Anti-TREM2 antibodies and methods of use thereof

Abstract

In one aspect, antibodies are provided that specifically bind to a triggering receptor 2 (TREM2) protein expressed by human myeloid cells. In some embodiments, the antibody reduces the level of soluble TREM2 (sTREM2). In some embodiments, the antibody enhances TREM2 activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 808,141, filed on February 20, 2019, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Background Art

[0003] Triggering receptor expressed on myeloid cells-2 (TREM2) is a transmembrane receptor expressed on microglia and believed to play a role in regulating phagocytosis, cell survival and proinflammatory cytokine production. Mutations in TREM2 have been identified in neurodegenerative diseases including Alzheimer's disease, Na-Harris 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 and with TREM2 mutations.

[0004] There remains a need for therapeutic agents that modulate TREM2 activity or sTREM2 levels. Summary of the invention

[0005] In one aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to triggering receptor expressed on human myeloid cells 2 (TREM2) is provided. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TREM2 comprises:

[0006] (a) a CDR-H1 sequence comprising the sequence GFSIEDFYIH (SEQ ID NO: 29);

[0007] (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;

[0008] (c) a CDR-H3 sequence comprising the sequence HADHGNYGSTMDY (SEQ ID NO: 31);

[0009] (d) a CDR-L1 sequence comprising the sequence HASQHINVWLS (SEQ ID NO: 32);

[0010] (e) a CDR-L2 sequence comprising the sequence KASNLHT (SEQ ID NO: 33); and

[0011] (f) A CDR-L3 sequence comprising the sequence QQGQTYPRT (SEQ ID NO: 34).

[0012] In some embodiments, the CDR-H2 sequence is selected from SEQ ID NO: 30, 39, 41 and 43.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises:

[0014] (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

[0015] (b) 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:39, 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

[0016] (c) 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:41, 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

[0017] (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.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence having 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, V H The sequence has at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, V H The sequence has at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, V 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. In some embodiments, V H The sequence has at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, V 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. In some embodiments, V H The sequence has at least 95% sequence identity to SEQ ID NO: 45. In some embodiments, V H The sequence comprises SEQ ID NO:45.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence having at least 85% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 36. L In some embodiments, V L The sequence has at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, V L The sequence has at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, V 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. In some embodiments, V L The sequence has at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, V L The sequence comprises SEQ ID NO:36.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises:

[0021] (a) comprising V of SEQ ID NO: 27 H Sequence and V comprising SEQ ID NO:28 L sequence; or

[0022] (b) comprising V of SEQ ID NO: 35 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0023] (c) V comprising SEQ ID NO: 37 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0024] (d) V comprising SEQ ID NO: 38 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0025] (e) V comprising SEQ ID NO: 40 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0026] (f) V comprising SEQ ID NO:42 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0027] (g) V comprising SEQ ID NO: 44 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0028] (h) V comprising SEQ ID NO: 45 H Sequence and V comprising SEQ ID NO:36 L sequence; or

[0029] (i) V comprising SEQ ID NO: 46 H Sequence and V comprising SEQ ID NO:36 L sequence.

[0030] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 comprises:

[0031] (a) a CDR-H1 sequence comprising the sequence GFTFT-α6-FYMS (SEQ ID NO: 48), wherein α6 is D or N;

[0032] (b) CDR-H2 sequence, which comprises the sequence VIRN-β5-β6-N-β8-YT-β 11 -β 12 -YNPSVKG (SEQ ID NO: 49), wherein β5 is K or R; β6 is A or P; β8 is G or A; β 11 is A or T; and β 12 is G or D;

[0033] (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;

[0034] (d) CDR-L1 sequence, which comprises the sequence QSSKSLLHS-δ 10 -GKTYLN (SEQ ID NO: 51), wherein δ 10 is N or T;

[0035] (e) a CDR-L2 sequence comprising the sequence WMSTRAS (SEQ ID NO: 8); and

[0036] (f) A CDR-L3 sequence comprising the sequence QQFLE-ϕ6-PFT (SEQ ID NO:52), wherein ϕ6 is Y or F.

[0037] 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.

[0038] In some embodiments, the antibody or antigen-binding fragment comprises:

[0039] (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

[0040] (b) 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:23, 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

[0041] (c) 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:25, 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

[0042] (d) 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:25, 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:23, 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

[0043] (e) 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

[0044] (f) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:13, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, 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

[0045] (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.

[0046] In some embodiments, the antibody or antigen-binding fragment comprises 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, V H The sequence has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, V H The sequence has at least 95% sequence identity to SEQ ID NO: 15. In some embodiments, V 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. In some embodiments, V H The sequence has at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, V 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. In some embodiments, V H The sequence has at least 95% sequence identity to SEQ ID NO: 79. In some embodiments, V H The sequence comprises SEQ ID NO:79.

[0047] In some embodiments, the antibody or antigen-binding fragment comprises 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, V L The sequence has at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, V L The sequence has at least 95% sequence identity to SEQ ID NO: 16. In some embodiments, V L The sequence comprises SEQ ID NO: 16. In some embodiments, V LThe sequence has at least 90% sequence identity to SEQ ID NO: 22. In some embodiments, V L The sequence has at least 95% sequence identity to SEQ ID NO: 22. In some embodiments, V 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. In some embodiments, V L The sequence has at least 95% sequence identity to SEQ ID NO: 68. In some embodiments, V L The sequence comprises SEQ ID NO:68.

[0048] In some embodiments, the antibody or antigen-binding fragment comprises:

[0049] (a) comprising V of SEQ ID NO: 15 H Sequence and V comprising SEQ ID NO: 16 L sequence; or

[0050] (b) V comprising SEQ ID NO: 19 H Sequence and V comprising SEQ ID NO:20 L sequence; or

[0051] (c) V comprising SEQ ID NO: 21 H Sequence and V comprising SEQ ID NO:20 L sequence; or

[0052] (d) V comprising SEQ ID NO: 19 H Sequence and V comprising SEQ ID NO:22 L sequence; or

[0053] (e) V comprising SEQ ID NO: 79 H Sequence and V comprising SEQ ID NO:22 L sequence; or

[0054] (f) V comprising SEQ ID NO: 24 H Sequence and V comprising SEQ ID NO:20 L sequence; or

[0055] (g) V comprising SEQ ID NO: 26 H Sequence and V comprising SEQ ID NO:20 L sequence; or

[0056] (h) V comprising SEQ ID NO: 24 H Sequence and V comprising SEQ ID NO:22 L sequence; or

[0057] (i) V comprising SEQ ID NO: 26 H Sequence and V comprising SEQ ID NO:22 L sequence; or

[0058] (j) V comprising SEQ ID NO: 2 H Sequence and V comprising SEQ ID NO:3 L sequence; or

[0059] (k) V comprising SEQ ID NO: 10 H Sequence and V comprising SEQ ID NO:11 L sequence; or

[0060] (l) V comprising SEQ ID NO: 24 H Sequence and V comprising SEQ ID NO:68 L sequence.

[0061] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to TREM2 comprises:

[0062] (a) a CDR-H1 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12 and 29;

[0063] (b) a CDR-H2 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 5, 13, 25, 30, 39, 41 and 43;

[0064] (c) a CDR-H3 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 6, 14, 17 and 31;

[0065] (d) a CDR-L1 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 7, 23 and 32;

[0066] (e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 8 and 33; and

[0067] (f) a CDR-L3 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 9, 18 and 34.

[0068] In some embodiments, the antibody or antigen-binding fragment comprises:

[0069] (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

[0070] (b) 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

[0071] (c) 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:23, 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

[0072] (d) 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:25, 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

[0073] (e) 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:25, 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:23, 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

[0074] (f) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, 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;

[0075] (g) 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

[0076] (h) 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:39, 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

[0077] (i) 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:41, 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

[0078] (j) 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:43, 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

[0079] (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.

[0080] 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.

[0081] In some embodiments, the antibody or antigen-binding fragment comprises:

[0082] (a) a V having at least 85% sequence identity to SEQ ID NO: 2 H A sequence having at least 85% sequence identity to SEQ ID NO: 3 L sequence; or

[0083] (b) a V having at least 85% sequence identity to SEQ ID NO: 10 H A sequence having at least 85% sequence identity to SEQ ID NO: 11 L sequence; or

[0084] (c) a V having at least 85% sequence identity to SEQ ID NO: 15 H A sequence having at least 85% sequence identity to SEQ ID NO: 16 L sequence; or

[0085] (d) a V having at least 85% sequence identity to SEQ ID NO: 19 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0086] (e) a V having at least 85% sequence identity to SEQ ID NO: 21 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0087] (f) a V having at least 85% sequence identity to SEQ ID NO: 19 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0088] (g) a V having at least 85% sequence identity to SEQ ID NO: 79 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0089] (h) a V having at least 85% sequence identity to SEQ ID NO: 24 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0090] (i) a V having at least 85% sequence identity to SEQ ID NO: 26 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0091] (j) a V having at least 85% sequence identity to SEQ ID NO: 24 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0092] (k) a V having at least 85% sequence identity to SEQ ID NO: 26 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0093] (l) a V having at least 85% sequence identity to SEQ ID NO: 27 H A sequence having at least 85% sequence identity to SEQ ID NO: 28 L sequence; or

[0094] (m) a V having at least 85% sequence identity to SEQ ID NO: 35 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0095] (n) a V having at least 85% sequence identity to SEQ ID NO: 37H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0096] (o) a V having at least 85% sequence identity to SEQ ID NO: 38 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0097] (p) a V having at least 85% sequence identity to SEQ ID NO: 40 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0098] (q) a V having at least 85% sequence identity to SEQ ID NO:42 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0099] (r) a V having at least 85% sequence identity to SEQ ID NO:44 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0100] (s) a V having at least 85% sequence identity to SEQ ID NO:45 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0101] (t) a V having at least 85% sequence identity to SEQ ID NO:46 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0102] (u) V having at least 85% sequence identity to SEQ ID NO: 24 H A sequence having at least 85% sequence identity to SEQ ID NO: 68 L sequence.

[0103] In some embodiments, the epitope recognized by the antibody or antigen-binding fragment thereof that specifically binds to TREM2 is the same or substantially the same as the epitope recognized by an antibody clone 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 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 CL0020188-18 188-6, clone CL0020188-7, clone CL0020188-8, clone CL0020307, ​​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.

[0104] In some embodiments, the epitope recognized by the antibody or antigen-binding fragment thereof is the same or substantially the same as the 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 specific 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 present disclosure provides 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 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 epitope recognized by the antibody or antigen-binding fragment thereof is the same or substantially the same as the 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 a specific embodiment, 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 present disclosure provides 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 143-149 of SEQ ID NO:1 (FPGESES (SEQ ID NO:69)).

[0105] In some embodiments, an antibody or antigen-binding fragment as disclosed herein reduces the level of soluble TREM2 protein (sTREM2). In some embodiments, an antibody or antigen-binding fragment as 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 sequence combination selected from the group consisting of: SEQ ID NO: 73 and 74; SEQ ID NO: 75 and 76; and SEQ ID NO: 77 and 78. In some embodiments, the potency assay is performed substantially as described in Example 11. In some embodiments, an antibody or antigen-binding fragment as disclosed herein enhances TREM2 activity. In some embodiments, the antibody or antigen-binding fragment thereof enhances phagocytosis or enhances migration, differentiation, function, or survival of myeloid cells, microglia, or macrophages. In some embodiments, the antibody or antigen-binding fragment thereof enhances microglial function without increasing neuroinflammation. In some embodiments, the antibody or antigen-binding fragment thereof enhances Syk phosphorylation. In some embodiments, the antibody or antigen-binding fragment thereof enhances Syk phosphorylation in the presence of a TREM2 ligand. In some embodiments, the antibody or antigen-binding fragment thereof exhibits cross-reactivity with a cynomolgus monkey TREM2 protein.

[0106] In some embodiments, the antibodies or antigen-binding fragments as disclosed herein are monoclonal antibodies. In some embodiments, the antibodies or antigen-binding fragments as disclosed herein are chimeric antibodies. In some embodiments, the antibodies or antigen-binding fragments as disclosed herein are humanized antibodies. In some embodiments, the antibodies or antigen-binding fragments as disclosed herein are fully human antibodies. In some embodiments, the antibodies or antigen-binding fragments as disclosed herein are Fab, F(ab')2, scFv or bivalent scFv.

[0107] In another aspect, the present disclosure provides antibodies or antigen-binding fragments thereof that compete with an isolated anti-TREM2 antibody as disclosed herein for binding to a human TREM2 protein.

[0108] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment as disclosed herein that specifically binds to TREM2 and a pharmaceutically acceptable carrier.

[0109] In another aspect, the present disclosure provides a kit comprising: an antibody or antigen-binding fragment as disclosed herein that specifically binds to TREM2, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment; and instructions for use thereof.

[0110] In another aspect, the present disclosure provides a method of 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 as disclosed herein, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment as disclosed herein.

[0111] In some embodiments, the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with Parkinson's disease associated with chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / parkinsonism-dementia syndrome (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, and symptomatic dementia. dementia), Guadeloupe PSP, Hallevorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroidization (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, dementia with neurofibrillary tangle predominance, 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 dementia with tangles only.

[0112] In yet another aspect, the present disclosure provides methods of reducing sTREM2 levels 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 as disclosed herein, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment as disclosed herein.

[0113] In yet another aspect, the present disclosure provides a method of 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 as disclosed herein, or a pharmaceutical composition comprising an anti-TREM2 antibody or antigen-binding fragment as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 Included are representative flow cytometry histograms showing binding of exemplary anti-TREM2 antibodies to surface TREM2 on TREM2-expressing HEK cells.

[0115] Figure 2 Included are representative dose response curves for pSyk signaling activation by exemplary anti-TREM2 antibodies in primary human macrophages. Solid black circles (●) represent anti-TREM2 antibodies, and open white circles (º) represent isotype controls.

[0116] Figure 3A and Figure 3B Including pretreatment with exemplary anti-TREM2 antibodies for 5 minutes ( Figure 3A ) or 24 hours ( Figure 3B ), followed by administration of lipid vesicles and assessment of the liposome response in the cells.

[0117] Figure 4 Included are representative dose response curves of NFAT-luciferase reporter activity in response to stimulation with exemplary anti-TREM2 antibodies in Jurkat NFAT cells expressing human TREM2 / DAP12. Solid black circles (●) represent anti-TREM2 antibodies, and open white circles (º) represent isotype controls.

[0118] Figure 5 Representative dose-response curves for cell survival in human macrophages in response to treatment with exemplary anti-TREM2 antibodies are shown.

[0119] Figure 6 Representative soluble TREM2 levels (sTREM2) are shown as a function of anti-TREM2 antibody concentration for exemplary anti-TREM2 antibodies.

[0120] Figure 7 is a bar graph indicating the mean pHrodo fluorescence intensity per cell in human macrophages treated with an exemplary anti-TREM2 antibody.

[0121] Fig. 8ARepresentative microscopy images of lipid accumulation in iPSC microglia treated with myelin followed by incubation with an exemplary anti-TREM2 antibody or isotype control.

[0122] Figure 8B is Fig. 8A Representative bar graph of Nile red staining (indicating lipid accumulation) of iPSC microglia imaged in .

[0123] Figures 8C-8F Figure 3 shows cholesterol ester species in iPSC microglia treated with myelin followed by incubation with exemplary anti-TREM2 antibodies ( Figure 8C and Fig. 8E ) and triglyceride lipid species ( Fig.8D and Fig.8F ) is a bar graph of the quantitative levels of . Fig. 8E and Fig.8F Data for iPSC microglia including a myelin elution step prior to incubation with an exemplary anti-TREM2 antibody.

[0124] Fig. 9 Representative mouse plasma pharmacokinetic profiles of exemplary anti-TREM2 antibodies are included.

[0125] Fig. 10A and Fig. 10B Included are shown injections of TREM2 cDNA KI (hu Trem2 KI / KI ) Total soluble TREM2 (sTREM2) in mouse plasma by exemplary anti-TREM2 antibodies in mice ( Fig. 10A ) and antibody binding to TREM2 ( Fig. 10B ) changes in the bar graph.

[0126] Fig.11A and Fig. 11B Included are dose-response binding curves of exemplary humanized and sequence-optimized anti-TREM2 antibodies to human TREM2 in HEK cells.

[0127] Fig. 12A and Fig. 12B Included are dose-response curves for pSyk signaling activation produced by exemplary humanized and sequence-optimized anti-TREM2 antibodies in HEK293-H6 cells.

[0128] Fig.13 Shown are dose-response curves for cell survival in human macrophages in response to treatment with exemplary humanized and sequence-optimized anti-TREM2 antibodies.

[0129] Fig.14A and 14BIncluded are dose-response curves for lipid clearance in iPSC microglia in response to treatment with exemplary humanized and sequence-optimized anti-TREM2 antibodies. DETAILED DESCRIPTION

[0130] I. Introduction

[0131] TREM2 is a transmembrane receptor expressed on the cell surface of microglia, dendritic cells, macrophages and osteoclasts. Not bound by a specific theory, it is believed that after ligand binding, TREM2 forms a signaling complex with transmembrane adapter proteins, DNAX activated protein 12 (DAP12), which is tyrosine phosphorylated by protein kinase SRC. It is believed that the activated TREM2 / DAP12 signaling complex mediates intracellular signaling by recruiting and phosphorylating kinases such as Syk kinases. TREM2 / DAP12 signaling regulates the activity of cells such as microglia and macrophages, such as phagocytosis, cell growth and survival, proinflammatory cytokine secretion and migration. TREM2 undergoes regulated intramembrane proteolysis, in which membrane-related full-length TREM2 is cut into sTREM2 parts that fall off from cells and membranes that are further degraded by γ secretase by metalloproteinase ADAM10 to retain C-terminal fragments. The level of sTREM2 has been reported to change in patients with Alzheimer's disease or frontotemporal dementia and with TREM2 mutations. Additionally, TREM2 mutations are associated with functional alterations such as impaired phagocytosis and reduced microglial function.

[0132] As described in detail 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 present disclosure provides anti-TREM2 antibodies and antigen-binding fragments thereof. Thus, in one aspect, the present disclosure provides anti-TREM2 antibodies and antigen-binding portions thereof.

[0133] In some embodiments, the anti-TREM2 antibody enhances TREM2 activity, such as enhancing phagocytosis or enhancing differentiation, function, migration, or survival of myeloid cells, microglia, or macrophages. Thus, in another aspect, a method of enhancing TREM2 activity in a subject, such as one with a neurodegenerative disease, is provided.

[0134] In some embodiments, the anti-TREM2 antibody reduces sTREM2 shedding. Thus, in another aspect, a method of reducing sTREM2 levels in a subject, e.g., with a neurodegenerative disease, is provided.

[0135] II. Definitions

[0136] As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" optionally includes a combination of two or more such molecules, and so forth.

[0137] As used herein, the terms "about" and "approximately" when used to modify a quantity specified as a value or range indicate the value and reasonable deviations from the value known to those skilled in the art, e.g., ± 20%, ± 10%, or ± 5%, are within the intended meaning of the value.

[0138] As used herein, the term "TREM2 protein" refers to a protein expressed by the gene TREM2 Encoded myeloid cell-expressed triggering receptor 2. As used herein, "TREM2 protein" refers to a native (i.e., wild-type) TREM2 protein of any vertebrate, such as, but not limited to, humans, non-human primates (e.g., cynomolgus monkeys), rodents (e.g., mice, rats), and other mammals. In some embodiments, the TREM2 protein is a human TREM2 protein having a sequence identified in UniprotKB accession number Q9NZC2 (SEQ ID NO: 1).

[0139] As used herein, the term "anti-TREM2 antibody" refers to an antibody that specifically binds to a TREM2 protein (e.g., human TREM2).

[0140] As used herein, the term "antibody" refers to a protein with an immunoglobulin fold that specifically binds to an antigen via its variable region. The term includes complete polyclonal antibodies, complete monoclonal antibodies, single-chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term "antibody" also includes antibody fragments that retain binding specificity via its variable region, including but not limited to Fab, F(ab')2, Fv, scFv, and bivalent scFv. Antibodies may contain light chains that are classified as κ or λ. Antibodies may contain heavy chains that are classified as γ, μ, α, δ, or ε, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0141] Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed primarily of two pairs of identical 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 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VL) are used interchangeably. H ) refer to these light and heavy chains, respectively.

[0142] The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is derived from a germline variable (V) gene, a diversity (D) gene, or a joining (J) gene (rather than from a constant (Cμ and Cδ) gene segment) and that confers specificity for binding to an antigen. Typically, an antibody variable region comprises four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions."

[0143] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for binding of the antibody to the epitope of the antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, which are numbered sequentially from the N-terminus and are also usually identified by the chain in which the particular CDR is located. Therefore, V H CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which the CDR is present, while V L CDR1 or CDR-L1 is CDR1 from the variable region of the light chain of an antibody in which the CDR is present.

[0144] The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework region of an antibody is the combined framework region of the constituent light and heavy chains, which is used to position and align the CDRs in three-dimensional space. The framework sequence can be obtained from a public DNA database or published reference that includes germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequences of human and mouse sequences.

[0145] The amino acid sequences of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics Database (IMGT), AbM, and observed antigen contacts ("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 defined by the Kabat, Chothia and / or Contact CDR definitions.

[0146] The terms "antigen binding portion" or "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 region. Examples of antigen binding fragments include, but are not limited to, Fab fragments (composed of VL, V H, CL and CH1 domains), F(ab')2 fragments (a bivalent fragment comprising 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).

[0147] The term "epitope" refers to the region or area of ​​an antigen to which the CDR of an antibody specifically binds and may include some amino acids or portions of some amino acids, such as 5 or 6 or more, such as 20 or more amino acids or portions of those amino acids. For example, when the target is a protein, the epitope may comprise continuous amino acids (e.g., a linear epitope) or amino acids from different parts of the protein that are close to each other by protein folding (e.g., a discontinuous or conformational epitope). In some embodiments, the epitope is phosphorylated at one amino acid (e.g., at a serine or threonine residue).

[0148] As used herein, the phrase "recognizing an epitope" as used with respect to an anti-TREM2 antibody means that the antibody CDR interacts with or specifically binds to an antigen (i.e., a TREM2 protein) at the epitope or a portion of the antigen containing the epitope.

[0149] As used herein, the term "multispecific antibody" refers to an antibody comprising two or more different antigen-binding portions, wherein each antigen-binding portion 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 via its variable region. As used herein, the term "bispecific antibody" refers to an antibody comprising two different antigen-binding portions, wherein each antigen-binding portion comprises a different variable region that recognizes a different antigen, or a fragment or portion of an antibody that binds to two different antigens via its variable region.

[0150] "Monoclonal antibody" refers to an antibody that is produced by a single clone of cells or a single cell line and consists or consists essentially of antibody molecules that are identical in primary amino acid sequence.

[0151] "Polyclonal antibody" refers to an antibody obtained from a heterogeneous population of antibodies, wherein different antibodies in the population bind to different epitopes of the antigen.

[0152] "Chimeric antibody" refers to an antibody molecule in which a constant region or a portion thereof is changed, replaced or exchanged so that an antigen binding site (i.e., a variable region, CDR, or a portion thereof) is connected to a constant region with a different or changed class, effector function, and / or species, or a variable region or a portion thereof is changed, replaced or exchanged by a variable region (e.g., CDR and framework regions from different species) with different or changed antigenic specificity. In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human). Methods for producing chimeric antibodies are described in the art.

[0153] "Humanized antibodies" are chimeric immunoglobulins derived from non-human sources (e.g., murine) that contain minimal sequences derived from non-human immunoglobulins outside the CDRs. In general, a humanized antibody will comprise at least one (e.g., two) antigen-binding variable regions, wherein the CDR regions substantially correspond to those of non-human immunoglobulins and the framework regions substantially correspond to those of human immunoglobulin sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin sequence. Methods for antibody humanization are known in the art.

[0154] "Human antibody" or "fully human antibody" is an antibody with human heavy and light chain sequences, which are generally derived from human germline genes. In some embodiments, antibodies are produced by human cells, by non-human animals utilizing human antibody repertoires (e.g., genetically engineered transgenic mice expressing human antibody sequences), or by phage display platforms.

[0155] The term "specific binding" refers to binding to an epitope or target with greater affinity, greater avidity, and / or greater duration of binding to an epitope or target in a sample than a molecule (e.g., an antibody or antigen binding portion thereof) binds to another epitope or non-target compound (e.g., a structurally different 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 times, e.g., at least 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times, 100 times, 1,000 times, 10,000 times, or greater affinity than other epitopes or non-target compounds. As used herein, the term "specific binding", "specifically binds to" a particular epitope or target, or "specific for" a particular epitope or target can be, for example, by having an equilibrium dissociation constant K of the epitope or target to which it binds. D The numerator is expressed as: 10 -4 M or smaller, e.g. 10 -5 M, 10 -6M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M. The skilled artisan will recognize 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 the target (e.g., a TREM2 protein).

[0156] The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules, such as an antibody (or antigen-binding portion thereof) and an antigen. Thus, for example, unless the context indicates otherwise or is obvious 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 can be measured by measuring the equilibrium dissociation constant (K D ), the equilibrium dissociation constant refers to the dissociation rate constant (k d ,time -1 ) divided by the association rate constant (k a ,time -1 M -1 ). D The kinetics of complex formation and dissociation can be determined, for example, by measuring the kinetics of complex formation and dissociation using: surface plasmon resonance (SPR) methods, such as the Biacore™ system; kinetic exclusion assays, such as KinExA ® ; and biolayer interferometry (e.g., using ForteBio ® Octet platform). As used herein, "binding affinity" includes not only formal binding affinities, such as those reflecting a 1:1 interaction between an antibody (or antigen-binding portion thereof) and an antigen, but also apparent affinities, such as those of K D Values ​​are calculated and may reflect affinity binding.

[0157] As used herein, the term "cross-reactivity" refers to the ability of an antibody to bind to an antigen other than the antigen from which the antibody was generated. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from another species other than the antigen from which the antibody was generated. As a non-limiting example, an anti-TREM2 antibody as described herein and 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).

[0158] The term "isolated" as used with respect to nucleic acids or proteins (e.g., antibodies) means that the nucleic acids or proteins are substantially free of other cellular components with which they are associated in the 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, the isolated nucleic acids or proteins (e.g., antibodies) are at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0159] 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 naturally occurring amino acids. Naturally occurring amino acids are those amino acids 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. Naturally occurring stereoisomers of 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 analogs" refers to compounds having the same basic chemical structure as naturally occurring amino acids (i.e., the alpha carbon is bound to a hydrogen, a carboxyl group, an amino group, and an R group), for example, 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 naturally occurring amino acids. "Amino acid mimetics" refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0160] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may contain complete L-amino acids, complete D-amino acids, or a mixture of L and D amino acids.

[0161] As used herein, the term "protein" refers to a dimer (ie, two) or multimer (ie, three or more) of a polypeptide or a single-chain polypeptide. The single-chain polypeptides of a protein may be linked by covalent bonds (eg, disulfide bonds) or non-covalent interactions.

[0162] The terms "polynucleotide" and "nucleic acid" refer interchangeably to nucleotide chains of any length, and include DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any matrix that can be incorporated into the chain by DNA or RNA polymerase. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides encompassed herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA.

[0163] The terms "conservative substitution" and "conservative mutation" refer to changes that result in one amino acid being substituted with another amino acid that can be classified as having similar characteristics. Examples of the categories of conservative amino acid groups defined in this manner may include: "charged / polar group", including 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); "aromatic group", including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "aliphatic group", including 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 the following: a "positively charged subgroup" comprising Lys, Arg and His; a "negatively charged subgroup" comprising Glu and Asp; and a "polar subgroup" comprising Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including the following: a "nitrogen ring subgroup" comprising Pro, His and Trp; and a "phenyl subgroup" comprising Phe and Tyr. In another other example, the aliphatic group can be subdivided into subgroups, such as an "aliphatic non-polar subgroup" comprising Val, Leu, Gly and Ala; and an "aliphatic weakly polar subgroup" comprising Met, Ser, Thr and Cys. Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to: substitution of Arg with Lys or vice versa, so that a positive charge can be maintained; substitution of Asp with Glu or vice versa, so that a negative charge can be maintained; substitution of Thr with Ser or vice versa, so that a free -OH can be maintained; and substitution of Asn with Gln or vice versa, so that a free -NH2 can be maintained. In some embodiments, a hydrophobic amino acid replaces a naturally occurring hydrophobic amino acid, such as in an active site, to maintain hydrophobicity.

[0164] In the context of two or more polypeptide sequences, the terms "identical" or "percent identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues over a specified 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 designated region using a sequence comparison algorithm or as measured by manual alignment and visual inspection.

[0165] For the sequence comparison of polypeptide, usually an amino acid sequence serves as a reference sequence for comparison with a candidate sequence. Alignment can be performed using the various methods available to those skilled in the art, such as visual alignment or using publicly available software to achieve maximum alignment using a known algorithm. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art. For the sequence comparison of the polypeptide sequence performed for this application purpose, the BLASTP algorithm standard protein BLAST is used for aligning two protein sequences with default parameters.

[0166] As used herein, the terms "subject," "individual," and "patient" 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.

[0167] The terms "treating / treatment" and similar terms are generally used herein to indicate obtaining a desired pharmacological and / or physiological effect. "Treatment" may refer to any marker of successful treatment or improvement of a neurodegenerative disease (e.g., Alzheimer's disease or another neurodegenerative disease described herein), including any objective or subjective parameter, such as relief, alleviation, improvement in patient survival, increased survival time or survival rate, alleviation of symptoms, or making the disease more tolerable to the patient, slowing down the rate of degeneration or debilitation, or improving the patient's physical or mental health. The treatment or improvement of symptoms may be based on objective or subjective parameters. The therapeutic effect may be compared with an individual or a group of individuals who have not received treatment, or with the same patient at different time points before or during treatment.

[0168] The term "pharmaceutically acceptable excipient" refers to an inactive drug ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, a buffer, a carrier, or a preservative.

[0169] As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent (e.g., an antibody as described herein) is an amount of the agent that treats, alleviates, removes, or reduces the severity of a subject's disease symptoms. A "therapeutic amount" of an agent (e.g., an antibody as described herein) can improve patient survival, increase survival time or survival rate, reduce symptoms, make an injury, disease, or disorder (e.g., a neurodegenerative disease) more tolerable, slow the rate of degeneration or debilitation, or improve the patient's physical or mental health.

[0170] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired biological site of action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, transdermal delivery, intramuscular delivery, intrathecal delivery, colon delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, an antibody as described herein is administered intravenously.

[0171] The term "control" or "control value" refers to a reference value or baseline value. An appropriate control can be determined by one of skill in the art. In some cases, a control value can be determined relative to a baseline within the same subject or experiment, for example, a sTREM2 measurement obtained 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, a control value can be determined relative to a control subject (e.g., a healthy control or a disease control) or an average value in a control subject population (e.g., a healthy control or a disease control, such as a population of 10, 20, 50, 100, 200, 500, 1000 or more control subjects), for example, a measurement of a subject's sTREM2 level at baseline or after treatment can be compared to a healthy control value.

[0172] III. Anti-TREM2 Antibodies

[0173] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to a TREM2 protein is provided. In some embodiments, the antibody specifically binds to a human TREM2 protein. In some embodiments, the anti-TREM2 antibody is selective for TREM2 relative to other TREM-like receptors (e.g., TREM1).

[0174] In some embodiments, the anti-TREM2 antibody is an antibody comprising one or more complementary determining regions (CDRs), heavy chain variable regions, and / or light chain variable region sequences as disclosed herein. In some embodiments, the anti-TREM2 antibody comprises one or more CDRs, heavy chain variable regions, and / or light chain variable region sequences as disclosed herein and further comprises one or more functional features as disclosed herein, such as antibodies that enhance TREM2 activity (e.g., enhance phagocytosis or enhance migration, differentiation, function, or survival of cells such as myeloid cells, microglia, or macrophages) or antibodies that reduce sTREM2 levels.

[0175] Anti-TREM2 antibody sequences

[0176] In some embodiments, the anti-TREM2 or antigen-binding fragment thereof comprises a heavy chain sequence or portion thereof and / or a light chain sequence or portion thereof derived from any of the following anti-TREM2 antibodies described herein: clone CL0020306, clone CL0020188, clone CL0020307, ​​and clone CL0020123. The CDRs, heavy chain variable regions, and light chain variable region amino acid sequences of these clones are set forth in the informal 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.

[0177] In some embodiments, the anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of:

[0178] (a) a heavy chain CDR1 (CDR-H1) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4, 12 and 29 or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 4, 12 and 29;

[0179] (b) a heavy chain CDR2 (CDR-H2) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 13, 25, 30, 39, 41 and 43 or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 5, 13, 25, 30, 39, 41 and 43;

[0180] (c) a heavy chain CDR3 (CDR-H3) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 6, 14, 17 and 31 or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 6, 14, 17 and 31;

[0181] (d) a light chain CDR1 (CDR-L1) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 7, 23 and 32 or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 7, 23 and 32;

[0182] (e) a light chain CDR2 (CDR-L2) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8 and 33 or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 8 and 33; and

[0183] (f) a light chain CDR3 (CDR-L3) sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 9, 18 and 34 or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 9, 18 and 34.

[0184] 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 at most two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, the CDR with at most two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, at most two amino acid substitutions are conservative substitutions.

[0185] In some embodiments, the anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of:

[0186] (a) a CDR-H1 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12 and 29;

[0187] (b) a CDR-H2 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 5, 13, 25, 30, 39, 41 and 43;

[0188] (c) a CDR-H3 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 6, 14, 17 and 31;

[0189] (d) a CDR-L1 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 7, 23 and 32;

[0190] (e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 8 and 33; and

[0191] (f) a CDR-L3 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 9, 18 and 34.

[0192] 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).

[0193] In some embodiments, the anti-TREM2 antibody comprises:

[0194] (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

[0195] (b) 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

[0196] (c) 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:23, 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

[0197] (d) 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:25, 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

[0198] (e) 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:25, 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:23, 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

[0199] (f) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:13, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, 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

[0200] (g) 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

[0201] (h) 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:39, 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

[0202] (i) 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:41, 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

[0203] (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.

[0204] 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 antibody comprises a heavy chain variable region comprising an 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.

[0205] 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.

[0206] 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, anti-TREM2 comprises: a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NO: 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 NO: 3, 11, 16, 20, 22, 28, 36, and 68.

[0207] In some embodiments, the anti-TREM2 antibody comprises:

[0208] (a) a V having at least 85% sequence identity to SEQ ID NO: 2 H A sequence having at least 85% sequence identity to SEQ ID NO: 3 L sequence; or

[0209] (b) a V having at least 85% sequence identity to SEQ ID NO: 10 H A sequence having at least 85% sequence identity to SEQ ID NO: 11 L sequence; or

[0210] (c) a V having at least 85% sequence identity to SEQ ID NO: 15 H A sequence having at least 85% sequence identity to SEQ ID NO: 16 L sequence; or

[0211] (d) a V having at least 85% sequence identity to SEQ ID NO: 19 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0212] (e) a V having at least 85% sequence identity to SEQ ID NO: 21 HA sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0213] (f) a V having at least 85% sequence identity to SEQ ID NO: 19 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0214] (g) a V having at least 85% sequence identity to SEQ ID NO: 79 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0215] (h) a V having at least 85% sequence identity to SEQ ID NO: 24 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0216] (i) a V having at least 85% sequence identity to SEQ ID NO: 26 H A sequence having at least 85% sequence identity to SEQ ID NO: 20 L sequence; or

[0217] (j) a V having at least 85% sequence identity to SEQ ID NO: 24 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0218] (k) a V having at least 85% sequence identity to SEQ ID NO: 26 H A sequence having at least 85% sequence identity to SEQ ID NO: 22 L sequence; or

[0219] (l) a V having at least 85% sequence identity to SEQ ID NO: 27 H A sequence having at least 85% sequence identity to SEQ ID NO: 28 L sequence; or

[0220] (m) a V having at least 85% sequence identity to SEQ ID NO: 35 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0221] (n) a V having at least 85% sequence identity to SEQ ID NO: 37 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0222] (o) a V having at least 85% sequence identity to SEQ ID NO: 38 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0223] (p) a V having at least 85% sequence identity to SEQ ID NO: 40 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0224] (q) a V having at least 85% sequence identity to SEQ ID NO:42 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0225] (r) a V having at least 85% sequence identity to SEQ ID NO:44 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0226] (s) a V having at least 85% sequence identity to SEQ ID NO:45 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0227] (t) a V having at least 85% sequence identity to SEQ ID NO:46 H A sequence having at least 85% sequence identity to SEQ ID NO: 36 L sequence; or

[0228] (u) V having at least 85% sequence identity to SEQ ID NO: 24 H A sequence having at least 85% sequence identity to SEQ ID NO: 68 L sequence.

[0229] In some embodiments, the anti-TREM2 antibody comprises one or more sequences covered by the consensus sequence disclosed herein. As a non-limiting example, the consensus sequence can be identified by aligning the heavy or light chain sequences (e.g., CDRs) of antibodies from the same (or similar) germline. In some embodiments, the consensus sequence can be generated by antibodies containing sequences having the same (or similar) length and / or having at least one highly similar CDR (e.g., highly similar CDR3). In some embodiments, such sequences in these antibodies can be aligned and compared to identify conservative amino acids or motifs (i.e., where changes in the sequence can change protein function) and / or regions where changes in the sequence occur (i.e., where changes in the sequence are unlikely to significantly affect protein function). Alternatively, the consensus sequence can be identified by aligning the heavy or light chain sequences (e.g., CDRs) of antibodies bound to the same or similar (e.g., overlapping) epitopes to determine conservative amino acids or motifs (i.e., where changes in the sequence can change protein function) and regions where changes occur in sequence alignments (i.e., where changes in the sequence are unlikely to significantly affect protein function). In some embodiments, one or more consensus sequences can be identified in 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 SEQ ID NOs: 47-52, capital letters represent amino acid residues that are absolutely conserved in the compared sequences (e.g., compared CDR sequences), while "X" or Greek letters (e.g., "α", "β", "γ", "δ", "ε" or "ε") represent amino acid residues that are absolutely conserved in the compared sequences (e.g., compared CDR sequences). ”) indicates an amino acid residue that is not absolutely conserved within the aligned sequences. It should be understood that when an amino acid is selected for insertion at a position marked by an “X” or a Greek letter, in some embodiments, the amino acid is selected from those found at the corresponding position in the aligned sequence.

[0230] Clone CL0020123 and variants of CL0020123

[0231] In some embodiments, the anti-TREM2 antibody or antigen-binding fragment thereof comprises:

[0232] (a) a CDR-H1 sequence comprising the sequence GFSIEDFYIH (SEQ ID NO: 29);

[0233] (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;

[0234] (c) a CDR-H3 sequence comprising the sequence HADHGNYGSTMDY (SEQ ID NO: 31);

[0235] (d) a CDR-L1 sequence comprising the sequence HASQHINVWLS (SEQ ID NO: 32);

[0236] (e) a CDR-L2 sequence comprising the sequence KASNLHT (SEQ ID NO: 33); and

[0237] (f) A CDR-L3 sequence comprising the sequence QQGQTYPRT (SEQ ID NO: 34).

[0238] In some embodiments, the anti-TREM2 antibody comprises a CDR-H2 sequence selected from SEQ ID NOs: 30, 39, 41, and 43.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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, 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, the 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.

[0245] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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 having 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.

[0246] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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, 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, the 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.

[0251] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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) with SEQ ID NO: 42. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 having 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) with SEQ ID NO: 36.

[0252] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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, 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, the 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.

[0257] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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) with SEQ ID NO: 45. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 having 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) with SEQ ID NO: 36.

[0258] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0259] Clones CL0020188, CL0020306, CL0020307, ​​and variants of CL0020188

[0260] In some embodiments, the anti-TREM2 antibody or antigen-binding fragment thereof comprises:

[0261] (a) a CDR-H1 sequence comprising the sequence GFTFT-α6-FYMS (SEQ ID NO: 48), wherein α6 is D or N;

[0262] (b) CDR-H2 sequence, which comprises the sequence VIRN-β5-β6-N-β8-YT-β 11 -β 12 -YNPSVKG (SEQ ID NO: 49), wherein β5 is K or R; β6 is A or P; β8 is G or A; β 11is A or T; and β 12 is G or D;

[0263] (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;

[0264] (d) CDR-L1 sequence, which comprises the sequence QSSKSLLHS-δ 10 -GKTYLN (SEQ ID NO: 51), wherein δ 10 is N or T;

[0265] (e) a CDR-L2 sequence comprising the sequence WMSTRAS (SEQ ID NO: 8); and

[0266] (f) A CDR-L3 sequence comprising the sequence QQFLE-ϕ6-PFT (SEQ ID NO:52), wherein ϕ6 is Y or F.

[0267] 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 selected from SEQ ID NOs: 9 and 18.

[0268] 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.

[0269] 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.

[0270] Clone CL0020188 and variants of CL0020188

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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, 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, the 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.

[0275] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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 having 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.

[0276] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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, 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, the 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.

[0281] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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) with SEQ ID NO: 79. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 having 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) with SEQ ID NO: 22.

[0282] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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: 22. 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: 22.

[0287] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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) with SEQ ID NO: 24. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 having 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) with SEQ ID NO: 22.

[0288] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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) with SEQ ID NO: 24. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 having 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) with SEQ ID NO: 68.

[0294] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as 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).

[0295] Clone CL0020306

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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) with SEQ ID NO: 2. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising light chain CDR1-3 having 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) with SEQ ID NO: 3.

[0301] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NO: 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).

[0302] Clone CL0020307

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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, 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, the 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.

[0307] In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising heavy chain CDR1-3 having 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 having 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.

[0308] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of SEQ ID NO: 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).

[0309] Binding characteristics of anti-TREM2 antibodies

[0310] In some embodiments, an antibody that specifically binds to a TREM2 protein as described herein binds to TREM2 expressed on a cell (e.g., a primary cell or cell line that endogenously expresses TREM2, such as a human macrophage, or a primary cell or cell line that is engineered to express TREM2, e.g., as described in the Examples section below). In some embodiments, an antibody that specifically binds to a TREM2 protein as described herein binds to a purified or recombinant TREM2 protein or a portion thereof, or to a chimeric protein comprising TREM2 or a portion thereof (e.g., an Fc-fusion protein comprising TREM2 or an Fc-fusion protein comprising an extracellular domain of TREM2).

[0311] In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with one or more other TREM2 proteins of another species. In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with a cynomolgus monkey ("cyno") TREM2 protein. In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with a mouse TREM2 protein. In some embodiments, an anti-TREM2 antibody exhibits cross-reactivity with human TREM2, cynomolgus monkey TREM2, and mouse TREM2.

[0312] 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)), dynamic exclusion assays (e.g., KinExA ® ), flow cytometry, fluorescence activated cell sorting (FACS), bio-layer interferometry (e.g., Octet™ (FortéBio, 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, dynamic exclusion assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, bio-layer interferometry is used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0313] Epitopes recognized by anti-TREM2 antibodies

[0314] In some embodiments, the epitope of human TREM2 recognized by the anti-TREM2 antibody is the same or substantially the same as the epitope recognized by the antibody clone as described herein. As used herein, as used with respect to the epitope recognized by the antibody clone as described herein, the term "substantially the same" means that the epitope recognized by the anti-TREM2 antibody is the same as the epitope recognized by the antibody clone as described herein, or within the epitope, or is almost the same as the epitope (e.g., having at least 90% sequence identity with the epitope or having one, two or three amino acid substitutions relative to the epitope, such as conservative substitutions), or has significant overlap with the epitope (e.g., having at least 50%, 60%, 70%, 80%, 90%, or 95% overlap).

[0315] In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that is identical or substantially identical to an epitope recognized by an antibody clone selected from the group consisting of clone CL0020306, clone CL0020188, clone CL0020307, ​​and clone CL0020123.

[0316] In some embodiments, the anti-TREM2 antibody binds to the TREM2 handle region of the epitope of human TREM2. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2, the epitope comprising residues 129-172 or residues 131-169 of SEQ ID NO:1, within the residues, or consisting of the residues. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2, the epitope comprising residues 129-148 of SEQ ID NO:1, within the residues, or consisting of the residues. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2, the epitope comprising amino acid residues 143-149 of SEQ ID NO:1, within the residues, or consisting of the residues. In some embodiments, the anti-TREM2 antibody is an agonist in the TREM2 handle region that activates TREM2 / DAP12 signaling (e.g., by inducing phosphorylation of kinases such as Syk) and binds to an epitope of human TREM2. In some embodiments, the anti-TREM2 antibody binds to the TREM2 stalk region of an epitope of human TREM2 and inhibits cleavage of TREM2 by a protease (e.g., ADAM17).

[0317] In some embodiments, the anti-TREM2 antibody binds to the TREM2 Ig variable (IgV) domain of the epitope of human TREM2. In some embodiments, the anti-TREM2 antibody is an agonist that activates TREM2 / DAP12 signaling (e.g., by inducing phosphorylation of kinases such as Syk) and binds to the TREM2 IgV domain of the epitope of human TREM2. In some embodiments, the anti-TREM2 antibody binds to an epitope of human TREM2 that comprises one or more of the following 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)).

[0318] Functional characterization of anti-TREM2 antibodies

[0319] In some embodiments, an anti-TREM2 antibody (e.g., an antibody as disclosed having one or more CDRs, heavy chain variable regions, and / or light chain variable region sequences) acts in one or more TREM2 activities as disclosed herein. For example, in some embodiments, an anti-TREM2 antibody is an antibody that modulates sTREM2 protein levels (e.g., sTREM2 levels shed from the cell surface into an extracellular sample), modulates recruitment or phosphorylation of kinases (e.g., Syk kinases) that interact with the TREM2 / DAP12 signaling complex, and / or modulates one or more activities downstream of the signaling complex (e.g., phagocytosis, cell growth, cell survival, cell differentiation, cytokine secretion, or cell migration). In some embodiments, an anti-TREM2 antibody as 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 essentially as described in Example 11.

[0320] In some embodiments, the anti-TREM2 antibody enhances one or more TREM2 activities induced by a ligand (e.g., those described herein). In some embodiments, the ligand is a lipid ligand. Examples of TREM2 lipid ligands include, but are not limited to, 1-palmitoyl-2-(5'-oxo-pentanoyl)-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-galactosylceramide (KRN7000), bis(monoacylglycerol) phosphate (BMP), cardiolipin (CL), ceramide, ceramide-1-phosphate (C1P), cholesterol ester (CE), cholesterol phosphate (CP), diacylglycerol 34:1 (DG 34:1), diacylglycerol 38:4 (DG 38:4), diacylglycerol pyrophosphate (DGPP), dihydroceramide (DhCer), dihydrosphingomyelin (DhSM), ether phosphatidylcholine (PCe), free cholesterol (FC), galactosylceramide (GalCer), galactosylsphingosine (GalSo), ganglioside GM1, ganglioside GM3, glucosylganglioside (GlcSo), Hank's Balanced Salt Solution (HBSS), Kdo2-lipid A (KLA), lactosylceramide (LacCer), lysoalkylacylglycerophosphocholine (LPAF), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysosynthesin (LSM), lysophosphatidylserine (LPS), N-acyl-phosphatidylethanolamine (NAPE), N-acyl-serine (NSer), oxygen The proteins included phosphatidylcholine (oxPC), palmitic acid-9-hydroxystearate (PAHSA), phosphatidylethanolamine (PE), phosphatidylethanol (PEtOH), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), dihydrosphingosine, dihydrosphingosine-1-phosphate (Sa1P), sphingomyelin (SM), sphingosine, sphingosine-1-phosphate (So1P), and sulfatide.

[0321] Regulation of sTREM2 shedding

[0322] In some embodiments, the anti-TREM2 antibodies alter sTREM2 protein levels in a sample, such as sTREM2 levels shed from the cell surface to an extracellular sample. In some embodiments, the anti-TREM2 antibodies reduce sTREM2 levels.

[0323] In some embodiments, 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%, at least 90% or more compared to the control value, then the anti-TREM2 antibody reduces the sTREM2 level. In some embodiments, if the amount of sTREM2 in the treated sample is reduced by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more compared to the control value, then the anti-TREM2 antibody reduces the sTREM2 level. In some embodiments, the control value is an untreated sample (e.g., a supernatant from a TREM2-expressing cell that has 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 treated with an appropriate non-TREM2 binding antibody.

[0324] In some embodiments, sTREM2 shedding is measured using a sample comprising a fluid, such as blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises cerebrospinal fluid. In some embodiments, the sample comprises supernatant from a cell culture (e.g., supernatant from primary cells or cell lines endogenously expressing TREM2, such as human macrophages, or primary cells or cell lines engineered to express TREM2, e.g., as described in the Examples section below).

[0325] 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 (EIA), such as enzyme multifactor immunoassays (EMIA), enzyme-linked immunosorbent assays (ELISA), microparticle enzyme immunoassays (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassays (CEIA), radioimmunoassays (RIA), immunofluorescence, chemiluminescent immunoassays (CL), and electrochemiluminescent immunoassays (ECL). In some embodiments, sTREM2 levels are measured using an ELISA assay. In some embodiments, sTREM2 levels are measured using an ELISA assay as described in the Examples section below.

[0326] Regulation of kinase recruitment or phosphorylation

[0327] In some embodiments, anti-TREM2 antibodies induce phosphorylation of kinases (e.g., but not limited to Syk, ZAP70, PI3K, Erk, AKT, or GSK3b) that interact with the TREM2 / DAP12 signaling complex. In some embodiments, anti-TREM2 antibodies induce phosphorylation of kinases that interact with the TREM2 / DAP12 signaling complex without blocking the binding of natural TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance phosphorylation induced by TREM2 ligands (e.g., lipid ligands) of kinases that interact with the TREM2 / DAP12 signaling complex. In some embodiments, anti-TREM2 antibodies induce or enhance phosphorylation of Syk. In some embodiments, if the level of Syk phosphorylation in a sample treated with an anti-TREM2 antibody increases 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%, at least 90% or more compared to a control value, then anti-TREM2 antibodies induce or enhance Syk phosphorylation. In some embodiments, if the level of Syk phosphorylation in a sample treated with an anti-TREM2 antibody increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a control value, then the anti-TREM2 antibody induces Syk phosphorylation. 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 treated with an appropriate non-TREM2 binding antibody.

[0328] In order to detect and / or quantify phosphorylation (e.g., Syk phosphorylation) in a sample, in some embodiments, an immunoassay is used. In some embodiments, the immunoassay is an enzyme immunoassay (EIA), an enzyme multifactor immunoassay (EMIA), an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA), an immunohistochemistry (IHC), an immunocytochemistry, a capillary electrophoresis immunoassay (CEIA), a radioimmunoassay (RIA), an immunofluorescence, a chemiluminescent immunoassay (CL), or an electrochemiluminescent immunoassay (ECL). In some embodiments, an immunoassay utilizing amplified luminescent proximity homogenous assay (AlphaLISA®, PerkinElmer Inc.) is used to detect and / or quantify phosphorylation.

[0329] In some embodiments, phosphorylation is measured using a sample comprising one or more cells, such as 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 are engineered to express TREM2, e.g., as described in the Examples section below). In some embodiments, the sample comprises a fluid, such as 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 a cell from such a tissue. In some embodiments, the sample comprises an endogenous fluid, tissue, or cell (e.g., from a human or non-human subject).

[0330] Regulation of phagocytosis

[0331] In some embodiments, anti-TREM2 antibodies enhance phagocytosis of dead cell debris, tissue debris, amyloid beta particles, or foreign matter. In some embodiments, anti-TREM2 antibodies enhance phagocytosis without blocking the binding of natural TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance phagocytosis induced by TREM2 ligands (e.g., lipid ligands). In some embodiments, if the level of phagocytosis in a sample treated with an anti-TREM2 antibody increases 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%, at least 90% or more compared to a control value, then the anti-TREM2 antibody enhances phagocytosis. In some embodiments, if the phagocytosis in a sample treated with an anti-TREM2 antibody increases by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more compared to a control value, then the anti-TREM2 antibody enhances phagocytosis. In some embodiments, the control value is the level of phagocytosis in an untreated sample, a sample that has been treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with an appropriate non-TREM2 binding antibody.

[0332] In some embodiments, phagocytosis is measured using a phagocytosis assay performed with a labeled matrix. Phagocytosis assays are known in the art. In some embodiments, a phagocytosis assay is performed on a sample comprising cells endogenously expressing TREM2, such as human macrophages or microglia. In some embodiments, a phagocytosis assay is performed on a sample comprising cells that have been engineered to express TREM2. In some embodiments, phagocytosis is measured using a human macrophage phagocytosis assay as described in the Examples section below.

[0333] Regulation of cell differentiation, function, migration and survival

[0334] In some embodiments, the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation (e.g., with respect to 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, anti-TREM2 antibodies enhance cell migration of one or more cell types (e.g., myeloid cells, macrophages, or microglial cells). In some embodiments, anti-TREM2 antibodies enhance cell survival of one or more cell types (e.g., myeloid cells, macrophages, or microglial cells). In some embodiments, anti-TREM2 antibodies enhance cell function of one or more cell types (e.g., myeloid cells, macrophages, or microglial cells). In some embodiments, anti-TREM2 antibodies enhance cell differentiation of one or more cell types (e.g., myeloid cells, macrophages, or microglial cells). In some embodiments, anti-TREM2 antibodies enhance migration, survival, function, and / or differentiation of myeloid cells. In some embodiments, anti-TREM2 antibodies enhance migration, survival, function, and / or differentiation of macrophages. In some embodiments, anti-TREM2 antibodies enhance migration, survival, function, and / or differentiation of microglial cells. In some embodiments, anti-TREM2 antibodies enhance microglial activation. In some embodiments, anti-TREM2 antibodies enhance migration, survival, function and / or differentiation of disease-associated microglial cells. In some embodiments, anti-TREM2 antibodies enhance cell migration, cell survival, cell function or cell differentiation without blocking the binding of natural TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance cell migration, cell survival, cell function or cell differentiation induced by TREM2 ligands (e.g., lipid ligands).

[0335] In some embodiments, if the level of activity (e.g., migration, survival, function, or differentiation) in a sample treated with an anti-TREM2 antibody increases 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%, at least 90% or more compared to a control value, then the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation. In some embodiments, if the level of activity (e.g., migration, survival, function, or differentiation) in a sample treated with an anti-TREM2 antibody increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more compared to a control value, then the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation. In some embodiments, the control value is an untreated sample (e.g., a sample that has not been treated with an anti-TREM2 antibody), a sample that has been treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with an appropriate non-TREM2 binding antibody.

[0336] In some embodiments, cell migration is measured using a chemotaxis assay. Chemotaxis assays are known in the art. In some embodiments, a cell migration assay (e.g., a chemotaxis assay) is performed on a sample comprising cells endogenously expressing TREM2, such as human macrophages. In some embodiments, a cell migration assay (e.g., a chemotaxis assay) is performed on a sample comprising cells that have been engineered to express TREM2. In some embodiments, cell migration is measured using a human macrophage chemotaxis assay as described in the Examples section below.

[0337] In some embodiments, cell viability is measured using a cell viability assay. Cell viability assays are known in the art. In some embodiments, a cell viability assay (e.g., a cell viability assay) is performed on a sample comprising cells endogenously expressing TREM2, such as human macrophages. In some embodiments, a cell viability assay (e.g., a cell viability assay) is performed on a sample comprising cells that have been engineered to express TREM2. In some embodiments, cell survival is measured using a human macrophage viability assay as described in the Examples section below.

[0338] In some embodiments, cell function is measured using a functional assay appropriate for the cell. For example, in some embodiments, macrophage function is assessed using a phagocytosis assay (e.g., as described in the Examples section below).

[0339] In some embodiments, cell differentiation is measured by assessing the ability of cells that endogenously express TREM2 to differentiate. For example, 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 below).

[0340] 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.

[0341] In some embodiments, anti-TREM2 antibodies enhance microglial function without increasing neuroinflammation. Neuroinflammation levels can be determined by measuring the levels 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 multifactor immunoassays (EMIA), enzyme-linked immunosorbent assays (ELISA), microparticle enzyme immunoassays (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassays (CEIA), radioimmunoassays (RIA), immunofluorescence, chemiluminescent immunoassays (CL), or electrochemiluminescent immunoassays (ECL).

[0342] IV. Preparation of Antibodies

[0343] In some embodiments, antibodies are prepared by immunizing one or more animals (e.g., mice, rabbits, or rats) with an antigen or antigen mixture for inducing an antibody response. In some embodiments, an antigen or antigen mixture and an adjuvant (e.g., Freund's adjuvant) are administered. After the initial immunization, one or more subsequent booster injections of the one or more antigens may be administered to increase antibody production. After immunization, antigen-specific B cells are harvested, for example, from spleen and / or lymphoid tissue. In order to generate monoclonal antibodies, B cells are fused with myeloid cells and subsequently screened for antigen specificity. Methods for preparing antibodies are also described in the following examples section.

[0344] The genes encoding the heavy and light chains of the target antibody can be cloned from cells, for example, the genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. The gene library encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Alternatively, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to the selected antigen. Antibodies can also be made bispecific, i.e., they can recognize two different antigens. Antibodies can also be heteroconjugates, such as two covalently linked antibodies or immunotoxins.

[0345] 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, such as a hybridoma or CHO cell expression system. Many such systems are widely available from commercial suppliers. H and V L In the embodiment of the region, V H and V L The regions may be expressed using a single vector, for example as a bicistronic expression unit or under the control of different promoters. H and V L The V region can be expressed using a separate vector. H or V L The region may optionally comprise a methionine at the N-terminus.

[0346] In some embodiments, the antibody is a chimeric antibody. Methods for making chimeric antibodies are known in the art. For example, a chimeric antibody can be made in which the antigen binding region (heavy chain variable region and light chain variable region) from one species, such as a mouse, is fused to the effector region (constant domain) of another species, such as a human. As another example, a "class switching" chimeric antibody can be made in which the effector region of the antibody is replaced by the effector region of a different immunoglobulin class or subclass.

[0347] In some embodiments, antibody is a humanized antibody.In general, non-human antibodies are humanized to reduce their immunogenicity.Humanized antibodies generally comprise one or more non-human (e.g., derived from mouse variable region sequences) variable regions (e.g., CDR) or portions thereof and possibly some non-human framework regions or portions thereof, and also comprise one or more constant regions derived from human antibody sequences.The method for making non-human antibody humanization is 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 making antibody humanization include, for example, variable domain resurfacing, CDR transplantation, transplantation specificity determining residues (SDR), guide selection and framework reorganization.

[0348] As an alternative to humanization, fully human antibodies can be generated. As a non-limiting example, the following transgenic animals (e.g., mice) can be produced, which can produce a complete library of human antibodies in the absence of endogenous immunoglobulins once they are immunized. For example, the homozygous deletion of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice has been described to completely suppress endogenous antibody production. The human germline immunoglobulin gene array is transferred to the germline mutant mice, which will result in the production of human antibodies when antigens are stimulated. As another example, human antibodies can be produced by a hybridoma-based method, for example, by using primary human B cells of a cell line for generating human monoclonal antibodies.

[0349] Human antibodies can also be produced using phage display or yeast display technology. In phage display, variable heavy chain and variable light chain gene pools are amplified and expressed in phage display vectors. In some embodiments, antibody libraries are natural libraries amplified from human sources. In some embodiments, antibody libraries are synthetic libraries made by cloning heavy chain and light chain sequences and recombining to generate larger antibody pools with different antigen specificities. Phages usually display antibody fragments (e.g., Fab fragments or scFv fragments), which are then screened for their combination with the target antigen.

[0350] In some embodiments, antibody fragments (e.g., Fab, Fab', F(ab')2, scFv, V H or V HH ). Various techniques for producing antibody fragments have been developed. Traditionally, these fragments are obtained via 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 method, 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.

[0351] In some embodiments, the antibody or antibody fragment is conjugated to another molecule, such as polyethylene glycol (PEGylation) or serum albumin, to provide a prolonged half-life in vitro.

[0352] In some embodiments, a multispecific antibody, e.g., a bispecific antibody, is provided comprising an anti-TREM2 antibody (or antigen-binding fragment thereof) as described herein. A multispecific antibody is an antibody having binding specificity to at least two different sites. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) has binding specificity to TREM2 and has binding specificity to 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) is capable of inducing TREM2 aggregation at the cell surface. Illustrative methods for measuring receptor aggregation using confocal FRET microscopy are described in Wallrabe et al., Biophys. J. , 2003, 85:559-571. Methods for making 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, "knob-into-hole" engineering, intramolecular trimerization, and fusing an antibody fragment to the N-terminus or C-terminus of another antibody, such as tandem variable domains.

[0353] V. Nucleic Acids, Vectors and Host Cells

[0354] In some embodiments, recombinant methods are used to prepare anti-TREM2 antibodies as disclosed herein. Thus, in some aspects, the disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding any anti-TREM2 antibody as described herein (e.g., any one or more of the CDRs, heavy chain variable regions, and light chain variable regions described herein); a vector comprising such nucleic acid; and a host cell introducing the nucleic acid for replication of the nucleic acid encoding the antibody and / or expression of the antibody.

[0355] In some embodiments, the polynucleotide (e.g., an isolated polynucleotide) comprises a nucleotide sequence encoding an antibody or antigen-binding portion thereof as described herein (e.g., as described in the section entitled "Anti-TREM2 Antibody Sequences" above). In some embodiments, the polynucleotide comprises a nucleotide sequence encoding one or more amino acid sequences disclosed in the following informal sequence table (e.g., CDR, heavy chain or light chain sequence). 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) with a sequence disclosed in the following informal sequence table (e.g., CDR, heavy chain or light chain sequence). In some embodiments, the polynucleotide as described herein is operably linked to a heterologous nucleic acid, such as a heterologous promoter.

[0356] Suitable vectors containing polynucleotides encoding antibodies of the present disclosure or fragments thereof include cloning vectors and expression vectors. Although the selected cloning vector may vary depending on the host cell intended for use, useful cloning vectors are generally capable of self-replication, may have a single target for a specific restriction endonuclease, and / or may carry genes for markers 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.

[0357] An expression vector is typically a replicable polynucleotide construct containing a nucleic acid of the present disclosure. The expression vector can be replicated in a host cell, either in episomal form or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), and any other vectors.

[0358] Host cells suitable for cloning or expressing polynucleotides or vectors as described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell, 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.

[0359] In another aspect, a method of making an anti-TREM2 antibody as described herein is provided. In some embodiments, the method comprises culturing a host cell as described herein (e.g., a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expressing the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0360] VI. Therapeutic approaches using anti-TREM2 antibodies

[0361] In another aspect, methods of treatment using an anti-TREM2 antibody as disclosed herein (e.g., an anti-TREM2 antibody as described in Section III above) are provided. In some embodiments, methods of treating a neurodegenerative disease are provided. In some embodiments, methods of modulating one or more TREM2 activities (e.g., in a subject with a neurodegenerative disease) are provided.

[0362] In some embodiments, a method for treating a neurodegenerative disease is provided. In some embodiments, the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with Parkinson's disease associated with chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / Parkinson's disease-dementia syndrome (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, familial British dementia, familial Danish dementia , GSS's disease, globular glial tauopathy, Guadeloupe Parkinsonism with dementia, Guadeloupe PSP, Hallerwarden-Spatz disease, hereditary diffuse leukoencephalopathy with globulin (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Na-Ha disease, neurofibrillary tangle dominant dementia, Niemann-Pick disease type C, globus pallidus-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. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neurodegenerative disease is Na-Ha 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 comprises administering to the subject an isolated antibody or antigen-binding fragment thereof that specifically binds to a human TREM2 protein (e.g., an anti-TREM2 antibody as described herein) or a pharmaceutical composition comprising an anti-TREM2 antibody as described herein.

[0363] In some embodiments, an anti-TREM2 antibody (or antigen binding portion thereof, or pharmaceutical composition) as described herein is 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, such as Alzheimer's disease characterized by an R47H mutation in TREM2.

[0364] In some embodiments, methods of regulating one or more TREM2 activities in a subject (e.g., a subject with a neurodegenerative disease) are provided. In some embodiments, the method includes regulating the level of sTREM2; regulating the recruitment or phosphorylation of a kinase (e.g., Syk kinase) that interacts with the TREM2 / DAP12 signaling complex; regulating phagocytosis (e.g., phagocytosis of cell debris, amyloid beta particles, etc.); regulating cell migration (e.g., migration of myeloid cells, macrophages, microglia, and disease-associated microglia); and / or regulating cell differentiation (e.g., about myeloid cells, macrophages, microglia, and disease-associated microglia). In some embodiments, methods of enhancing one or more TREM2 activities in a subject with a neurodegenerative disease are provided. In some embodiments, methods of reducing sTREM2 levels in a subject with a neurodegenerative disease are provided. In some embodiments, a method of modulating one or more TREM2 activities in a subject comprises administering to the subject an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a human TREM2 protein (e.g., an anti-TREM2 antibody as described herein) or a pharmaceutical composition comprising an anti-TREM2 antibody as described herein.

[0365] In some embodiments, the subject to be treated is a human, such as a human adult or a human child.

[0366] In some embodiments, a method for reducing plaque accumulation in a subject with a neurodegenerative disease is provided. In some embodiments, the method includes administering an antibody or pharmaceutical composition as described herein to a subject. In some embodiments, the subject suffers from Alzheimer's disease. In some embodiments, the subject is an animal model of a neurodegenerative disease (e.g., 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 anti-TREM2 antibodies 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 a subject before administration of anti-TREM2 antibodies).

[0367] In some embodiments, a therapeutically effective amount or dose of an anti-TREM2 antibody is administered to a subject. 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 can be used. However, the dosage may vary according to several factors, including the selected route of administration, the formulation of the composition, the patient's response, the severity of the illness, the subject's weight, and the judgment of the prescribing physician. The dosage may increase or decrease over time as required by individual patients. In some cases, a low dose is initially given to the patient and then increased to an effective dose that the patient can tolerate. The determination of the effective amount is entirely within the capabilities of those skilled in the art.

[0368] The route of administration of an anti-TREM2 antibody as described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, enteral, ocular or otic delivery, or any other method known in the art. In some embodiments, the antibody is administered orally, intravenously or intraperitoneally.

[0369] In some embodiments, an anti-TREM2 antibody (and optionally another therapeutic agent) is administered to a subject for an extended period of time, for example, for at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 days, or longer.

[0370] VII. Pharmaceutical Compositions and Kits

[0371] In another aspect, pharmaceutical compositions and kits are provided comprising antibodies that specifically bind to human TREM2 proteins. In some embodiments, the pharmaceutical compositions and kits are used to treat neurodegenerative diseases. In some embodiments, the pharmaceutical compositions and kits are used to modulate (e.g., enhance or inhibit) one or more TREM2 activities, such as Syk phosphorylation. In some embodiments, the pharmaceutical compositions and kits are used to modulate (e.g., reduce) sTREM2 levels.

[0372] Pharmaceutical composition

[0373] In some embodiments, a pharmaceutical composition comprising an anti-TREM2 antibody or an antigen-binding fragment thereof is provided. In some embodiments, the anti-TREM2 antibody is an antibody or an antigen-binding fragment thereof described in Section III above.

[0374] In some embodiments, the pharmaceutical composition comprises an anti-TREM2 antibody as described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating agent that is physiologically compatible and does not interfere with or otherwise inhibit the activity of the active agent. Various pharmaceutically acceptable excipients are well known in the art.

[0375] In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical or subcutaneous administration. The pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds, which are used, for example, to stabilize the composition or increase or decrease the absorption of the active agent. The physiologically acceptable compound may include, for example, a carbohydrate, such as glucose, sucrose or dextran; an antioxidant, such as ascorbic acid or glutathione; a chelating agent; a low molecular weight protein; a composition that reduces the clearance or hydrolysis of the active agent; or an excipient; or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are well known in the art.

[0376] The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, for example, by means of conventional mixing, dissolving, granulating, dragee making, emulsifying, encapsulating, embedding or lyophilizing processes. The following methods and excipients are exemplary only and are by no means limiting.

[0377] For oral administration, the anti-TREM2 antibody can be formulated by combining it with a pharmaceutically acceptable carrier well known in the art. Such carriers can formulate the compound into tablets, pills, lozenges, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the treated patient. Pharmaceutical preparations for oral use can be obtained by mixing the compound with a solid excipient, optionally grinding the resulting mixture, and processing the granular mixture after adding a suitable auxiliary agent as needed to obtain a tablet or lozenge core. 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, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrazolidone (PVP). If necessary, a disintegrant, such as cross-linked polyvinylpyrazolidone, agar, or alginic acid or a salt thereof such as sodium alginate, may be added.

[0378] Anti-TREM2 antibodies can be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. For injection, one or more compounds can be formulated into a formulation by dissolving, suspending or emulsifying the one or more compounds in an aqueous or non-aqueous solvent (e.g., vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol); and, if necessary, using conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the compound can be formulated in an aqueous solution, such as in a physiologically compatible buffer (e.g., Hanks's solution, Ringer's solution, or saline buffer). Formulations for injection can be presented in unit dosage form, such as in ampoules or multi-dose containers, with the addition of a preservative. The composition can be in the form of a suspension, solution, or emulsion, for example, in an oily or aqueous vehicle, and can contain a preparatant, such as a suspending agent, a stabilizer, and / or a dispersant.

[0379] In some embodiments, the anti-TREM2 antibody is prepared for delivery in a sustained release, controlled release, extended release, timed release, or delayed release formulation, for example, in a semipermeable matrix of a solid hydrophobic polymer containing an active agent. Various types of sustained release materials have been established and are well known to those skilled in the art. Current extended release formulations include film-coated tablets, multi-particulate or spherical systems, matrix technologies using hydrophilic or lipophilic materials, and wax-based tablets with pore-forming excipients. Sustained release delivery systems can release the compound over a time course of hours or days, such as 4, 6, 8, 10, 12, 16, 20, 24 hours or longer, depending on their design. Typically, sustained release formulations can be prepared using naturally occurring or synthetic polymers, such as polymerized vinyl pyrazolidones, such as polyvinyl pyrazolidones (PVP); carboxyvinyl hydrophilic polymers; hydrophobic and / or hydrophilic hydrocolloids, such as methylcellulose, ethylcellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; and carboxypolymethylene.

[0380] Typically, pharmaceutical compositions for in vivo administration are sterile. Sterilization can be accomplished according to methods known in the art, such as heat sterilization, steam sterilization, sterile filtration or irradiation.

[0381] The dosage and required drug concentration of the pharmaceutical composition of the present disclosure may vary according to the intended specific use. The determination of an appropriate dosage or route of administration is entirely within the skill of those skilled in the art. Suitable dosages are also described in Section VI above.

[0382] Pill Box

[0383] In some embodiments, a kit comprising an anti-TREM2 antibody or an antigen-binding fragment thereof is provided. In some embodiments, the anti-TREM2 antibody is an antibody or an antigen-binding fragment thereof described in Section III above.

[0384] In some embodiments, the medicine box also includes one or more additional therapeutic agents. For example, in some embodiments, the medicine box includes an anti-TREM2 antibody as described herein and also includes one or more additional therapeutic agents for treating neurodegenerative diseases such as Alzheimer's disease. In some embodiments, the therapeutic agent is an agent (e.g., an antidepressant, a dopamine agonist, or an antipsychotic) for treating cognitive or behavioral symptoms of neurodegenerative diseases. In some embodiments, the therapeutic agent is a neuroprotectant (e.g., carbidopa / levodopa, anticholinergics, dopaminergics, monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, glutamate, histone deacetylase (HDAC) inhibitors, caspase inhibitors, melatonin, anti-inflammatory agents, hormones (e.g., estrogen or progesterone), or vitamins).

[0385] In some embodiments, the kit comprises an anti-TREM antibody as described herein and further comprises one or more reagents for measuring sTREM2 levels. In some embodiments, the kit comprises an anti-TREM antibody as described herein and further comprises one or more reagents for measuring TREM2 activity (e.g., for measuring Syk phosphorylation).

[0386] In some embodiments, the kit also includes illustrative materials (e.g., instructions for using the kit to perform the methods of treatment described in Section VI above) containing guidelines (i.e., protocols) for practicing the methods described herein. Although illustrative materials typically include written or printed materials, they are not limited to such materials. The present disclosure encompasses any medium capable of storing such instructions and conveying the instructions to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CD-ROMs), etc. Such media may include Internet websites that provide such illustrative materials.

[0387] VIII. Examples

[0388] The present invention will be described in more detail with the aid of specific examples.The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way.

[0389] Example 1. Generation and initial characterization of anti-TREM2 antibodies

[0390] Recombinant expression and purification of mouse Fc-fused human TREM2 ECD

[0391] The extracellular domain (residues 19-172) of human TREM2 (UniProtKB ID - Q9NZC2) was subcloned into the pRK vector with a secretion signal from mouse IgG κ chain V-III, amino acids 1-20 (UniProtKB ID – P01661) were subcloned in the N-terminal region, and the mouse Fc tag was subcloned between the TREM2 ECD and Fc in the C-terminal region with GGGGS (SEQ ID NO:64).

[0392] Purified plasmids were transfected into Expi293F™ cells (Thermo Fisher) using the Expi293F™ expression system kit according to the manufacturer's instructions. To inhibit the maturation of N-linked glycans and reduce glycosylation heterogeneity, the high mannosidase I inhibitor kifunensine (Sigma) was added to the culture medium at a concentration of 1 μg / mL immediately after transfection. Transfected cells were incubated in a 6% CO2 humidified atmosphere at 125 rpm and 37°C on an orbital shaker (Infors HT Multitron). ExpiFectamine™ 293 transfection enhancers 1 and 2 were added to the cells 16 hours after transfection, and the culture supernatant was harvested 96 hours after transfection. EDTA-free protease inhibitors (Roche) were supplemented to the clarified supernatant and stored at -80°C.

[0393] For rhTREM2-Fc separation, the clarified culture supernatant was loaded on 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. 1M Tris-HCl buffer pH 8.0 was added to the protein solution immediately after elution 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 SEC mobile phase buffer was maintained at 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 50 mM arginine, which is also a protein storage buffer. All chromatography steps were performed on an AKTA pure or AKTA Avant system (GE Healthcare Life Sciences).

[0394] Recombinant expression and purification of His-tagged TREM2 ECD

[0395] The extracellular domain (residues 19-172) of TREM2 (UniProtKB - Q9NZC2) was subcloned into the pRK vector with a secretion signal from mouse Ig kappa chain V-III, amino acids 1-20 (UniProtKB ID – P01661) were subcloned in the N-terminal region, and a 6X-His tag (SEQ ID NO:65) was subcloned in the C-terminal region. The insert was verified by sequencing and maxi-prep plasmid purification was performed.

[0396] 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 in a 6% CO2 humidified atmosphere at 125 rpm and 37°C on an orbital shaker (Infors HT Multitron). ExpiFectamine™ 293 transfection enhancers 1 and 2 were added to the cells 16 hours after transfection, and the culture supernatant was harvested 96 hours after transfection.

[0397] 1M imidazole pH 8.0 to 10 mM final concentration is supplemented in the harvest culture medium and filtered using a Nalgene Rapid-Flow disposable filter unit (Thermo Fisher) with a pore size of 0.4 microns. HisPur Ni-NTA resin (Thermo Fisher) is washed with MQ water and balanced with loading buffer (20 mM Tris pH 8.0, 150 mM NaCl and 10 mM imidazoles). Affinity purification is carried out using a gravity flow method. The harvest culture medium is loaded onto the resin and non-specific binding proteins are washed with the loading buffer supplemented with 50 mM and 100 mM imidazoles. The His-tagged TREM2 extracellular domain bound is eluted with 20 mM Tris pH 8.0, 150 mM NaCl and 200 mM imidazoles. The eluted protein is concentrated using an Amicon 10 kDa concentrator and further purified using an AKTA Avant system (GE Healthcare Life Sciences) by gel filtration chromatography. The protein was loaded onto a HiLoad Superdex 200 16 / 600 (GE Healthcare Life Sciences) column balanced with 1x PBS and eluted and graded using 1x PBS as an electrophoresis buffer. The eluted fractions were analyzed by electrophoresis on a polyacrylamide (PAGE) gel under denaturing and native conditions. The eluted fractions were further characterized by analytical size exclusion chromatography and intact protein mass determination. The results from PAGE and analytical characterization were used to merge the highly glycosylated protein fractions and these fractions were aliquoted and stored at -80°C.

[0398] Antibody production

[0399] Rodents (mice and rats) were immunized using rhTREM2-Fc immunogens or BWZ cells expressing the full-length Trem2 receptor using standard protocols. Titers were measured using serum collected at different time points throughout the immunization. Flow cytometry was used to detect antigen-specific immune responses using rhTREM2-Fc immunogens and live BWZ cells expressing full-length TREM2. The selection criteria for candidate antibodies included rodent antibody production and specificity for binding to TREM2, as detected by flow cytometry. Antibody-secreting cells were isolated from animal immune tissues, including spleen, lymph nodes, and bone marrow.

[0400] Analyze single cell suspension to determine the binding properties of secreted antibodies. Antibody secreting cells are loaded into microfluidic devices and separated in nanoliter volume reaction chambers so that fluorescence and microscopy based on bright field images can be used to detect secreted antibodies (see, e.g., U.S. Patent No. 9,188,593). Binding assays are performed, which involve the binding of detection antibodies to antigen-coated microbeads, the binding of detection soluble fluorescently labeled antigens to antibodies fixed on beads, and the binding of detection antibodies to cell surface expressed antigens. Cell surface expressed antigens include recombinant and native forms of antigens presented on cell surfaces.

[0401] Image analysis is used to identify the chambers showing positive fluorescent signals, which indicates the presence of single cells producing antibodies with the desired properties, and the chamber contents are recovered and cracked in 384-well plates (see, for example, U.S. Patent No. 10,087,408). The single cell lysate is then subjected to RT-PCR to amplify heavy chain and light chain variable region sequences. The resulting amplicon is then sequenced to determine the cDNA sequence of the paired heavy chain and light chain variable region from the selected single cell. The resulting sequence is manually inspected and analyzed to determine sequence diversity and somatic hypermutation. Sequences are selected for expression based on screening data and sequence diversity. The expressed antibodies are tested to confirm antigen binding specificity.

[0402] Primary screening of anti-TREM2 antibodies

[0403] Primary screening of antibodies was performed in TREM2-expressing HEK 293 cells, wild-type iPSCs, and TREM2 knockout iPSCs as follows.

[0404] 1. Screening for TREM2 Binding in HEK Cells Expressing TREM2

[0405] HEK 293 cell lines stably expressing human TREM2 / DAP12 were generated by transfecting cells with vectors expressing wild-type human TREM2 and DAP12 and DAP12 alone. Stable expression clones were selected and cell surface TREM2 expression was assessed by flow cytometry. Surface TREM2 expression was detected using APC-bound rat anti-human / mouse TREM2 monoclonal antibody (R&D, catalog number MAB17291). The clone showing the highest wild-type TREM2 expression level was selected and named "HEK293-H6". Clones stably expressing DAP12 were analyzed by Western blotting, and the selected clones were named "HEK293-DAP12#1".

[0406] HEK 293 overexpressing human TREM2 (HEK293-H6) and HEK 293 overexpressing GFP (B5) were harvested by 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 plated at 10% for each cell line. 6 / mL density was resuspended in FACS buffer with human TrustainFcX solution (Biolegend, catalog number 422302). The mixed cell line was seeded in a 96-well round bottom plate at 200,000 cells / well and incubated at room temperature for 20 minutes. After incubation, the cells were centrifuged and incubated on ice for 45 minutes with anti-TREM2 antibodies titrated at about 0 – 200 nM. After incubation, the cells were centrifuged and washed three times with FACS buffer. The cells were then incubated on ice for 30 minutes with secondary antibodies (Alexa Fluor 647 AffiniPure F(ab')2 fragment goat anti-human IgG (H+L), Jackson ImmunoResearch Laboratories, catalog number 109-606-088, 1:800 dilution). After incubation, cells were washed three times with FACS buffer, resuspended in 100 μL FACS buffer, and analyzed by flow cytometry (BD FACSCanto II, San Jose, CA), acquiring 30,000 events for each sample. The mean fluorescence intensity of each cell was calculated by FlowJo software and used to generate dose-response binding curves.

[0407] Figure 1 Representative results are shown for exemplary antibodies binding to the cell surface receptor TREM2 in HEK293-H6 cells.

[0408] Assessment of activation of TREM2-dependent pSyk signaling

[0409] The activation of TREM2-dependent pSyk signaling was measured using the commercial AlphaLisa assay from Perkin-Elmer in human macrophages or HEK293-H6 cells.

[0410] For all experiments involving the use of lipid vesicles containing 70% DOPC and 30% POPS, lipid vesicles were prepared within two weeks of the experiment as follows: 7 mg DOPC (1,2-dioleoyl- s -glycero-3-phosphocholine) and 3 mg POPS (1-palmitoyl-2-oleoyl- s-glycerol-3-phospho-L-serine) was combined in chloroform in a glass vial and dried under a stream of N2 gas for 1-2 hours or until completely dry. The lipid mixture was resuspended in 1 mL HBSS (final lipid concentration was about 10 mg / mL) and vortexed for 2-3 minutes. Subsequently, the lipid suspension was extruded using an Avanti micro extruder constructed with a 100-nm pore size membrane to form 10 mg / mL small unilamellar vesicles.

[0411] 1. Administration of Antibodies into Cells

[0412] The day before the assay, human macrophages or HEK293-H6 cells were seeded on 96-well plates coated with poly-D-lysine at 100,000 cells / well or 40,000 cells / well, respectively. The antibody was diluted into PBS starting at 300 nM and titrated at 10-point serial dilutions, with 3-fold dilutions between each point. For the antagonist dose-response curve, lipid vesicles containing 70% DOPC and 30% POPS at a final concentration of 1 mg / mL were also included in the antibody / PBS mixture. The cells were washed 3 times with HBSS using a Biotek 405 / 406 plate washer, after which 50 μL of antibody / PBS (with or without vesicles) was added to each well using a Hamilton Nimbus liquid handler. The plate was then transferred to a 37°C incubator for 5 minutes. The liposome / antibody solution was removed by tapping the plate, and 40 μL of lysis buffer (Cell Signaling Technologies, CST) containing 1 μM PMSF was added using a liquid handler. Lysates were then either frozen at -80°C or immediately assayed with the AlphaLisa assay.

[0413] Human macrophages are prepared for following determination. According to RosetteSep human monocyte enrichment mixed scheme (Stemcell Technologies, REF#15068), human monocytes are separated from fresh blood. The isolated monocytes are washed with washing buffer (PBS+2% FBS) and resuspended in 10 mL ACK lysis buffer (ThermoFisherScientific, catalog number A10492) to crack red blood cells. Add 20 mL washing buffer to stop cell lysis, and the sample is centrifuged and washed with culture medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids and 1% penicillin-streptomycin) once more. Then in 250-mL flasks, in culture medium, in the presence of 50 ng / mL mankind recombinant M-CSF (Gibco, catalog number PHC9501), human monocytes are differentiated into macrophages. Fresh human M-CSF was spiked on day 3 and human macrophages were subsequently harvested on day 5 and used for the assay.

[0414] 2. AlphaLisa Assay

[0415] The pSyk of cell lysate was determined using the standard protocol of Perkin Elmer pSyk AlphaLisa test kit. In brief, 10 μL lysate / well was transferred to a white opaque 384-well Optiplate (Perkin Elmer). Then, 5 μL Acceptor Mix (working solution containing acceptor beads) was added to each well, followed by sealing with a foil seal and incubating at room temperature for 1 hour. Subsequently, 5 μL Donor Mix (working solution containing donor beads) was added to each well under reduced illumination conditions. The plate was sealed again and incubated at room temperature for 1 hour. Finally, the plate was read using AlphaLisa settings on a Perkin Elmer EnVision plate reader.

[0416] Figure 2 Representative anti-TREM2 antibody dose-response curves for pSyk signaling activation in primary human macrophages are shown. Solid black circles (●) represent anti-TREM2 antibodies, and open white circles (º) represent isotype controls. Each curve represents the mean of three independent experiments, and the EC 50 The values ​​are provided in Table 1 below. The results indicate that anti-TREM2 antibodies are able to activate TREM2-DAP12 ITAM signaling in primary human macrophages.

[0417] Liposome response assay in iPSC-microglia

[0418] TREM2 agonist antibodies and phosphatidylserine-containing liposomes activate pSyk via TREM2. To understand the effect of anti-TREM2 antibodies on Syk signaling in the presence of liposomes, iPSC microglia were pretreated with anti-TREM2 antibodies and the liposome response in the cells was then assessed.

[0419] Before 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. Once the floating cells in the co-culture were primarily identified as mature microglia (>80%), the microglia were used for the assay.

[0420] Two days before the assay, human iPSC microglial cells were seeded on 96-well plates coated with poly-D-lysine at 30,000 cells / well. Antibodies were diluted to 100 nM in a culture medium containing IMDM, 10% Hyclone FBS and 1% penicillin-streptomycin, and antibody solutions were given to the cells for 24 hours or for 5 minutes at 37 ° C. Subsequently, cells were washed once with HBSS and then given 1 mg / mL lipid vesicles containing 70% DOPC and 30% POPS at 37 ° C for 5 minutes. Liposome solution was removed by tapping the plate, and 30 μL of lysis buffer (Cell Signaling Technologies, CST) containing 1 μM PMSF was added. The lysate was then frozen at -80 ° C or immediately assayed with AlphaLisa. The pSyk of cell lysates was determined using the standard protocol of the Perkin Elmer pSyk AlphaLisa kit as described above.

[0421] Figure 3A and Figure 3B Figure 2 shows pSyk signal activation in human iPSC microglia cells incubated with anti-TREM2 antibodies, followed by administration of lipid vesicles and assessment of liposome response in the cells. White bars indicate incubations performed using PBS instead of lipid vesicles as controls. Data represent the mean and standard error of 2-7 independent experiments. Figure 3A Data for iPSC microglia pretreated with antibodies for 5 minutes are shown, and Figure 3BData for iPSC microglia pretreated with antibodies for 24 hours are shown. The results show that pretreatment of human iPSC microglia with anti-TREM2 antibodies increased the phospho-Syk signal triggered by liposomes compared to isotype controls, indicating that anti-TREM2 antibodies do not interfere with, but rather enhance, lipid activation of pSyk signaling in cells.

[0422] Human TREM2 NFAT reporter assay

[0423] As follows, human TREM2 / DAP12 expression Jurkat NFAT cell line was generated. Jurkat NFAT reporter cells were infected with a lentiviral vector expressing human TREM2 and DAP12 and cultured in RPMI containing 10% Hyclone FBS and 1% avidin / streptomycin. Stably expressed clones were selected in the presence of puromycin and Zeocin. Cell surface TREM2 expression was assessed using biotinylated anti-TREM2 antibodies (SEQ ID NO: 66 and 67) by flow cytometry. The clone showing the highest wild-type TREM2 expression level was selected and named hTrem2 / NFAT Jurkat reporter cells for use in the assay described below.

[0424] One day before the assay, 96-well plates were pre-coated with anti-TREM2 antibodies or isotype controls at 0-500 nM dose titration (45 μL / well, 12 points total) and incubated overnight at 4°C. After overnight incubation, the pre-coated plates were washed twice with PBS and then loaded with hTrem2 / NFAT Jurkat reporter cells (10 6 Cells / well). The plates were incubated at 37°C for 24 hours, after which 50 μL / well 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 signal measurement by a photometer (Perkin Elmer Envision).

[0425] Figure 4 Included are representative anti-TREM2 antibody dose response curves for activation of NFAT as measured by detection of the reporter gene luciferase, and activated EC 50 The values ​​are provided in Table 1 below. Figure 4 The results in Figure 3 indicate that candidate anti-TREM2 antibodies are able to induce NFAT activation and sufficient downstream signaling to activate transcriptional responses relative to isotype controls.

[0426] Survival assay of human macrophages

[0427] Human monocytes were isolated according to the RosetteSep human monocyte enrichment hybrid protocol (Stemcell Technologies, catalog number 15068). The isolated monocytes were washed with wash buffer (PBS + 2% FBS) and resuspended in 10 mL ACK lysis solution (ThermoFisher Scientific, catalog number A10492) to lyse red blood cells. 20 mL of wash buffer was added to terminate the lysis. The cell suspension was centrifuged and washed once with culture medium (RPMI 1640 + 10% FBS + penicillin / streptomycin). The cells were incubated for 10 min at 4 °C for 2 h. 6 The cells were resuspended in culture medium at a density of 10 cells / mL and used in the survival assay described below.

[0428] One day before the assay, 96-well plates were pre-coated with anti-TREM2 antibodies or isotype controls at 0-200 nM dose titration (45 μL / well, 12 points total) and incubated overnight at 4°C. After overnight incubation, the pre-coated plates were washed twice with PBS and then loaded with human monocytes (10 5 cells / well). After 5 days at 37°C, the culture medium was aspirated and 100 μL PBS + 100 μL Celltiter-glo medium (Promega, catalog number G7571) was added to each well. After incubation for 10 minutes, the cell culture medium was transferred to a multi-well plate compatible for luminometer use and the luminescence of cell viability was recorded.

[0429] Figure 5 Representative anti-TREM2 antibody dose-response curves showing cell survival in human macrophages under low M-CSF conditions and surviving EC 50 The values ​​are provided below in Table 1. The results indicate that the TREM2 agonist antibodies have sufficient receptor activation capacity to induce transcriptional responses for modulating cellular function and promoting survival of human macrophages under M-CSF conditions.

[0430] Biacore kinetic measurements of antibodies

[0431] Surface plasmon resonance (Biacore™ 8K instrument) was used to measure the affinity of anti-TREM2 antibodies to human and cynomolgus TREM2 ECD. Anti-TREM2 antibodies were captured on a Biacore Series S CM5 sensor chip (GE Healthcare Life Sciences, catalog number 29149604) using a human Fab capture kit (GE Healthcare Life Sciences, catalog number 28958325). Serial 3-fold dilutions of recombinant human or cynomolgus TREM2 were injected at a flow rate of 30 µL / min. Antibody binding was monitored for 300 seconds, followed by antibody dissociation for 600+ seconds in HBS-EP+ electrophoresis buffer (GE Healthcare Life Sciences, catalog number BR100669). The binding response was corrected by subtracting the RU value from the blank flow cell. The k on and k off The kinetic analysis was performed by fitting a 1:1 Languir model. D The binding values ​​are provided in Table 1 below.

[0432] Table 1. In vitro characteristics of antibodies

[0433]

[0434] NB: No binding detected

[0435] ND: Not Determined

[0436] Example 2. Regulation of soluble TREM2 levels and phagocytosis behavior in human macrophages

[0437] Soluble TREM2 dose-response assay in human macrophages

[0438] Generate human macrophages as described above. The day before the assay, human macrophages were seeded on 96-well plates coated with poly-D-lysine at 100,000 cells / well. The antibody was diluted with human macrophage culture medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% penicillin-streptomycin), starting from 300nM and continuing with 10-point serial dilution titration, with 3 times dilution between each point. The antibody was given to the cells and incubated for 24 hours. After incubation with the antibody, the plate was centrifuged to remove debris, and the supernatant was collected for soluble TREM2 measurement.

[0439] Soluble TREM2 was measured as follows. In brief, MSD small spot streptomycin plates (Meso Scale Discovery) were coated with biotinylated anti-hTREM2 monoclonal antibodies (R&D Systems) at 4°C overnight. The plates were then blocked with 3% BSA / TBST for 1 hour at room temperature. Samples and standards were prepared with SDS buffer by heating to 95°C for 5 minutes. The prepared samples and standards were diluted with 3% BSA / TBST 1:10 in the assay plate after blocking. TREM2-His protein diluted with 3% BSA / TBST was used as a standard for absolute quantification. After incubation for two hours at room temperature, the plates were washed with TBST. The primary detection antibody was diluted with 3% BSA / TBST of goat anti-human TREM2 (R&D Systems) labeled with sulfo group, added to the plate, and incubated for one hour at room temperature. After washing with TBST, 2x MSD was used to read buffer T to develop the MSD plate, followed by detection using an MSD Sector plate reader. MSD values ​​were converted to absolute amounts of sTREM2 by fitting the standard curve using Prism 7.0 software (Graphpad). Regulation of TREM2 shedding was expressed as the ratio of soluble TREM2 from cells incubated with the test anti-TREM2 antibodies normalized to soluble TREM2 from cells not cultured in culture medium with the specific anti-TREM2 antibodies.

[0440] Figure 6 Representative soluble TREM2 levels (sTREM2) are shown as a function of anti-TREM2 antibody concentration. The results indicate that anti-TREM2 antibodies are able to reduce sTREM2 levels in human macrophages in a dose-dependent manner after overnight treatment.

[0441] Phagocytosis assay in human macrophages

[0442] Human macrophages were generated as described above. Two days before the assay, human macrophages were seeded on 96-well plates coated with poly-D-lysine at 80,000 cells / well. The antibodies were diluted to 100 nM in a medium containing RPMI, 10% HycloneFBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% penicillin-streptomycin. The antibody solution was then given to the cells at 37°C for 24 hours. The nuclei and cell membranes were then stained for 10 minutes, after which 5 µg / mL of pHrodo-myelin was added. The cells were then incubated at 37°C for 4 hours. The pHrodo fluorescence of each cell was measured on a high-capacity confocal microscope (OperaPhoenix), and the fluorescence intensity was quantified on the instrument software.

[0443] By using Safaiyan et al. (2016, Nature Neuroscience pHrodo-myelin was prepared by purifying myelin from wild-type C57Bl / 6 mouse brain (Jackson Laboratories) as described in BioRad (19(8):995-998). After purification, myelin was resuspended in PBS and adjusted to 1 mg / mL protein concentration using DC Protein Assay Kit 2 (BioRad, catalog number 5000112). Myelin was labeled with pHrodo-red using a microlabeling kit (ThermoFisher, catalog number P35363) according to the manufacturer's instructions. Excess labeling was removed by precipitating myelin at 10,000 g for 5 minutes, removing the supernatant, and repeating these steps 3-5 times.

[0444] Figure 7 Representative results of a phagocytosis assay in human macrophages are shown. Myelin phagocytosis was measured by detecting and quantifying pHrodo fluorescence in microscopic images of TREM2-treated macrophages and comparing the measurements to those of isotype controls. The results showed that human macrophages treated with exemplary TREM2 agonist antibodies had increased pHrodo-myelin phagocytosis relative to isotype controls, suggesting that anti-TREM2 antibodies can beneficially clear myelin debris from cells.

[0445] Example 3. Regulation of lipid accumulation in iPSC microglia

[0446] Lipid storage assay

[0447] Before 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 a plate containing primary human astrocytes and co-cultured for 14-21 days. Once the floating cells in the co-culture were primarily identified as mature microglial cells (>80%), the microglial cells were used for the assay.

[0448] Cells (iPSC microglia, 30,000 cells / well) were seeded in complete serum medium in PDL-coated 96-well plates. After 24 h at 37°C, purified unlabeled myelin (50 μg / mL final concentration, using Safaiyan et al. (2016, Nature Neuroscience19(8):995-998) was incorporated into the wells. After lipid treatment for 24 hours at 37°C, anti-TREM2 antibodies or RSV controls were incorporated into the wells to a final concentration of 100 nM. The cells were incubated at 37°C for an additional 48-72 hours before harvesting or imaging the cells. For myelin washout experiments, myelin was removed after a 24-hour incubation period and replaced with antibody-containing medium for a subsequent 24-48 hour incubation.

[0449] For Nile red imaging, supernatant was removed and cells were incubated at 37°C for 30 minutes in live cell imaging buffer (LifeTechnologies, catalog number A14291DJ) containing 1 μM Nile red (ThermoFisher, catalog number N1142) and 1 drop / mL Nucblue (ThermoFisher, catalog number R37605). After the incubation period, staining solution was removed and cells were fixed in 4% paraformaldehyde. Alexa 568 and DAPI irradiation settings were then used on Opera Phoenix high-capacity confocal imager to image cells. Liquid spots were analyzed using a spot finding algorithm using the Harmony software providing the instrument. Fig. 8A Included are representative microscopy images of iPSC microglia treated with vehicle or myelin (50 μg / mL final concentration) for 24 hours followed by incubation with isotype control or exemplary anti-TREM2 antibodies (CL0020123) for 72 hours. Figure 8B is used for Fig. 8A Representative bar graph of the same anti-TREM2 antibody in microscopy images of . Nile red staining was quantified by total spot intensity per cell and data are shown as the mean and standard deviation of three technical replicates within different fields of the same microscopy sample.

[0450] For lipidomics 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 a 96-well plate. The plate was agitated on a shaker at 4°C and 1200 rpm for 20 minutes and then centrifuged at 300 xg for 5 minutes. A 50 μL sample of the supernatant was transferred to an LCMS vial and kept at -80°C until analyzed on the instrument.

[0451] Liquid levels were analyzed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled to an electrospray mass spectrometer (QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 5 µL of sample was injected into a BEHC18 1.7 µm, 2.1 × 100 mm column (Waters Corporation, Milford, Massachusetts, USA) at 55 °C using a flow rate of 0.25 mL / min. For positive ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) and 10 mM ammonium formate + 0.1% formic acid; mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) and 10 mM ammonium formate + 0.1% formic acid. For negative ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) and 10 mM ammonium acetate; mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) and 10 mM ammonium acetate. The gradient was programmed as follows: 0.0–8.0 min from 45% B to 99% B, 8.0–9.0 min at 99% B, 9.0–9.1 min to 45% B, and 9.1–10.0 min at 45% B. Electrospray ionization was performed in positive or negative ion mode using the following settings: curtain gas at 30 °C; collision gas at mid setting; ion spray voltage at 5500 (positive mode) or 4500 (negative mode); temperature at 250 °C (positive mode) or 600 °C (negative mode); ion source gas 1 at 50 °C; ion source gas 2 at 60 °C. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM) with the following parameters: residence time (msec) and collision energy (CE); declustering potential (DP) at 80°; entrance potential (EP) at 10° (positive mode) or -10° (negative mode); and collision cell exit potential (CXP) at 12.5° (positive mode) or -12.5° (negative mode). Liquids were quantified using a mixture of non-endogenous internal standards. Liquids were identified based on their retention time and the MRM characteristics of commercially available reference standards (Avanti Polar Lipids, Birmingham, AL, USA).

[0452] Figure 8C and Fig.8D Cholesteryl esters (CE) as detected by mass spectrometry are shown in cell lysates of iPSC microglia treated with an exemplary anti-TREM2 antibody for 72 hours following a 24-hour myelin treatment ( Figure 8C ) and triacylglycerol (TAG) lipid species ( Fig.8D ) level. Fig. 8E and Figure 8F Figure 3 shows cholesteryl esters (CE) as detected by mass spectrometry in cell lysates of iPSC microglia subjected to a myelin elution experiment using an exemplary anti-TREM2 antibody ( Fig. 8E ) and triacylglycerol (TAG) lipid species ( Fig.8F ) level. Figures 8C-8F The LC / MS data generated in were normalized to an internal standard for CE data and to a myelin + isotype control for each individual lipid species for TAG data.

[0453] Lipid accumulation in iPSC microglia was induced by myelin treatment, as reflected by increased neutral lipid staining (Nile Red) and by LC / MS for detection of specific lipid species in cell lysates. Figures 8A-8F The data shown in the generally indicate that treatment of iPSC microglia with an exemplary anti-TREM2 antibody after myelin challenge reduces the accumulation of lipid species, as indicated by a reduction in neutral lipid staining in the cells and by a reduction in CE and TAG lipid species measured by LC / MS. Reductions in lipid levels caused by antibody treatment were observed at various time points ranging from 24 hours to 72 hours. To eliminate the possibility that the reduction in lipid levels was caused by blocking lipid uptake, myelin elution experiments were performed in which myelin was removed before adding anti-TREM2 antibodies. Fig.8F It was shown that anti-TREM2 antibodies also reduced lipid levels relative to isotype controls in iPSC microglia with myelin washout prior to antibody treatment.

[0454] Example 4. Functional epitope partitioning of antibodies

[0455] TREM2 antibody epitope bins were determined by competitive binding on TREM2 protein. Epitope partitioning experiments were performed on CarterraLSA instruments at 25°C using the classic sandwich epitope partitioning configuration method. All test antibodies were fixed to the HC30M chip by amine coupling. The test antibodies were then subjected to multiple cycles of sandwich competitive binding. Each cycle consisted of an antigen (His-tagged TREM2 ECD) injected into the fixed antibody, followed by an analyte antibody injected into the fixed antibody. At the end of each cycle, the surface of the fixed antibody was regenerated by injecting a low pH buffer (pH = 3) containing 1.25 M NaCl. Epitope binding data were evaluated by Carterra software to create a competition matrix and epitope partitioning. The results are provided in Table 2.

[0456] Two agonist subregions were identified by epitope partitioning of anti-TREM2 antibodies: (1) stalk-binding agonists and (2) IgV domain-binding agonists. Antibodies with the same subregion demonstrated the same function, such as inhibition of TREM2-DAP12 pSyk activation by lipid ligands (antagonist antibodies), pSyk activation using individual antibodies (stalk-binding agonists, IgV domain-binding agonists).

[0457] Table 2. Anti-TREM2 antibody panels annotated by functional category

[0458]

[0459] Example 5. Binding of Antibodies to TREM2 Stalk Peptide

[0460] TREM2 antibodies were evaluated for binding to human and mouse TREM2 stalk peptides. Test peptides included: (1) full-length stalk (amino acids 129-172 of human TREM2, UniProtKB Q9NZC2; amino acids 131-169 of mouse TREM2, UniProtKBQ99NH8) and (2) truncated stalk peptides containing the ADAM10 / 17 cleavage site (amino acids 149-163 of human / mouse TREM2). Binding of antibodies to 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 hour. Antibodies diluted in 1% BSA / PBS were then added and incubated for 1 hour. Antibodies bound to the peptides were detected using an anti-human κ-HRP secondary antibody (Bethyl Laboratories, Inc.) diluted in 1% BSA / PBS. The plates were assayed by reacting with detection reagent (One-step TMB Ultra, Thermo) and measuring the absorbance at 450 nm (A450) by a standard spectrophotometric instrument (BioTek®). The results are provided in Table 3 below. The data are shown as normalized values ​​(fold relative to background, where background = isotype control).

[0461] Table 3. Binding of anti-TREM2 antibodies to TREM2 handle peptides

[0462]

[0463] Table 3 depicts human and mouse stalk region peptide-antibody binding interactions that support the epitope partitioning data in Table 2. Based on the data in Table 3, certain anti-TREM2 antibodies appear to bind to a site corresponding to amino acids 129-148 of the TREM2 extracellular stalk region.

[0464] The ability of anti-TREM2 antibodies to inhibit ADAM17 cleavage of TREM2 handle peptides was also analyzed using fluorescence polarization assays. TREM2 handle peptides were first prepared with an assay buffer (25 mM Tris pH 7.5, 2.5 μM ZnCl2, 0.005% Brij-35) with streptomycin. Anti-TREM2 antibodies were then pre-incubated with TREM2 handle peptides for 30 minutes at room temperature. After the pre-incubation period, ADAM17 (R&D systems, catalog number 930-ADB) was added and incubated with the peptide at 37°C for 20 hours. The next day, the sample was further diluted with assay buffer and transferred to a black, light-tight 384-well plate. Fluorescence polarization was subsequently measured on a Perkin Elmer EnVision plate reader. The fluorescence polarization of the TREM2 handle peptide pre-incubated with anti-TREM2 antibodies was compared with the fluorescence polarization of the full-length TREM2 handle peptide and the enzyme control (full-length TREM2 handle peptide with ADAM17).

[0465] TREM2 stalk binding antibodies significantly enhanced fluorescence polarization, indicating partial inhibition of stalk peptide cleavage by ADAM17 (clones CL0020141, CL0020188, CL0020313, CL0020308). 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.

[0466] Example 6. Pharmacokinetic Analysis of Anti-TREM2 Antibodies

[0467] The pharmacokinetic profile of anti-TREM2 antibodies 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) studies and target engagement studies. Human Trem2 cDNA KI homozygous mice (hu Trem2 KI / KI ) for 24 h target engagement studies. The generation and breeding of human Trem2 cDNA KI mice is described below.

[0468] Generation of human Trem2 cDNA KI mouse model

[0469] Human TREM2 cDNA KI mice (hu Trem2 KI / KI). The human Trem2 cDNA-pA sequence was inserted at the endogenous ATG start site of mouse Trem2. Insertion of the human Trem2 cDNA-pA resulted in replacement of the exon 1 sequence of mouse Trem2, which allowed expression of the human Trem2 cDNA driven by the endogenous mouse promoter and disrupted expression of endogenous mouse Trem2. hu was generated using homologous recombination in a C57BL / 6 genetic background Trem2 KI / KI Mouse.

[0470] For hu Trem2 KI / KI Targeting vector, long homology arm (LA) extends about 3.6 kb in the 5' end upstream of mankind Trem2 cDNA-pA sequence, and short homology arm (SA) extends about 2.3 kb in the 3' downstream of FRT side joint Neo box.Long homology arm and short homology arm are all amplified from C57BL / 6 BAC clone (RP23: 358G22) and then subcloned into about 2.4kb pSP72 (Promega) backbone vector containing ampicillin selection box.The neomycin box of hUBS-gb2 FRT side joint is just inserted into the hTrem2-pA box downstream, thereby producing a targeting vector of about 13.6 kb in size.10 μg targeting vector is linearized with restriction enzyme NotI (New England Biolabs) and subsequently transfected into FLP C57Bl / 6 (B6) embryonic stem cell (ES) by electroporation.After selecting with G418 antibiotic, amplification survival clone is used for PCR analysis to identify positive recombinant ES clone. The primer sequences used 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 a region from the bghpA element downstream of the short homology arm (SA) outside the 3' region. Five clones were identified as positive and selected for further amplification. The Neo cassette was removed by the Flp transgene during ES clone expansion.

[0471] First, genomic DNA extracted from five positive clones was characterized by sequencing analysis. A 1.19-kb product was amplified and sequenced by 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' genome / neo box linker region and the 3' KI box linker region. The sequencing results confirmed that the human Trem2 cDNA-pA sequence was introduced into all five clones.

[0472] Five positive clones were further characterized by Southern Blotting analysis using the probe of targeting short arm and long arm. The ES cell genomic DNA digested with Ssp I and Bam HI was hybridized with short arm and long arm probe respectively. Confirm that all five ES clones carry the homologous recombination event of correction in both long arm and short arm. The primer sequence for amplifying short arm probe (658 bp) is (SEQ ID NO:57=5'-ACA GGA GGG ACC TAC CTT CAG 3'; SEQ ID NO:58=5'-GCC TGC CTT TCA GAG ACC TCA GTC -3'). The primer sequence for amplifying long arm probe (681 bp) is (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').

[0473] Then all five ES cell clones were injected into C57BL / 6 blastocysts. Germline transmission was confirmed by PCR genotyping from a cloned pup. Primers for genotyping were (SEQ ID NO:61 = pr1: 5'- CGC CTA CCCTAG TCC TGA CTG TTG -3', SEQ ID NO:62 = pr2: 5'- AAA GCC TAC AGC ATC CTC ACCTC -3'; and SEQ ID NO:63 = pr3: 5'- GCA TCA TGG GGT TGT AGA TTC CG -3'). The PCR product of pr1 / pr2 on the wild type was 658 bp. The PCR product of pr1 / pr3 on the KI allele was 469 bp.

[0474] Antibody administration and plasma / CSF collection

[0475] For PK analysis, 10 mg / kg anti-TREM2 antibody or control IgG was given to C57BL / 6J mice by intravenous (IV) tail vein injection (dosing volume was approximately 200 μL / mouse, n = 3 per group). Blood samples were collected 1 hour, 24 hours, 4 days and 7 days after administration. Blood samples of the first three time points were collected via submandibular bleeding using a 3-mm lancet (GoldenRod animal lancet). The last and final blood samples were collected by cardiac puncture on the 7th day. Blood was collected in EDTA tubes (Sarstedt Microvette 500 K3E, catalog number 201341102), which were slowly inverted to mix 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.

[0476] For 24-hour target engagement studies, mouse surrogate anti-TREM2 antibodies were tested using C57BL / 6J mice and hu Trem2 KI / KI Mice tested human anti-TREM2 antibodies. Mice were given 100 mg / kg anti-TREM2 antibody or control IgG by intravenous (IV) tail vein injection (dosing volume was approximately 200 μL / mouse, n = 5 per group). Blood samples were collected 24 hours before dosing to determine TREM2 baseline levels. Final blood and CSF samples were collected 24 hours after dosing. Plasma preparations were prepared as described above. To collect CSF samples, a sagittal incision was made on the back of the skull, and subcutaneous tissue and muscle were separated to expose the cisterna magna. Pre-stretched glass capillaries were used to puncture the cisterna magna and collect CSF samples. The CSF was then centrifuged at 4°C to remove blood residues, and the CSF supernatant was transferred to a 0.5-mL Low Protein LoBind Eppendorf tube (Eppendorf, catalog number 022431064) for storage at -80°C until analysis.

[0477] Analysis of anti-TREM2 antibody plasma levels in vivo

[0478] For anti-TREM2 antibody PK analysis, a general human anti-Fc sandwich ELISA was used to quantify total antibody concentration in mouse plasma. 384-well MaxiSorp plates were coated overnight with 1 μg / mL anti-huFc donkey polyclonal antibody (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 a detection reagent. A standard curve for each individual antibody from 2 nM to 2.7 pM was generated using 3-fold dilutions using five-parameter logistic regression.

[0479] Fig. 9 Representative mouse PK profiles of certain anti-TREM2 antibodies are shown. Antibody clearance (CL [mL / day / kg]) for each representative antibody over a 7-day period is provided and compared to a normal no-effector isotype control. Fig. 9 Each antibody shown exhibited comparable clearance relative to the isotype control.

[0480] In vivo target engagement: sTREM2 plasma levels

[0481] To measure soluble TREM2 (sTREM2) plasma levels, human TREM2 cDNA KI mice (hu Trem2 KI / KI ) bleeding and then treated intravenously with 100 mg / kg test anti-TREM2 antibody or isotype control. Mice were bled 24 hours after administration. Plasma was obtained from blood samples and evaluated in the MSD assay performed as follows. The MSD SECTOR plate was coated with 1 μg / mL capture antibody (R+D anti-TREM2 antibody, catalog number MAB17291-100) diluted with PBS and incubated overnight at 4 ° C. The sample wells were blocked with undiluted MSD blocking agent A for 1 hour. The plasma samples were diluted with 25% MSD blocking agent A 1:20 in Tris buffered saline (TBST) containing 0.05% Tween-20 and added to each sample well on the plate, which was then incubated at room temperature for 2 hours. Subsequently, the detection antibody (MSD sulfo-labeled goat anti-human antibody, catalog number R32AJ-1, 1:1000) was added to each sample well, and the plate was incubated at room temperature for 1 hour. Each sample well was washed with TBST using a Biotek plate washer. Detection reagent (MSD Read Buffer) was added and measured using an MSD MesoSector S600 reader to obtain results for sTREM2 binding to the antibody.

[0482] Fig. 10A and Fig. 10BShown in hu Trem2 KI / KI Total sTREM2 levels of exemplary anti-TREM2 antibodies in plasma ( Fig. 10A ) and antibody binding to sTREM2 levels ( Fig. 10B ). For total sTREM2 and bound sTREM2 assays, data were normalized to pre-dose sTREM2 baseline levels. The results indicated that total circulating sTREM2 levels did not significantly affect the change in mice treated with anti-TREM2 antibodies compared to isotype controls 24 hours after treatment with the antibodies, suggesting that total circulating sTREM2 levels were unaffected at early time points after antibody administration. In contrast, antibody-bound sTREM2 levels were higher in mice injected with anti-TREM2 antibodies relative to isotype controls.

[0483] Example 7. Sequence Optimization and Humanization of Anti-TREM2 Antibodies

[0484] Exemplary anti-TREM2 antibodies were sequence optimized and humanized, followed by characterization of binding kinetics and binding specificity.

[0485] Sequence optimization was performed by searching for residues within the CDR sequences that were susceptible to chemical modification (e.g., asparagine deamidation motifs (NG), aspartate isomerization motifs (DS), and potential oxidation residues (tryptophan (W) and methionine (M)) and making amino acid substitutions with conservative and germline residues to remove such sequence libraries. The binding kinetics of humanized and sequence-optimized variants of anti-TREM2 antibodies were then analyzed using Biacore and dose-titered cell binding to HEK293-H6 cells (see Example 1 for representative protocols).

[0486] The results of analyzing the binding characteristics of humanized and sequence-optimized variants of antibody CL0020188 are provided in Table 4. The NG motif in the CL0020188 CDR-H2 sequence (SEQ ID NO: 5) and CDR-L1 sequence (SEQ ID NO: 7) was modified, grafted to the human framework region, and analyzed. Table 4 provides the K values ​​in HEK293-H6 cells as measured by Biacore. D Values ​​and EC as measured by dose titration binding assay 50 value.

[0487] Table 4. Binding characteristics of sequence-optimized and humanized variants of CL0020188

[0488]

[0489] 3m = V H A24G / L45P / V48L

[0490] As shown in Table 4, the humanized and sequence-optimized clones of CL00201088 exhibited affinity values ​​for hTREM2 similar to that of the parent antibody (K D = 9.5 nM), as measured by Biacore. This is consistent with the cell binding results in HEK293-H6 cells shown in Table 4. 50 = 0.44 nM), the humanized and sequence-optimized clones exhibited comparable and subnanomolar affinity for TREM2 expressed in HEK293-H6 cells. Overall, the results indicate comparable binding kinetics between the parental antibody and the humanized and sequence-optimized variants.

[0491] The results of analyzing the binding characteristics of humanized and sequence-optimized variants of antibody CL0020123 are provided in Table 5. The NG and DS motifs in the CL0020123 CDR-H2 sequence (SEQ ID NO: 30) were modified, grafted to the human framework regions, and analyzed. Table 5 provides the K values ​​in HEK293-H6 cells as measured by Biacore. D Values ​​and EC as measured by dose titration binding assay 50 value.

[0492] Table 5. Binding characteristics of sequence-optimized and humanized variants of CL0020123

[0493]

[0494] 1m = V H R71A

[0495] 2m = V H V67A / R71A

[0496] with the parent antibody (K D = 0.10 nM), the humanized and sequence-optimized clone exhibited approximately 4-fold higher K for binding to hTREM2. D Values, as measured by Biacore. On the other hand, in a dose-titering cell binding assay in HEK293-H6 cells, the humanized and sequence-optimized clones exhibited comparable and subnanomolar affinities for TREM2.

[0497] Example 8. In vitro characterization of sequence-optimized, humanized anti-TREM2 antibodies

[0498] Exemplary sequence-optimized and humanized anti-TREM2 antibodies (Example 7) were evaluated by in vitro methods as described in Examples 1 and 3. The antibodies were assessed for TREM2 binding in HEK cells expressing TREM2, TREM2-dependent pSyk signaling in HEK-H6 cells, the ability to promote human macrophage survival, and the ability to modulate lipid accumulation in iPSC microglia. Figures 11 to 14 show the results of representative anti-TREM2 antibodies. The anti-TREM2 antibodies showed good cell binding in an assay using HEK293-H6 cells expressing TREM2 (EC50 value of 0.34 nM ( Fig.11A ) and 0.08 nM ( Fig. 11B ). Anti-TREM2 antibodies also activated pSyk phosphorylation in HEK293-H6 cells expressing TREM2 ( Fig. 12A and Fig. 12B In addition, anti-TREM2 antibodies induced macrophage survival, with better survival activity observed for the CL0020188 variant antibody ( Fig.13 Finally, anti-TREM2 antibodies demonstrated the ability to reduce lipid accumulation in myelin-treated iPSC microglia ( Fig.14A and Fig. 14B ).

[0499] Example 9. Mouse PK of sequence-optimized, humanized anti-TREM2 antibodies

[0500] The pharmacokinetic characteristics of certain sequence-optimized, humanized anti-TREM2 antibodies (Example 7) were evaluated in wild-type mice in a 7-day pharmacokinetic (PK) study similar to that described in Example 6. Each dose group contained n=3 mice. Table 6 provides the PK properties of exemplary sequence-optimized and humanized anti-TREM2 antibodies (Example 7).

[0501] Table 6. Mouse PK properties of anti-TREM2 antibodies

[0502]

[0503] Example 10. PK of sequence-optimized, humanized anti-TREM2 antibodies in cynomolgus monkeys

[0504] The pharmacokinetic characteristics of anti-TREM2 antibodies were evaluated in natural cynomolgus monkeys. Anti-TREM2 antibodies were injected into natural cynomolgus monkeys aged 2-4 years (weighing about 2-3 kg) via intravenous push. The dosages administered included 3 mg / kg and 25 mg / kg, with n=3 monkeys in each dosage group. Blood samples (about 1 mL) were collected before and after administration at 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours and 24 hours, and 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days and 28 days. The samples were frozen at about 5 ° C and then centrifuged to obtain plasma, and the obtained plasma was kept on dry ice and then stored at -70 ° C until analysis. The anti-TREM2 antibody levels of plasma samples were analyzed as follows.

[0505] For anti-TREM2 antibody PK analysis, the total antibody concentration in monkey plasma was quantified using a general anti-human IgG sandwich electrochemiluminescent immunoassay (ECLIA) on the Meso Scale Discovery (MSD) platform. In brief, 1% casein-based PBS blocking buffer (Thermo Scientific, MA) was added to MSD GOLD 96-well small spot streptomycin coated microtiter plates (Meso Scale Discovery, MD) and incubated for about 1 h. After the plate was blocked and washed, 0.5 μg / mL of biotinylated anti-human IgG goat antibody (SouthernBiotech, AL) in the working solution was added to the assay plate and incubated for 1-2 h. After incubation and washing steps, plasma test samples (i.e., samples with anti-TREM2 humanized antibodies) were added to the assay plate and incubated for 1-2 h. Note that the test sample must be diluted with 0.5% casein-based PBS assay buffer (Thermo Scientific, MA) at a minimum required dilution (MRD) of 1:100 to obtain a final 1% plasma matrix before being added to the assay plate. After capturing the anti-TREM2 antibody analyte and washing steps, 0.4 µg / mL of the secondary ruthenium (SULFO-TAG) anti-human IgG goat antibody (Meso Scale Discovery, MD) in the working solution was added to the assay plate and incubated for about 1 h. Finally, after incubation and washing steps, the assay read buffer (1X MSDRead Buffer T) was added to the assay plate to generate the assay sample signal. The sample signal reading from the MSD plate reader is in the form of an electrochemiluminescence (ECL) signal and is expressed in ECL units (ECLU). All previously described assay reaction steps were performed at ambient temperature and with shaking on a plate oscillator (where appropriate). The assay has a dynamic calibration standard range of 19.5-2500 ng / mL (or 0.195-25 ng / mL at 1:100 post-MRD) with 8 standard points serially diluted 1:2, plus a blank plasma sample. Plasma sample concentrations were back-calculated from the assay calibration standard curve using a weighted four-parameter nonlinear logistic regression fit. Table 7 provides pharmacokinetic (PK) properties of exemplary sequence-optimized and humanized anti-TREM2 antibodies (Example 7).

[0506] Table 7. PK properties of anti-TREM2 antibodies in cynomolgus monkeys

[0507]

[0508] CL0020188 exhibited similar low clearance levels between different dose levels and a linear pharmacokinetic profile in cynomolgus monkeys. In addition, there were no clinical pathological findings associated with the administration of the variants in cynomolgus monkeys (data not shown).

[0509] Example 11. Comparison of anti-TREM2 antibodies

[0510] The affinity of anti-TREM2 antibodies to human TREM2 and cynomolgus monkey TREM2 was measured by Biacore (described in Example 1). The efficacy of anti-TREM2 antibodies in healthy human volunteer CSF samples (Innovative Research) and healthy cynomolgus monkey CSF samples (Worldwide Primates) was measured by MSD assay. The efficacy of each antibody was determined by its EC50. In brief, MSD GOLD 96-well small spot streptomycin plates (MSD, catalog number L45SA) coated with capture antibodies (biotinylated goat anti-human TREM2, R&D Systems, catalog number BAF1828) were incubated with biofluid samples diluted 1:3 with assay buffer (25% (v / v) MSD sealer A (MSD, catalog number R93BA-A), 75% (v / v) TBST) at room temperature for one hour. After rinsing the wells with TBST, sulfo-labeled anti-TREM2 antibodies were added to the wells of the plate in serial dilutions (4x dilution between 11 points) and incubated at room temperature for one hour. The wells were diluted with TBST, and MSD read buffer (MSD, catalog number R92TC-3) was added to the wells. The signal from the sample was measured using an MSD Meso Sector S600 device. The EC50 value of each antibody was determined by four-parameter variable slope nonlinear regression. Reference antibodies #1 and #2 correspond to 4C5 and 6E7 described in WO 2018 / 195506. Reference antibody #3 corresponds to AL2p-58 described in WO 2019 / 028292. The variable region of reference antibody #1 is represented by SEQ ID NOs: 73 and 74. The variable region of reference antibody #2 is represented by SEQ ID NOs: 75 and 76. The variable region of reference antibody #3 is represented by SEQ ID NOs: 77 and 78. The results are provided in Table 8.

[0511] As shown in Table 8, the CL0020188 and CL0020123 variants as disclosed herein have a stronger affinity for human TREM2 relative to the reference antibody and more effectively bind to sTREM2 in CSF samples isolated from healthy human volunteer subjects. In addition, the CL0020188 variant has a stronger affinity for cynomolgus monkey TREM2 relative to the reference antibody and more effectively binds to sTREM2 in CSF samples isolated from healthy cynomolgus monkey subjects. This is demonstrated by the relative amount of antibody required to reach the half-maximal effective concentration (EC50) for binding a given amount of sTREM2 under the same conditions. As shown in Table 8, higher relative amounts are required for reference antibodies #1, #2, and #3 to reach EC50 compared to the CL0020188 and CL0020123 antibodies.

[0512] Table 8. Comparison of properties of anti-TREM2 antibodies

[0513]

[0514] Example 12. Epitope Mapping of Anti-TREM2 Antibodies

[0515] In order to identify the epitope 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) for 0, 60, 600 and 3600 seconds at 20 ° C. Hydrogen / deuterium exchange was quenched by adding an equal volume of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). The quenched sample was then subjected to pepsin / protease XIII digestion and LC-MS analysis. In brief, the quenched sample was injected into a packed pepsin / protease XIII column (2.1 x30 mm) maintained at 20 ° C, and the resulting peptides were analyzed using a UPLC-MS system comprising a Waters Acquity UPLC (Waters Corporation) coupled to a Q Exactive™ HF-Hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher). The peptides were separated on a 50 mm x 1 mm C8 column maintained at -6°C using a gradient from 2% to 31% solvent B in 16.5 minutes (solvent B: 0.2% formic acid in acetonitrile; solvent A: 0.2% formic acid in water). Mass spectra were recorded in MS mode only. HDX Workbench (Pascal et al. 2012. Journal of The American Society for Mass Spectrometry23:1512-1521) to process raw MS data. Deuterium levels were calculated using the average mass difference between deuterated peptides and their native form at t0. Peptide identification was performed by searching MS / MS data against human TREM2 sequences using Mascot software (Matrix Science). The mass tolerances for precursor and product ions were 10 ppm and 0.02 Da, respectively.

[0516] Based on the HDX mass spectrometry results, CL0020188 and variants of CL0020188 bind to amino acid residues 143-149 (FPGESES (SEQ ID NO:69)) of human TREM2 (SEQ ID NO:1), while CL0020123 and variants of CL0020123 bind to (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)) of human TREM2.

[0517] IX. Informal Sequence Listing

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525] Sequence Listing <110> DENALI THERAPEUTICS INC. <120> Anti-TREM2 antibodies and methods of use thereof <130> 102342-003310PC-1173316 <140> <141> <150> 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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> twenty one <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty one 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> twenty two <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty two 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> twenty three <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty three Gln Ser Ser Lys Ser Leu Leu His Ser Thr Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> twenty four <211> 118 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty four 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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> VARIANT <222> (6) <223> / alternative="Gln" <220> <221> VARIANT <222> (8) <223> / Alternative="Glu" <220> <221> SITE <222> (1)..(17) <223> / Annotation="The variant residues given in the sequence are relative to the variant position No preference for residues in annotation" <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> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> VARIANT <222> (6) <223> / Alternative="Asn" <220> <221> SITE <222> (1)..(10) <223> / Annotation="The variant residues given in the sequence are relative to the variant position No preference for residues in annotation" <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> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> VARIANT <222> (5) <223> / Alternative="Arg" <220> <221> VARIANT <222> (6) <223> / Alternative="Pro" <220> <221> VARIANT <222> (8) <223> / Alternative="Ala" <220> <221> VARIANT <222> (11)..(11) <223> / Alternative="Thr" <220> <221> VARIANT <222> (12)..(12) <223> / Alternative="Asp" <220> <221> SITE <222> (1)..(19) <223> / Annotation="The variant residues given in the sequence are relative to the variant position No preference for residues in annotation" <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> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> VARIANT <222> (1) <223> / Alternative="Thr" <220> <221> VARIANT <222> (4) <223> / Alternative="Ser" <220> <221> SITE <222> (1)..(9) <223> / Annotation="The variant residues given in the sequence are relative to the variant position No preference for residues in annotation" <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> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> VARIANT <222> (10)..(10) <223> / Alternative="Thr" <220> <221> SITE <222> (1)..(16) <223> / Annotation="The variant residues given in the sequence are relative to the variant position No preference for residues in annotation" <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> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> VARIANT <222> (6) <223> / alternative="Phe" <220> <221> SITE <222> (1)..(9) <223> / Annotation="The variant residues given in the sequence are relative to the variant position No preference for residues in annotation" <400> 52 Gln Gln Phe Leu Glu Tyr Pro Phe Thr 1 5 <210> 53 <211> twenty one <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 53 aggaatgtgg ggagcacgga g 21 <210> 54 <211> twenty four <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 54 tgcatcgcat tgtctgagta ggtg 24 <210> 55 <211> twenty two <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="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> / Comment="Description of artificial sequence: synthetic primer" <400> 56 tataggaact tcgcgacacg gacac 25 <210> 57 <211> twenty one <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 57 acaggaggga cctaccttca g 21 <210> 58 <211> twenty four <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 58 gcctgccttt cagagacctc agtc 24 <210> 59 <211> twenty four <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 59 cctctccggc tgctcatctt actc 24 <210> 60 <211> twenty four <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 60 gtctctcagc cctggcagag tttg 24 <210> 61 <211> twenty four <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 61 cgcctaccct agtcctgact gttg 24 <210> 62 <211> twenty three <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic primer" <400> 62 aaagcctaca gcatcctcac ctc 23 <210> 63 <211> twenty three <212> DNA <213> Artificial Sequence <220> <221> source <223> / Comment="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> / Comment="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> / Annotation="Description of artificial sequence: synthetic 6xHis tag" <400> 65 His His His His His 1 5 <210> 66 <211> 140 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="Description of artificial sequence: synthetic peptide" <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> / Comment="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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <400> 83 Gln Gly Glu Ser 1

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2, wherein the antibody or antigen-binding fragment thereof comprises: (a) CDR-H1 sequence consisting of the sequence GFTFTDFYMS (SEQ ID NO:4); (b) CDR-H2 sequence, which consists of the sequence VIRN-β5-β6-N-β8-YT-β 11 -β 12 -YNPSVKG (SEQ ID NO: 49), wherein β5 is K; β6 is A; β8 is G or A; β 11 is A; and β 12 It is G; (c) a CDR-H3 sequence consisting of the sequence ARLTYGFDY (SEQ ID NO: 17); (d) CDR-L1 sequence, which consists of the sequence QSSKSLLHS-δ 10 -GKTYLN (SEQ ID NO: 51), wherein δ 10 is N or T; (e) a CDR-L2 sequence consisting of the sequence WMSTRAS (SEQ ID NO: 8); and (f) CDR-L3 sequence consisting of the sequence QQFLEYPFT (SEQ ID NO: 18).

2. The isolated antibody or antigen-binding fragment of claim 1, wherein the CDR-H2 sequence is selected from any one of SEQ ID NO: 5 and 25.

3. The isolated antibody or antigen-binding fragment of claim 1, wherein the CDR-L1 sequence is selected from any one of SEQ ID NO: 7 and 23.

4. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises: (a) CDR-H1 consisting of the amino acid sequence of SEQ ID NO:4, CDR-H2 consisting of the amino acid sequence of SEQ ID NO:5, CDR-H3 consisting of the amino acid sequence of SEQ ID NO:17, CDR-L1 consisting of the amino acid sequence of SEQ ID NO:7, CDR-L2 consisting of the amino acid sequence of SEQ ID NO:8, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO:18; or (b) CDR-H1 consisting of the amino acid sequence of SEQ ID NO:4, CDR-H2 consisting of the amino acid sequence of SEQ ID NO:5, CDR-H3 consisting of the amino acid sequence of SEQ ID NO:17, CDR-L1 consisting of the amino acid sequence of SEQ ID NO:23, CDR-L2 consisting of the amino acid sequence of SEQ ID NO:8, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO:18; or (c) CDR-H1 consisting of the amino acid sequence of SEQ ID NO:4, CDR-H2 consisting of the amino acid sequence of SEQ ID NO:25, CDR-H3 consisting of the amino acid sequence of SEQ ID NO:17, CDR-L1 consisting of the amino acid sequence of SEQ ID NO:7, CDR-L2 consisting of the amino acid sequence of SEQ ID NO:8, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO:18; or (d) CDR-H1 consisting of the amino acid sequence of SEQ ID NO:4, CDR-H2 consisting of the amino acid sequence of SEQ ID NO:25, CDR-H3 consisting of the amino acid sequence of SEQ ID NO:17, CDR-L1 consisting of the amino acid sequence of SEQ ID NO:23, CDR-L2 consisting of the amino acid sequence of SEQ ID NO:8 and CDR-L3 consisting of the amino acid sequence of SEQ ID NO:

18.

5. The isolated antibody or antigen-binding fragment of claim 1, comprising a V sequence consisting of any one of SEQ ID NOs: 15, 19, 21, 24, 26 and 79. H sequence.

6. The isolated antibody or antigen-binding fragment of claim 5, wherein the V H The sequence consists of SEQ ID NO:

15.

7. The isolated antibody or antigen-binding fragment of claim 5, wherein the V H The sequence consists of SEQ ID NO:

24.

8. The isolated antibody or antigen-binding fragment of claim 5, wherein the V H The sequence consists of SEQ ID NO:

79.

9. The isolated antibody or antigen-binding fragment of claim 1, comprising a V consisting of any one of SEQ ID NOs: 16, 20 and 22. L sequence.

10. The isolated antibody or antigen-binding fragment of claim 9, wherein the V L The sequence consists of SEQ ID NO:

16.

11. The isolated antibody or antigen-binding fragment of claim 9, wherein the V L The sequence consists of SEQ ID NO:

22.

12. The isolated antibody or antigen-binding fragment of claim 9, wherein the V L The sequence consists of SEQ ID NO:

20.

13. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises: (a) V consisting of SEQ ID NO: 15 H Sequence and V consisting of SEQ ID NO:16 L sequence; or (b) V consisting of SEQ ID NO: 19 H Sequence and V consisting of SEQ ID NO:20 L sequence; or (c) V consisting of SEQ ID NO: 21 H Sequence and V consisting of SEQ ID NO:20 L sequence; or (d) V consisting of SEQ ID NO: 19 H Sequence and V consisting of SEQ ID NO:22 L sequence; or (e) V consisting of SEQ ID NO: 79 H Sequence and V consisting of SEQ ID NO:22 L sequence; or (f) V consisting of SEQ ID NO: 24 H Sequence and V consisting of SEQ ID NO:20 L sequence; or (g) V consisting of SEQ ID NO: 26 H Sequence and V consisting of SEQ ID NO:20 L sequence; or (h) V consisting of SEQ ID NO: 24 H Sequence and V consisting of SEQ ID NO:22 L sequence; or (i) V consisting of SEQ ID NO: 26 H Sequence and V consisting of SEQ ID NO:22 L sequence.

14. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof reduces the level of soluble TREM2 protein (sTREM2).

15. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof enhances TREM2 activity.

16. The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the antibody or antigen-binding fragment thereof enhances phagocytosis or enhances migration, differentiation, function or survival of myeloid cells, microglia or macrophages.

17. The isolated antibody or antigen-binding fragment thereof of claim 16, wherein the antibody or antigen-binding fragment thereof enhances microglial function without increasing neuroinflammation.

18. The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the antibody or antigen-binding fragment thereof enhances Syk phosphorylation.

19. The isolated antibody or antigen-binding fragment thereof of claim 18, wherein the antibody or antigen-binding fragment thereof enhances Syk phosphorylation in the presence of a TREM2 ligand.

20. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof exhibits cross-reactivity with cynomolgus monkey TREM2 protein.

21. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monoclonal antibody.

22. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a chimeric antibody.

23. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a humanized antibody.

24. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a fully human antibody.

25. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is a Fab, a F(ab')2, a scFv, or a bivalent scFv.

26. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

27. A medicine kit, comprising: The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26; and Its instruction manual.

28. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 25 or the pharmaceutical composition of claim 26 in the preparation of a medicament for a subject having a neurodegenerative disease, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia.

29. The method of claim 28, wherein the Parkinson's disease is frontotemporal dementia with Parkinson's disease associated with chromosome 17 or post-encephalitic Parkinson's disease.

30. A polynucleotide comprising a nucleic acid sequence encoding the isolated antibody or antigen-binding fragment thereof of claim 1.

31. A vector comprising the polynucleotide of claim 30.

32. A host cell comprising the polynucleotide of claim 30 or the vector of claim 31.

33. A method for preparing an anti-TREM2 antibody, comprising culturing the host cell of claim 32 under conditions suitable for expression of the antibody.

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