept on ice. A 70 μL volume of 9:1 methanol:water solution containing 1:100 internal standard was added to the cells in the 96-well plate. The plate was agitated at 1200 rpm on a shaker at 4° C. for 20 min, then centrifuged at 300×g for 5 min. A 50 μL sample of the supernatant was transferred to an LCMS vial and kept at −80° C. until analysis on the instrument.

[0254] Lipid levels were analyzed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled with electrospray mass spectrometry (QTRAP 6500+Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected onto a BEH C18 1.7 μm, 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.25 mL / min at 55° C. For positive ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium formate + 0.1% formic acid, and mobile phase B consisted of 90:10 isopropyl alcohol / acetonitrile (v / v) containing 10 mM ammonium formate + 0.1% formic acid. For negative ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium acetate, and mobile phase B consisted of 90:10 isopropyl alcohol / acetonitrile (v / v) containing 10 mM ammonium acetate. The gradient was programmed as follows: 45% B to 95% B in 0.0–8.0 min, 99% B in 8.0–9.0 min, 45% B in 9.0–9.1 min, and 45% B in 9.1–10.0 min. Electrospray ionization was performed in either positive or negative ion mode by applying the following settings: curtain gas: 30, collision gas: set to medium, ion spray voltage: 5500 (positive mode) or 4500 (negative mode), temperature: 250 °C (positive mode) or 600 °C (negative mode), ion source gas 1:50, ion source gas 2:60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM) with the following parameters: dwell time (msec) and collision energy (CE), declustering potential (DP): 80, entrance potential (EP): 10 (positive mode) or -10 (negative mode), and collision cell exit potential (CXP): 12.5 (positive mode) or -12.5 (negative mode). Lipids were quantified using a mixture of non-endogenous internal standards.Lipids were identified based on their retention time and MRM characteristics of commercially available reference standards (Avanti Polar Lipids, Birmingham, Ala., USA).

[0255] Figures 8C and 8D show levels of cholesterol ester (CE) (Figure 8C) and triacylglyceride (TAG) lipid species (Figure 8D) detected by mass spectrometry in cell lysates of iPSC-microglial cells treated with an exemplary anti-TREM2 antibody for 72 hours after 24 hours of myelin treatment. Figures 8E and 8F show levels of cholesterol ester (CE) (Figure 8E) and triacylglyceride (TAG) lipid species (Figure 8F) detected by mass spectrometry in cell lysates of iPSC-microglial cells subjected to a myelin washout experiment with an exemplary anti-TREM2 antibody. The LC / MS data generated in Figures 8C-8F were normalized to internal standards for the CE data and to myelin plus isotype controls for each individual lipid species for the TAG data.

[0256] Lipid accumulation in iPSC-microglia was induced by myelin treatment, as reflected by increased neutral lipid staining (Nile Red) and by LC / MS to detect specific lipid species in cell lysates. The data shown in Figures 8A-8F collectively show that treatment of iPSC-microglia cells after myelin exposure with an exemplary anti-TREM2 antibody reduced lipid species accumulation, as shown by decreased neutral lipid staining in the cells, as well as decreased CE and TAG lipid species levels measured by LC / MS. Reduction in lipid levels as a result of antibody treatment was observed at various time points ranging from 24 h to 72 h. To exclude the possibility that reduced lipid levels were caused by a blockade of lipid uptake, a myelin washout experiment was performed in which myelin was removed prior to addition of anti-TREM2 antibody. Figure 8F shows that anti-TREM2 antibody also reduced lipid levels in iPSC-microglia that were myelin washed out prior to antibody treatment, compared to isotype control.

[0257] Example 4. Functional epitope binning of antibodies TREM2 antibody epitope bins were determined by competitive binding on the TREM2 protein. Epitope binning experiments were performed on a Carterra LSA instrument at 25°C using the classical sandwich epitope binning configuration method. All test antibodies were immobilized on a HC30M chip by amine coupling. Multiple cycles of sandwich competitive binding were then performed on the test antibodies. Each cycle consisted of an injection of antigen (His-tagged TREM2 ECD) followed by an injection of analyte antibody onto the immobilized antibody. At the end of each cycle, the immobilized antibody surface was regenerated by injecting a low pH buffer (pH=3) containing 1.25 M NaCl. The epitope binding data was evaluated by Carterra software to generate a competitive matrix and epitope bins. The results are shown in Table 2.

[0258] Epitope binning of anti-TREM2 antibodies identified two agonist bins: (1) stalk-binding agonists and (2) IgV domain-binding agonists. Antibodies in the same bin exhibit the same function, e.g., inhibition of TREM2-DAP12 pSyk activation by lipid ligands (antagonist antibodies) and activation of pSyk by antibodies alone (stalk-binding agonists, IgV domain-binding agonists).

[0259] (Table 2) Anti-TREM2 antibody bins annotated by functional class TIFF2024164116000002.tif22128

[0260] Example 5. Antibody binding to TREM2 stalk peptide TREM2 antibodies were evaluated for binding to human and mouse TREM2 stalk region peptides. Peptides tested included (1) the full-length stalk region (amino acids 129-172 of human TREM2, UniProtKB Q9NZC2; amino acids 131-169 of mouse TREM2, UniProtKB Q99NH8), and (2) a truncated stalk peptide containing the ADAM10 / 17 cleavage site (amino acids 149-163 of human / mouse TREM2). Antibody binding to the TREM2 stalk peptides was detected using a standard sandwich ELISA. Briefly, 96-well half-area ELISA plates were coated with streptavidin overnight at 4°C. The next day, biotinylated TREM2 stalk peptides diluted in 1% BSA / PBS were added to the plates and incubated for 1 h. Antibodies diluted in 1% BSA / PBS were then added and incubated for 1 h. Antibody bound to the peptide was detected with anti-human kappa-HRP secondary antibody (Bethyl Laboratories, Inc.) diluted in 1% BSA / PBS. Plates were assayed by reaction with detection reagent (One-step TMB Ultra, Thermo) and measurement of absorbance at 450 nm (A450) with a standard spectrophotometer (BioTek®). Results are shown in Table 3 below. Data are presented as normalized values ​​(fold over background, background = isotype control).

[0261] (Table 3) Binding of anti-TREM2 antibodies to the TREM2 stalk peptide TIFF2024164116000003.tif56128

[0262] Table 3 shows peptide-antibody binding interactions in the human and mouse stalk regions that support the epitope binning data in Table 2. Based on the data in Table 3, the likely binding site of a particular anti-TREM2 antibody corresponds to amino acids 129-148 in the TREM2 extracellular stalk region.

[0263] The ability of anti-TREM2 antibodies to inhibit TREM2 stalk peptide cleavage by ADAM17 was also analyzed using a fluorescence polarization assay. First, the TREM2 stalk peptide was prepared in assay buffer (25 mM Tris pH 7.5, 2.5 μM ZnCl2, 0.005% Brij-35) containing streptavidin. The anti-TREM2 antibodies were then pre-incubated with the TREM2 stalk peptide for 30 min at room temperature. After the pre-incubation period, ADAM17 (R&D systems, Cat. No. 930-ADB) was added and incubated with the peptide for 20 h at 37°C. The next day, samples were further diluted in assay buffer and transferred to an opaque black 384-well plate. Fluorescence polarization was then measured on a Perkin Elmer EnVision plate reader. Fluorescence polarization of the TREM2 stalk peptide pre-incubated with anti-TREM2 antibody was compared to that of the full-length TREM2 stalk peptide and an enzyme control (full-length TREM2 stalk peptide containing ADAM17).

[0264] TREM2 stalk binding antibodies significantly increased fluorescence polarization and demonstrated partial inhibition of stalk peptide cleavage by ADAM17 (clones CL0020141, CL0020188, CL0020313, CL0020308). The IgV binding antibody CL0020107 did not bind to the TREM2 stalk region peptide and therefore showed no effect on peptide cleavage in the fluorescence polarization assay.

[0265] Example 6. Pharmacokinetic analysis of anti-TREM2 antibodies The pharmacokinetic profile of the anti-TREM2 antibody was evaluated in mice. C57BL / 6J mice were purchased from Jackson Laboratory (stock number 000664) at 2 months of age and used for 7-day pharmacokinetic (PK) and target binding studies. Human Trem2 cDNA KI homozygous mice (huTrem2 KI / KI ) were used for 24-hour target binding assays at 3 months of age. The generation and breeding of human Trem2 cDNA KI mice are described below.

[0266] Generation of human TREM2 cDNA KI mouse model Human TREM2 cDNA KI mouse (huTrem2 KI / KI ) was generated as follows: the human Trem2 cDNA-pA sequence was inserted into the mouse Trem2 endogenous ATG start site. The insertion of human Trem2 cDNA-pA replaces the exon 1 sequence of mouse Trem2, allowing expression of the human Trem2 cDNA driven by the endogenous mouse promoter and disrupting expression of endogenous mouse Trem2. huTrem2 KI / KI Mice were generated on a C57BL / 6 genetic background using homologous recombination.

[0267] huTrem2 KI / KIFor the targeting vector, the long homology arm (LA) extends approximately 3.6 kb upstream of the 5' end of the human Trem2 cDNA-pA sequence, and the short homology arm (SA) extends approximately 2.3 kb downstream of the 3' to the FRT-flanked Neo cassette. Both the long and short homology arms were amplified from a C57BL / 6 BAC clone (RP23:358G22) and then subcloned into a 2.4 kb pSP72 (Promega) backbone vector containing an ampicillin selection cassette. A targeting vector approximately 13.6 kb in size was obtained by inserting the hUBS-gb2 FRT-flanked neomycin cassette immediately downstream of the hTrem2-pA cassette. 10 μg of the targeting vector was linearized with the restriction enzyme NotI (New England Biolabs) and then transfected into FLP C57Bl / 6 (B6) embryonic stem (ES) cells by electroporation. After selection with G418 antibiotic, surviving clones were expanded for PCR analysis to identify positive recombinant ES clones. The primer sequences for PCR screening (SEQ ID NO: 53 = 5'-AGG AAT GTG GGG AGC ACG GAG-3' and SEQ ID NO: 54 = 5'-TGC ATC GCA TTG TCT GAG TAG GTG-3') amplified a 2.81 kb fragment containing the region from the bghpA element to the downstream of the short homology arm (SA) outside the 3' region. Five clones were identified as positive and selected for further expansion. The Neo cassette was removed by the Flp transgene during ES clone expansion.

[0268] Genomic DNA extracted from five positive clones was first characterized by sequencing analysis. A 1.19 kb product was amplified and sequenced with primers (SEQ ID NO:55=5'-ACC CTA GTC CTG ACT GTT GCT C-3', SEQ ID NO:56=5'-TAT AGG AAC TTC GCG ACA CGG ACA C-3') to confirm the 5' genomic / neo cassette junction and the 3' KI cassette junction. The sequencing results confirmed that the human Trem2 cDNA-pA sequence was inserted into all five clones.

[0269] Five positive clones were further characterized by Southern blotting analysis using probes targeting the short and long arms. ES cell genomic DNA digested with Ssp I and Bam HI was hybridized with the short and long arm probes, respectively. It was confirmed that all five ES clones retained the correct homologous recombination events in both the long and short arms. The primer sequences for amplifying the short arm probe (658 bp) are (SEQ ID NO:57=5'-ACA GGA GGG ACC TAC CTT CAG3', SEQ ID NO:58=5'-GCC TGC CTT TCA GAG ACC TCA GTC-3). The primer sequences for amplifying the long arm probe (681 bp) are (SEQ ID NO:59=5'-CCT CTC CGG CTG CTC ATC TTA CTC-3', SEQ ID NO:60=5'-GTC TCT CAG CCC TGG CAG AGT TTG-3').

[0270] All five ES cell clones were then injected into C57BL / 6 blastocysts. Offspring from one clone were confirmed for germline transmission by PCR genotyping. Genotyping primers were (SEQ ID NO:61=pr1: 5'-CGC CTA CCC TAG TCC TGA CTG TTG-3', SEQ ID NO:62=pr2: 5'-AAA GCC TAC AGC ATC CTC ACC TC-3', and SEQ ID NO:63=pr3: 5'-GCA TCA TGG GGT TGT AGA TTC CG-3'). The PCR product of wild type pr1 / pr2 is 658 bp. The PCR product of KI allele pr1 / pr3 is 469 bp.

[0271] Antibody administration and plasma / CSF collection For PK analysis, C57BL / 6J mice were dosed with anti-TREM2 antibodies or control IgG at 10 mg / kg via intravenous (IV) tail vein injection (dose volume approximately 200 μL / mouse, n=3 per group). Blood samples were collected 1 h, 24 h, 4 days, and 7 days after dosing. Blood samples at the first three time points were collected by submandibular bleeding using a 3 mm lancet (GoldenRod animal lancet). A final terminal blood sample on day 7 was collected by cardiac puncture. Blood was collected into EDTA tubes (Sarstedt Microvette 500 K3E, catalog number 201341102), mixed by gentle inversion, and centrifuged at 4°C. The plasma (top) layer was transferred to a 1.5 mL Eppendorf tube and stored at -80°C until analysis.

[0272] In a 24-hour target binding study, C57BL / 6J mice were used to test the murine surrogate anti-TREM2 antibodies, huTrem2 KI / KI Mice were used to test human anti-TREM2 antibodies. Mice were dosed with anti-TREM2 antibodies or control IgG at 100 mg / kg via intravenous (IV) tail vein injection (dose volume approximately 200 μL / mouse, n=5 per group). Blood samples were collected 24 hours prior to dosing to determine baseline levels of TREM2. Terminal blood and CSF samples were collected 24 hours after dosing. Plasma preparations were prepared as previously described. To collect CSF samples, a sagittal incision was made behind the skull and the subcutaneous tissue and muscle were separated to expose the cisterna magna. A pre-drawn glass capillary tube was used to puncture the cisterna magna and collect CSF samples. The CSF was then centrifuged at 4°C to remove blood debris and the CSF supernatant was transferred to a 0.5 mL Low Protein LoBind Eppendorf tube (Eppendorf, catalog number 022431064) and stored at -80°C until analysis.

[0273] Analysis of plasma levels of in vivo anti-TREM2 antibodies For PK analysis of anti-TREM2 antibodies, a common human anti-Fc sandwich ELISA was used to quantify total antibody concentrations in mouse plasma. 384-well MaxiSorp plates were coated overnight with 1 μg / mL anti-huFc donkey polyclonal (Jackson Immunoresearch). After incubation with plasma diluted 1:2,000 or 1:20,000 in assay buffer (PBST, 1% BSA), anti-huFc donkey antibody conjugated to HRP (Jackson Immunoresearch) was added as detection reagent. Five-parameter logistic regression was used to generate standard curves ranging from 2 nM to 2.7 pM using 3-fold dilutions for each individual antibody.

[0274] Figure 9 shows representative mouse PK profiles of certain anti-TREM2 antibodies. Antibody clearance rates (CL [mL / day / kg]) are shown over a 7-day period for each antibody represented and compared to a normal effectorless isotype control. Each antibody shown in Figure 9 showed comparable clearance rates compared to the isotype control.

[0275] In vivo target binding: Plasma levels of sTREM2 To measure plasma levels of soluble TREM2 (sTREM2), we used human Trem2 cDNA KI mice (huTrem2 KI / KI) were bled and then treated intravenously with 100 mg / kg of anti-TREM2 test antibody or isotype control. Mice were bled 24 hours after dosing. Plasma was obtained from the blood samples and evaluated in the MSD assay performed as follows. MSD SECTOR plates were coated with 1 μg / mL of capture antibody (R+D anti-TREM2 antibody, catalog number MAB17291-100) diluted in PBS and incubated overnight at 4° C. Sample wells were blocked with undiluted MSD Blocker A for 1 hour. Plasma samples were diluted 1:20 in 25% MSD Blocker A in Tris-buffered saline containing 0.05% Tween-20 (TBST) and added to each sample well on the plate, which was then incubated at room temperature for 2 hours. Detection antibody (MSD sulfo-tagged goat anti-human, catalog number R32AJ-1, 1:1000) was then added to each sample well and the plate was incubated at room temperature for 1 hour. A TBST wash was performed for each sample well using a Biotek plate washer. Detection reagent (MSD read buffer) was added and measured using an MSD Meso Sector S600 reader to obtain the results of sTREM2 binding to the antibody.

[0276] 10A and 10B are schematic diagrams illustrating an exemplary anti-TREM2 antibody, huTrem2. KI / KI Total sTREM2 levels in plasma (FIG. 10A) and antibody-bound sTREM2 levels (FIG. 10B) are shown. For total and bound sTREM2 assays, data were normalized to baseline levels of sTREM2 before dosing. Results show that total circulating sTREM2 levels did not change significantly between mice treated with anti-TREM2 antibodies compared to isotype controls after 24 hours of treatment with antibodies, suggesting that total circulating sTREM2 levels are not affected at early time points after antibody dosing. In contrast, levels of antibody-bound sTREM2 were higher in mice injected with anti-TREM2 antibodies compared to isotype controls.

[0277] Example 7. Sequence optimization and humanization of anti-TREM2 antibodies Exemplary anti-TREM2 antibodies were sequence optimized, humanized, and characterized for binding kinetics and binding specificity.

[0278] Sequence optimization was performed by searching within the CDR sequences for residues susceptible to chemical modification (e.g., asparagine deamidation motif (NG), aspartate isomerization motif (DS)), as well as residues that may be oxidized (tryptophan (W) and methionine (M)), and by amino acid substitution with conservative and germline residues to remove the propensity of such sequences. The humanized and sequence-optimized anti-TREM2 antibody variants were then analyzed for binding kinetics using Biacore and titrated cellular binding to HEK293-H6 cells (see Example 1 for a representative protocol).

[0279] The analysis of the binding properties of the humanized and sequence-optimized variants of antibody CL0020188 is shown in Table 4. The NG motifs in the CL0020188 CDR-H2 sequence (SEQ ID NO: 5) and CDR-L1 sequence (SEQ ID NO: 7) were modified and grafted onto human framework regions and analyzed. Table 4 shows the K D values, and EC measured by titrated binding assay 50 Indicates the value.

[0280] (Table 4) Binding properties of sequence-optimized and humanized variants of CL0020188 TIFF2024164116000004.tif691643m=V H A24G / L45P / V48L in

[0281] As shown in Table 4, the humanized and sequence-optimized clones of CL00201088 were significantly higher than the parent antibody (K D = 9.5 nM) for hTREM2, which was consistent with the cell binding results for HEK293-H6 cells shown in Table 4. 50= 0.44 nM), the humanized and sequence-optimized clones showed comparable and sub-nanomolar affinity for TREM2 expressed in HEK293-H6 cells. Taken together, the results show comparable binding kinetics between the parent antibody and the humanized and sequence-optimized variants.

[0282] The results of the analysis of the binding properties of the humanized and sequence-optimized variants of the antibody CL0020123 are shown in Table 5. The NG and DS motifs in the CL0020123 CDR-H2 sequence (SEQ ID NO: 30) were modified and grafted onto human framework regions and analyzed. Table 5 shows the K D values, and EC measured by titrated binding assay 50 Indicates the value.

[0283] (Table 5) Binding properties of sequence-optimized and humanized variants of CL0020123 TIFF2024164116000005.tif1041641m=V H R71A in 2m=V H V67A / R71A in

[0284] Parent antibody (K D =0.10 nM), the humanized and sequence-optimized clone had approximately 4-fold higher K for binding to hTREM2 as measured by Biacore. D In contrast, in a titrated cellular binding assay in HEK293-H6 cells, the humanized and sequence-optimized clones showed equivalent and subnanomolar affinity for TREM2.

[0285] Example 8. In vitro characterization of sequence-optimized and humanized anti-TREM2 antibodies An exemplary sequence-optimized and humanized anti-TREM2 antibody (Example 7) was evaluated by the in vitro methods described in Examples 1 and 3. The antibodies were evaluated for TREM2 binding in TREM2-expressing HEK cells, TREM2-dependent pSyk signaling in HEK-H6 cells, the ability to promote human macrophage cell survival, and the ability to modulate lipid accumulation in iPSC-microglia. Figures 11-14 show the results of a representative anti-TREM2 antibody. The anti-TREM2 antibody showed good cell binding in assays using TREM2-expressing HEK293-H6 cells with EC50 values ​​of 0.34 nM (Figure 11A) and 0.08 nM (Figure 11B). The anti-TREM2 antibody also activated pSyk signaling in TREM2-expressing HEK293-H6 cells (Figures 12A and 12B). Furthermore, anti-TREM2 antibodies induced macrophage survival, with better survival activity observed with the CL0020188 variant antibody (Figure 13). Finally, anti-TREM2 antibodies demonstrated the ability to reduce lipid accumulation in myelin-treated iPSC-microglia (Figures 14A and 14B).

[0286] Example 9. Mouse PK of sequence-optimized and humanized anti-TREM2 antibodies The pharmacokinetic profile of certain sequence-optimized, humanized anti-TREM2 antibodies (Example 7) was evaluated in wild-type mice in a 7-day pharmacokinetic (PK) study similar to that described in Example 6. Each dose group included n=3 mice. Table 6 shows the PK characteristics of exemplary sequence-optimized, humanized anti-TREM2 antibodies (Example 7).

[0287] (Table 6) Mouse PK characteristics of anti-TREM2 antibodies TIFF2024164116000006.tif39164

[0288] Example 10. Cyno PK of sequence-optimized and humanized anti-TREM2 antibodies The pharmacokinetic profile of anti-TREM2 antibodies was evaluated in naive cynomolgus monkeys. Naive cynomolgus monkeys aged 2-4 years (weight approximately 2-3 kg) were injected with anti-TREM2 antibodies via intravenous bolus injection. Doses included 3 mg / kg and 25 mg / kg, with n=3 monkeys per dose group. Blood samples (approximately 1 mL) were collected pre-dose, and 10 min, 30 min, 1 h, 6 h, 12 h, and 24 h, as well as 3, 7, 10, 14, 17, 21, 24, and 28 days after dosing. Samples were cooled at approximately 5°C and then centrifuged to obtain plasma, which was kept on dry ice before being stored at -70°C until analysis. Plasma samples were analyzed for anti-TREM2 antibody levels as follows.

[0289] For anti-TREM2 antibody PK analysis, total antibody concentrations were quantified in monkey plasma using a generic anti-human_IgG sandwich electrochemiluminescence immunoassay (ECLIA) on the Meso Scale Discovery (MSD) platform. Briefly, 1% casein-based PBS blocking buffer (Thermo Scientific, MA) was added to MSD GOLD 96-well small spot streptavidin-coated microtiter plates (Meso Scale Discovery, MD) and incubated for approximately 1 hour. Following plate blocking and washing steps, biotinylated anti-human_IgG goat antibody (SouthernBiotech, AL) in 0.5 μg / mL working solution was added to the assay plate and allowed to incubate for 1-2 hours. Following incubation and washing steps, plasma test samples (i.e., samples containing anti-TREM2 humanized antibodies) were added to the assay plate and allowed to incubate for 1-2 hours. Please note that test samples had to be diluted at an assay minimum-required-dilution (MRD) of 1:100 in 0.5% casein-based PBS assay buffer (Thermo Scientific, MA) prior to addition to the assay plate, resulting in a final plasma matrix of 1%. Following the anti-TREM2 antibody analyte capture and wash steps, a secondary ruthenylated (SULFO-TAG) anti-human IgG goat antibody (Meso Scale Discovery, MD) was added to the assay plate at a working solution of 0.4 μg / mL and incubated for approximately 1 hour. Finally, following the incubation and wash steps, assay read buffer (1× MSD read buffer T) was added to the assay plate to generate the assay sample signal. The sample signal readout from the MSD plate reader was in the form of electrochemiluminescence (ECL) signal and was expressed in ECL units (ECLU). All the aforementioned assay reaction steps were performed at ambient temperature with shaking (if required) on a plate shaker.The assay has a dynamic calibration standard range of 19.5-2500 ng / mL (or 0.195-25 ng / mL after 1:100 MRD) with eight standard steps serially diluted 1:2 plus blank plasma samples. Plasma sample concentrations were back-calculated from an assay calibration standard curve fitted to a weighted four-parameter nonlinear logistic regression. Table 7 shows the pharmacokinetic (PK) properties of an exemplary sequence-optimized, humanized anti-TREM2 antibody (Example 7).

[0290] (Table 7) Cynomolgus monkey PK characteristics of anti-TREM2 antibody TIFF2024164116000007.tif21164

[0291] The CL0020188 variant showed similar low clearance levels across different dose levels and a linear pharmacokinetic profile in cynomolgus monkeys. Furthermore, no clinical pathology findings were associated with administration of the variant to cynomolgus monkeys (data not shown).

[0292] Example 11. Comparison of anti-TREM2 antibodies The affinity of anti-TREM2 antibodies to human TREM2 and cynomolgus monkey TREM2 was measured by Biacore (described in Example 1). The potency of anti-TREM2 antibodies was measured in healthy human volunteer CSF samples (Innovative Research) and healthy cynomolgus monkey CSF samples (Worldwide Primates) by MSD assay. The potency of each antibody was determined by its EC50. Briefly, MSD GOLD 96-well small spot streptavidin plates (MSD, Catalog No. L45SA) coated with capture antibody (biotinylated goat anti-human TREM2, R&D Systems, Catalog No. BAF1828) were incubated with biological fluid samples diluted 1:3 in assay buffer (25% (v / v) MSD Blocker A (MSD, Catalog No. R93BA-A), 75% (v / v) TBST) for 1 hour at room temperature. After rinsing the wells with TBST, sulfo-tagged anti-TREM2 antibodies were added to the wells of the plate in serial dilutions (4-fold dilutions over 11 steps) and incubated at room temperature for 1 hour. The wells were washed with TBST and MSD read buffer (MSD, Cat. No. R92TC-3) was added to the wells. The signal from the samples was measured using an MSD Meso Sector S600 instrument. The EC50 value of each antibody was determined by 4-parameter variable slope nonlinear regression. Reference antibodies #1 and #2 correspond to 4C5 and 6E7 as described in WO2018 / 195506. Reference antibody #3 corresponds to AL2p-58 as described in WO2019 / 028292. The variable regions of reference antibody #1 are represented in SEQ ID NOs: 73 and 74. The variable regions of reference antibody #2 are represented in SEQ ID NOs: 75 and 76. The variable regions of reference antibody #3 are represented in SEQ ID NOs: 77 and 78. The results are shown in Table 8.

[0293] As shown in Table 8, the CL0020188 variant and the CL0020123 variant disclosed herein have stronger affinity for human TREM2 compared to the reference antibody, and bind more strongly to sTREM2 in CSF samples isolated from healthy human volunteer subjects. Furthermore, the CL0020188 variant has stronger affinity for cynomolgus monkey TREM2 compared to the reference antibody, and binds more strongly to sTREM2 in CSF samples isolated from healthy cynomolgus monkey subjects. This is demonstrated by the relative amounts of antibody required to reach the half maximal effective concentration (EC50) for binding to a given amount of sTREM2 under the same conditions. As shown in Table 8, higher relative amounts of the reference antibodies #1, #2, and #3 are required to achieve EC50 compared to the CL0020188 and CL0020123 antibodies.

[0294] (Table 8) Comparison of anti-TREM2 antibody properties TIFF2024164116000008.tif56161

[0295] Example 12. Epitope mapping of anti-TREM2 antibodies To identify the epitopes of anti-TREM2 antibodies at the peptide level, hydrogen-deuterium exchange (HDX) mass spectrometry was used. Recombinant human TREM2 alone or mixed with anti-TREM2 antibodies was incubated with deuterium oxide labeling buffer (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.0) at 20°C for 0, 60, 600, and 3600 seconds. Hydrogen / deuterium exchange was quenched by adding an equal volume of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). Quenched samples were then subjected to pepsin / protease XIII digestion and LC-MS analysis. Briefly, the quenched samples were injected onto a Pepsin / Protease XIII packed column (2.1×30 mm) held at 20° C., and the resulting peptides were analyzed using a UPLC-MS system consisting of a Waters Acquity UPLC (Waters Corporation) coupled to a Q Exactive™ HF-Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher). Peptides were separated on a 50 mm×1 mm C8 column held at −6° C. using a 16.5 min gradient from 2% to 31% Solvent B (Solvent B: 0.2% formic acid in acetonitrile, Solvent A: 0.2% formic acid in water). Mass spectra were recorded in MS-only mode. Raw MS data were processed using HDX Workbench (Pascal et al. 2012. Journal of The American Society for Mass Spectrometry 23:1512-1521). Deuterium levels were calculated using the average mass difference at t0 between the deuterated peptides and their native forms. Peptide identification was performed by searching the MS / MS data against the human TREM2 sequence using Mascot softward (Matrix Science). Mass tolerances for precursor and product ions were 10 ppm and 0.02 Da, respectively.

[0296] Based on the results of HDX mass spectrometry, CL0020188 and variants of CL0020188 bind to human TREM2 (SEQ ID NO: 1) at amino acid residues 143-149 (FPGESES (SEQ ID NO: 69)), whereas CL0020123 and variants of CL0020123 bind to human TREM2 at (i) amino acid residues 55-63 (GEKGPCQRV (SEQ ID NO: 70)), (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQ ID NO: 71)), and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO: 72)).

[0297] IX. Abbreviated sequence listing TIFF2024164116000009.tif239170TIFF2024164116000010.tif235170TIFF2024164116 000011.tif239170TIFF2024164116000012.tif239170TIFF2024164116000013.tif88170

[0298] Sequence information SEQUENCE LISTING <110> DENALI THERAPEUTICS INC. <120> ANTI-TREM2 ANTIBODIES AND METHODS OF USE THEREOF <150> US 62 / 808,141 <151> 2019-02-20 <160> 83 <170> PatentIn version 3.5 <210> 1 <211> 230 <212> PRT <213> Homo sapiens <400> 1 Met Glu Pro Leu Arg Leu Leu Ile Leu Leu Phe Val Thr Glu Leu Ser 1 5 10 15 Gly Ala His Asn Thr Thr Val Phe Gln Gly Val Ala Gly Gln Ser Leu 20 25 30 Gln Val Ser Cys Pro Tyr Asp Ser Met Lys His Trp Gly Arg Arg Lys 35 40 45 Ala Trp Cys Arg Gln Leu Gly Glu Lys Gly Pro Cys Gln Arg Val Val 50 55 60 Ser Thr His Asn Leu Trp Leu Leu Ser Phe Leu Arg Arg Trp Asn Gly 65 70 75 80 Ser Thr Ala Ile Thr Asp Asp Thr Leu Gly Gly Thr Leu Thr Ile Thr 85 90 95 Leu Arg Asn Leu Gln Pro His Asp Ala Gly Leu Tyr Gln Cys Gln Ser 100 105 110 Leu His Gly Ser Glu Ala Asp Thr Leu Arg Lys Val Leu Val Glu Val 115 120 125 Leu Ala Asp Pro Leu Asp His Arg Asp Ala Gly Asp Leu Trp Phe Pro 130 135 140 Gly Glu Ser Glu Ser Phe Glu Asp Ala His Val Glu His Ser Ile Ser 145 150 155 160 Arg Ser Leu Leu Glu Gly Glu Ile Pro Phe Pro Pro Thr Ser Ile Leu 165 170 175 Leu Leu Leu Ala Cys Ile Phe Leu Ile Lys Ile Leu Ala Ala Ser Ala 180 185 190 Leu Trp Ala Ala Ala Trp His Gly Gln Lys Pro Gly Thr His Pro Pro 195 200 205 Ser Glu Leu Asp Cys Gly His Asp Pro Gly Tyr Gln Leu Gln Thr Leu 210 215 220 Pro Gly Leu Arg Asp Thr 225 230 <210> 2 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Glu Val Lys Leu Leu Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Pro Glu Trp Leu 35 40 45 Gly Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Leu Ser Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Val 100 105 110 Met Val Thr Val Ser Ser 115 <210> 3 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 3 Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly 1 5 10 15 Glu Ser Ala Ser Ile Thr Cys Gln Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 4 Gly Phe Thr Phe Thr Asp Phe Tyr Met Ser 1 5 10 <210> 5 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 5 Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 6 Ala Arg Leu Ser Tyr Gly Phe Asp Tyr 1 5 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 7 Gln Ser Ser Lys Ser Leu Leu His Ser Asn Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 8 Trp Met Ser Thr Arg Ala Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 9 Gln Gln Phe Leu Glu Phe Pro Phe Thr 1 5 <210> 10 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 10 Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Phe 20 25 30 Tyr Met Ser Trp Ile Arg Gln Pro Pro Gly Arg Ala Pro Glu Trp Leu 35 40 45 Gly Val Ile Arg Asn Arg Pro Asn Gly Tyr Thr Thr Asp Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile 65 70 75 80 Leu Tyr Leu Gln Met Ser Thr Leu Arg Ala Asp Asp Thr Ala Phe Tyr 85 90 95 Tyr Cys Thr Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Val 100 105 110 Met Val Thr Val Ser Ser 115 <210> 11 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 11 Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly 1 5 10 15 Glu Ser Ala Ser Ile Thr Cys Gln Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Val Val Gly Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 12 Gly Phe Thr Phe Thr Asn Phe Tyr Met Ser 1 5 10 <210> 13 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 13 Val Ile Arg Asn Arg Pro Asn Gly Tyr Thr Thr Asp Tyr Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 14 Thr Arg Leu Thr Tyr Gly Phe Asp Tyr 1 5 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 15 Glu Val Lys Leu Leu Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Pro Glu Trp Leu 35 40 45 Gly Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Val 100 105 110 Met Val Thr Val Ser Ser 115 <210> 16 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 16 Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly 1 5 10 15 Glu Ser Ala Ser Ile Thr Cys Gln Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 17 Ala Arg Leu Thr Tyr Gly Phe Asp Tyr 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 18 Gln Gln Phe Leu Glu Tyr Pro Phe Thr 1 5 <210> 19 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 20 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Gln Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 21 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 22 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 22 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Gln Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Thr Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 23 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 23 Gln Ser Ser Lys Ser Leu Leu His Ser Thr Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 24 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Arg Asn Lys Ala Asn Ala Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 25 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 25 Val Ile Arg Asn Lys Ala Asn Ala Tyr Thr Ala Gly Tyr Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 26 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Leu 35 40 45 Ser Val Ile Arg Asn Lys Ala Asn Ala Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 27 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 28 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Val Thr Ile Thr Cys His Ala Ser Gln His Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asp His Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 29 Gly Phe Ser Ile Glu Asp Phe Tyr Ile His 1 5 10 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 30 Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 31 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 31 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 32 His Ala Ser Gln His Ile Asn Val Trp Leu Ser 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 33 Lys Ala Ser Asn Leu His Thr 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 34 Gln Gln Gly Gln Thr Tyr Pro Arg Thr 1 5 <210> 35 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln His Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 37 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Gln Gly Asp Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Trp Ile Asp Pro Glu Gln Gly Asp Ser Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 40 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Glu Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Trp Ile Asp Pro Glu Asn Gly Glu Ser Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 42 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 42 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Gln Gly Glu Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 43 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Trp Ile Asp Pro Glu Gln Gly Glu Ser Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 44 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 44 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Gln Gly Asp Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 45 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 45 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Glu Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Ile Glu Asp Phe 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Gln Gly Glu Ser Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 His Ala Asp His Gly Asn Tyr Gly Ser Thr Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (6)..(6) <223> / replace="Gln" <220> <221> VARIANT <222> (8)..(8) <223> / replace="Glu" <220> <221> SITE <222> (1)..(17) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 47 Trp Ile Asp Pro Glu Asn Gly Asp Ser Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (6)..(6) <223> / replace="Asn" <220> <221> SITE <222> (1)..(10) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 48 Gly Phe Thr Phe Thr Asp Phe Tyr Met Ser 1 5 10 <210> 49 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (5)..(5) <223> / replace="Arg" <220> <221> VARIANT <222> (6)..(6) <223> / replace="Pro" <220> <221> VARIANT <222> (8)..(8) <223> / replace="Ala" <220> <221> VARIANT <222> (11)..(11) <223> / replace="Thr" <220> <221> VARIANT <222> (12)..(12) <223> / replace="Asp" <220> <221> SITE <222> (1)..(19) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 49 Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro Ser 1 5 10 15 Val Lys Gly <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (1)..(1) <223> / replace="Thr" <220> <221> VARIANT <222> (4)..(4) <223> / replace="Ser" <220> <221> SITE <222> (1)..(9) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 50 Ala Arg Leu Thr Tyr Gly Phe Asp Tyr 1 5 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (10)..(10) <223> / replace="Thr" <220> <221> SITE <222> (1)..(16) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 51 Gln Ser Ser Lys Ser Leu Leu His Ser Asn Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (6)..(6) <223> / replace="Phe" <220> <221> SITE <222> (1)..(9) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 52 Gln Gln Phe Leu Glu Tyr Pro Phe Thr 1 5 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 53 aggaatgtgg ggagcacgga g 21 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 54 tgcatcgcat tgtctgagta ggtg 24 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 55 accctagtcc tgactgttgc tc 22 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 56 tataggaact tcgcgacacg gacac 25 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 57 acaggaggga cctaccttca g 21 <210> 58 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 58 gcctgccttt cagagacctc agtc 24 <210> 59 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 59 cctctccggc tgctcatctt actc 24 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 60 gtctctcagc cctggcagag tttg 24 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 61 cgcctaccct agtcctgact gttg 24 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 62 aaagcctaca gcatcctcac ctc 23 <210> 63 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 63 gcatcatggg gttgtagatt ccg 23 <210> 64 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 64 Gly Gly Gly Gly Ser 1 5 <210> 65 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic 6xHis tag" <400> 65 His His His His His His 1 5 <210> 66 <211> 140 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 66 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ser Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ser Asp Pro Thr Thr Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Arg Thr Ser Gly Thr Gly Asp Tyr Trp Gly Gln Gly Thr Ser Leu 100 105 110 Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala 115 120 125 Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr 130 135 140 <210> 67 <211> 140 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 67 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Phe Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Thr 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe 130 135 140 <210> 68 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 68 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Gln Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 69 Phe Pro Gly Glu Ser Glu Ser 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 70 Gly Glu Lys Gly Pro Cys Gln Arg Val 1 5 <210> 71 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 71 Thr Leu Arg Asn Leu Gln Pro His Asp Ala Gly Leu 1 5 10 <210> 72 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 72 Val Glu Val Leu 1 <210> 73 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 73 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Val Gly Val Pro Leu Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Arg 85 90 95 Asn Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 74 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 74 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly His Ser Phe Thr Asn Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gln Arg Thr Phe Tyr Tyr Asp Ser Ser Gly Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 75 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 75 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asp Ser Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 76 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 76 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Gln Arg Thr Phe Tyr Tyr Asp Ser Ser Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 77 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 77 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr Arg Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 78 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 78 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Gln 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Gly Gly Asp Thr Asn Tyr Ala Gly Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Leu Arg Asn Gln Pro Gly Glu Ser Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 79 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Leu 35 40 45 Ser Val Ile Arg Asn Lys Ala Asn Gly Tyr Thr Ala Gly Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Thr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 80 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 80 Asn Gly Asp Ser 1 <210> 81 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 81 Gln Gly Asp Ser 1 <210> 82 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 82 Asn Gly Glu Ser 1 <210> 83 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 83 Gln Gly Glu Ser 1

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, (a) G-F-T-F-T-α ６ -F-Y-M-S (SEQ ID NO: 48), ６ is D or N; (b) VIRN-β ５ -β ６ -N-β ８ -Y-T-β １１ -β １２ -Y-N-P-S-V-K-G (SEQ ID NO: 49), ５ is K or R, and β ６ is A or P, and β ８ is G or A, and β １１ is A or T, and β １２ is G or D; (c) γ １ -R-L-γ ４ -Y-G-F-D-Y (SEQ ID NO: 50), １ is A or T, and γ ４ is T or S; (d) Q-S-S-K-S-L-LHS-δ １０ A CDR-L1 sequence comprising the sequence: δ-G-K-T-Y-L-N (SEQ ID NO:51), １０ is N or T; (e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); and (f) QQ-FLE-φ ６ -P-F-T (SEQ ID NO: 52), ６ is Y or F. The antibody or antigen-binding fragment thereof.

2. 2. The isolated antibody or antigen-binding fragment of claim 1, wherein the CDR-H1 sequence is selected from any one of SEQ ID NOs: 4 and 12.

3. 3. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the CDR-H2 sequence is selected from any one of SEQ ID NOs: 5, 13, and 25.

4. The isolated antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the CDR-H3 sequence is selected from any one of SEQ ID NOs: 6, 14, and 17.

5. The isolated antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the CDR-L1 sequence is selected from any one of SEQ ID NOs: 7 and 23.

6. The isolated antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the CDR-L3 sequence is selected from any one of SEQ ID NOs: 9 and 18.

7. (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

9.

7. The isolated antibody or antigen-binding fragment of any one of claims 1 to 6, comprising:

8. V having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, and 79 Ｈ 8. The isolated antibody or antigen-binding fragment of any one of claims 1 to 7, comprising the sequence:

9. The V Ｈ 9. The isolated antibody or antigen-binding fragment of claim 8, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

15.

10. The V Ｈ 10. The isolated antibody or antigen-binding fragment of claim 9, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

15.

11. The V Ｈ 11. The isolated antibody or antigen-binding fragment of claim 10, wherein the sequence comprises SEQ ID NO:

15.

12. The V Ｈ 9. The isolated antibody or antigen-binding fragment of claim 8, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

24.

13. The V Ｈ 13. The isolated antibody or antigen-binding fragment of claim 12, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

24.

14. The V Ｈ 14. The isolated antibody or antigen-binding fragment of claim 13, wherein the sequence comprises SEQ ID NO:

24.

15. The V Ｈ 9. The isolated antibody or antigen-binding fragment of claim 8, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

79.

16. The V Ｈ 16. The isolated antibody or antigen-binding fragment of claim 15, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

79.

17. The V Ｈ 17. The isolated antibody or antigen-binding fragment of claim 16, wherein the sequence comprises SEQ ID NO:

79.

18. V having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 68 Ｌ 18. The isolated antibody or antigen-binding fragment of any one of claims 1 to 17, comprising the sequence:

19. The V Ｌ 20. The isolated antibody or antigen-binding fragment of claim 18, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

16.

20. The V Ｌ 20. The isolated antibody or antigen-binding fragment of claim 19, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

16.

21. The V Ｌ 21. The isolated antibody or antigen-binding fragment of claim 20, wherein the sequence comprises SEQ ID NO:

16.

22. The V Ｌ 19. The isolated antibody or antigen-binding fragment of claim 18, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

22.

23. The V Ｌ 23. The isolated antibody or antigen-binding fragment of claim 22, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

22.

24. The V Ｌ 24. The isolated antibody or antigen-binding fragment of claim 23, wherein the sequence comprises SEQ ID NO:

22.

25. The V Ｌ 19. The isolated antibody or antigen-binding fragment of claim 18, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

68.

26. The V Ｌ 26. The isolated antibody or antigen-binding fragment of claim 25, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

68.

27. The V Ｌ 27. The isolated antibody or antigen-binding fragment of claim 26, wherein the sequence comprises SEQ ID NO:

68.

28. (a) V comprising SEQ ID NO: 15 Ｈ V comprising sequence SEQ ID NO:16 Ｌ Array, or (b) V comprising SEQ ID NO: 19 Ｈ V comprising the sequence SEQ ID NO: 20 Ｌ Array, or (c) V comprising SEQ ID NO: 21 Ｈ V comprising the sequence SEQ ID NO: 20 Ｌ Array, or (d) V comprising SEQ ID NO: 19 Ｈ V comprising sequence SEQ ID NO:22 Ｌ Array, or (e) V comprising SEQ ID NO: 79 Ｈ V comprising sequence SEQ ID NO:22 Ｌ Array, or (f) V comprising SEQ ID NO: 24 Ｈ V comprising the sequence SEQ ID NO: 20 Ｌ Array, or (g) V comprising SEQ ID NO: 26 Ｈ V comprising the sequence SEQ ID NO: 20 Ｌ Array, or (h) V comprising SEQ ID NO: 24 Ｈ V comprising sequence SEQ ID NO:22 Ｌ Array, or (i) V comprising SEQ ID NO: 26 Ｈ V comprising sequence SEQ ID NO:22 Ｌ Array, or (j) V containing SEQ ID NO: 2 Ｈ V comprising the sequence SEQ ID NO:3 Ｌ Array, or (k) V comprising SEQ ID NO: 10 Ｈ V comprising the sequence SEQ ID NO:11 Ｌ Array, or (l) V comprising SEQ ID NO: 24 Ｈ V comprising the sequence SEQ ID NO:68 Ｌ array 28. The isolated antibody or antigen-binding fragment of any one of claims 1 to 27, comprising:

29. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, (a) a CDR-H1 sequence comprising the sequence of GFSIEDFYIH (SEQ ID NO: 29); (b) W-I-D-P-E-β ６ -G-β ８ -SK-YA-PK-F-Q-G (SEQ ID NO: 47), ６ is N or Q, and β ８ is D or E; (c) a CDR-H3 sequence comprising the sequence HADHGNYGSTMDY (SEQ ID NO: 31); (d) a CDR-L1 sequence comprising the sequence HASQHINVWLS (SEQ ID NO: 32); (e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID NO: 33); and (f) a CDR-L3 sequence comprising the sequence QQGQTYPRT (SEQ ID NO: 34) The antibody or antigen-binding fragment thereof.

30. 30. The isolated antibody or antigen-binding fragment of claim 29, wherein the CDR-H2 sequence is selected from SEQ ID NOs: 30, 39, 41, and 43.

31. (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:30, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:34, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, CDR-H2 comprising the amino acid sequence of SEQ ID NO:41, CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:34, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

34.

31. The isolated antibody or antigen-binding fragment of claim 29 or 30, comprising:

32. V having at least 85% sequence identity to any one of SEQ ID NOs: 27, 35, 37, 38, 40, 42, 44, 45, and 46 Ｈ 32. The isolated antibody or antigen-binding fragment of any one of claims 29 to 31, comprising the sequence:

33. The V Ｈ 33. The isolated antibody or antigen-binding fragment of claim 32, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

27.

34. The V Ｈ 34. The isolated antibody or antigen-binding fragment of claim 33, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

27.

35. The V Ｈ 35. The isolated antibody or antigen-binding fragment of claim 34, wherein the sequence comprises SEQ ID NO:

27.

36. V having at least 85% sequence identity to SEQ ID NO: 28 or 36 Ｌ 36. The isolated antibody or antigen-binding fragment of any one of claims 29 to 35, comprising the sequence

37. The V Ｌ 37. The isolated antibody or antigen-binding fragment of claim 36, wherein the sequence has at least 90% sequence identity to SEQ ID NO:

28.

38. The V Ｌ 38. The isolated antibody or antigen-binding fragment of claim 37, wherein the sequence has at least 95% sequence identity to SEQ ID NO:

28.

39. The V Ｌ 39. The isolated antibody or antigen-binding fragment of claim 38, wherein the sequence comprises SEQ ID NO:

28.

40. (a) V comprising SEQ ID NO: 27 Ｈ V comprising the sequence SEQ ID NO:28 Ｌ Array, or (b) V comprising SEQ ID NO: 35 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (c) V comprising SEQ ID NO: 37 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (d) V comprising SEQ ID NO: 38 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (e) V comprising SEQ ID NO: 40 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (f) V comprising SEQ ID NO: 42 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (g) V comprising SEQ ID NO: 44 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (h) V comprising SEQ ID NO: 45 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ Array, or (i) V comprising SEQ ID NO: 46 Ｈ V comprising the sequence SEQ ID NO:36 Ｌ array 37. The isolated antibody or antigen-binding fragment of claim 36, comprising:

41. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, (a) a CDR-H1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 4, 12, and 29; (b) a CDR-H2 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 5, 13, 25, 30, 39, 41, and 43; (c) a CDR-H3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 6, 14, 17, and 31; (d) a CDR-L1 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 7, 23, and 32; (e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 8 and 33; and (f) a CDR-L3 sequence comprising any one of the amino acid sequences of SEQ ID NOs: 9, 18, and 34. The antibody or antigen-binding fragment thereof.

42. (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (b) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, CDR-H2 comprising the amino acid sequence of SEQ ID NO:30, CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:34, or (h) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:34, or (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, CDR-H2 comprising the amino acid sequence of SEQ ID NO:41, CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or (j) CDR-H1 comprising the amino acid sequence of SEQ ID NO:29, CDR-H2 comprising the amino acid sequence of SEQ ID NO:43, CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:34, or (k) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

9.

42. The isolated antibody or antigen-binding fragment of claim 41 , comprising:

43. 43. The isolated antibody or antigen-binding fragment of claim 41 or 42, comprising a heavy chain variable region having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45, 46, and 79.

44. 44. The isolated antibody or antigen-binding fragment of any one of claims 41 to 43, comprising a light chain variable region having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, 28, and 36.

45. (a) a V having at least 85% sequence identity to SEQ ID NO:2 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

3. Ｌ Array, or (b) a V having at least 85% sequence identity to SEQ ID NO:10 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

11. Ｌ Array, or (c) a V having at least 85% sequence identity to SEQ ID NO: 15 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

16. Ｌ Array, or (d) a V having at least 85% sequence identity to SEQ ID NO:19 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

20. Ｌ Array, or (e) a V having at least 85% sequence identity to SEQ ID NO:21 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

20. Ｌ Array, or (f) a V having at least 85% sequence identity to SEQ ID NO: 19 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

22. Ｌ Array, or (g) a V having at least 85% sequence identity to SEQ ID NO:79 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

22. Ｌ Array, or (h) a V having at least 85% sequence identity to SEQ ID NO:24 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

20. Ｌ Array, or (i) a V having at least 85% sequence identity to SEQ ID NO:26 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

20. Ｌ Array, or (j) a V having at least 85% sequence identity to SEQ ID NO:24 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

22. Ｌ Array, or (k) a V having at least 85% sequence identity to SEQ ID NO: 26 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

22. Ｌ Array, or (l) a V having at least 85% sequence identity to SEQ ID NO:27 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

28. Ｌ Array, or (m) a V having at least 85% sequence identity to SEQ ID NO:35 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (n) a V having at least 85% sequence identity to SEQ ID NO: 37 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (o) a V having at least 85% sequence identity to SEQ ID NO:38 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (p) a V having at least 85% sequence identity to SEQ ID NO: 40 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (q) a V having at least 85% sequence identity to SEQ ID NO: 42 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (r) a V having at least 85% sequence identity to SEQ ID NO:44 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (s) a V having at least 85% sequence identity to SEQ ID NO: 45 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (t) a V having at least 85% sequence identity to SEQ ID NO: 46 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

36. Ｌ Array, or (u) a V having at least 85% sequence identity to SEQ ID NO: 24 Ｈ and V having at least 85% sequence identity to SEQ ID NO:

68. Ｌ array 45. The isolated antibody or antigen-binding fragment of any one of claims 41 to 44, comprising:

46. An isolated antibody or antigen-binding fragment thereof that specifically binds to human myeloid cell expressed triggering receptor 2 (TREM2), comprising: clone CL0020306, clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020188-9, The antibody or antigen-binding fragment thereof recognizes the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of CL0307, ​​clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7, and clone CL0020123-8.

47. 47. The isolated antibody or antigen-binding fragment of claim 46, which recognizes the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of clone CL0020123, clone CL0020123-1, clone CL0020123-2, clone CL0020123-3, clone CL0020123-4, clone CL0020123-5, clone CL0020123-6, clone CL0020123-7, and clone CL0020123-8.

48. In SEQ ID NO:1 (i) amino acid residues 55-63 (GEKGPCQRV (SEQ ID NO:70)); (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residues 126 to 129 (VEVL (SEQ ID NO: 72)) 48. The isolated antibody or antigen-binding fragment of claim 47, which recognizes one or more of:

49. 47. The isolated antibody or antigen-binding fragment of claim 46, which recognizes the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of clone CL0020188, clone CL0020188-1, clone CL0020188-2, clone CL0020188-3, clone CL0020188-4, clone CL0020188-5, clone CL0020188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307, ​​and clone CL0020306.

50. 50. The isolated antibody or antigen-binding fragment of claim 49, which recognizes amino acid residues 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO:69)).

51. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, comprising the antibody of SEQ ID NO:1 (i) amino acid residues 55-63 (GEKGPCQRV (SEQ ID NO:70)); (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQ ID NO:71)), and (iii) amino acid residues 126 to 129 (VEVL (SEQ ID NO: 72)) The antibody or antigen-binding fragment thereof, which recognizes an epitope comprising or consisting of one or more of the following:

52. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof recognizes an epitope including or consisting of amino acid residues 143 to 149 of SEQ ID NO:1 (FPGESES (SEQ ID NO:69)).

53. 53. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 52, which reduces levels of soluble TREM2 protein (sTREM2).

54. 54. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 53, which enhances TREM2 activity.

55. 55. The isolated antibody or antigen-binding fragment thereof of claim 54, which enhances phagocytosis or enhances the migration, differentiation, function, or survival of bone marrow cells, microglia, or macrophages.

56. 56. The isolated antibody or antigen-binding fragment thereof of claim 55, which enhances microglial function without increasing neuroinflammation.

57. 55. The isolated antibody or antigen-binding fragment thereof of claim 54, which enhances Syk phosphorylation.

58. 58. The isolated antibody or antigen-binding fragment thereof of claim 57, which enhances Syk phosphorylation in the presence of a TREM2 ligand.

59. 59. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 58, which exhibits cross-reactivity with cynomolgus TREM2 protein.

60. 60. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a monoclonal antibody.

61. 60. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a chimeric antibody.

62. 60. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a humanized antibody.

63. 60. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, wherein the antibody is a fully human antibody.

64. The antigen-binding fragment is selected from the group consisting of Fab, F(ab') ２ 60. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 59, which is a single scFv, scFv, or a bivalent scFv.

65. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 64; A pharma- ceutically acceptable carrier and 13. A pharmaceutical composition comprising:

66. 65. An antibody or antigen-binding fragment thereof that competes with the isolated antibody of any one of claims 1-64 for binding to human TREM2 protein.

67. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 64, or a pharmaceutical composition according to claim 65; Its instruction manual and Including the kit.

68. 66. A method of treating a neurodegenerative disease in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 or a pharmaceutical composition according to claim 65.

69. The neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica (dementia pugilistica), pugilistica), diffuse neurofibrillary tangle disease with calcification, Down's syndrome, familial British dementia, familial Danish dementia, Gerstmann-Sträussler-Scheinker disease, globular glial tauopathy, Guadeloupian parkinsonism with dementia, PSP Guadeloupian type, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallidopontonigral degeneration (pallidopontonigral degeneration) 69. The method of claim 68, wherein the dementia is selected from the group consisting of dementia cerebrovascular disease, dementia cerebrovascular accident ...

70. A method for reducing the level of sTREM2 in a subject having a neurodegenerative disease, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 64, or a pharmaceutical composition described in claim 65.

71. A method for enhancing TREM2 activity in a subject having a neurodegenerative disease, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 64 or a pharmaceutical composition described in claim 65.

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