Anti-TREM2 antibody and method of use thereof

Abstract

In one aspect, an antibody is provided that specifically binds to human myeloid cell-expressed triggering receptor 2 (TREM2) protein. In some embodiments, the antibody reduces the level of soluble TREM2 (sTREM2). In some embodiments, the antibody enhances TREM2 activity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 960,663 filed on 13 January 2020, U.S. Provisional Patent Application No. 63 / 070,728 filed on 26 August 2020, and U.S. Provisional Patent Application No. 63 / 091,717 filed on 14 October 2020, the disclosures thereof incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] background Myelocyte-expressed trigger receptor 2 (TREM2) is a transmembrane receptor expressed in microglia and is thought to function in phagocytic regulation, cell survival, and inflammatory cytokine production. TREM2 mutations have been identified in neurodegenerative diseases including Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia. Furthermore, altered levels of soluble TREM2 (sTREM2) have been reported in the cerebrospinal fluid of Alzheimer's disease patients or patients with frontotemporal dementia who have TREM2 mutations.

[0003] There is still a need for therapeutic agents that modulate TREM2 activity or sTREM2 levels. [Overview of the Initiative]

[0004] overview In one embodiment, an antibody is provided that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2). In some embodiments, the antibody comprises a modified Fc polypeptide capable of binding to the transferrin receptor protein. In any embodiment disclosed herein, the antibody comprises a CDR, V following one of the exemplary sequences provided herein. H , and / or V L It may include, and may further include, a modified Fc polypeptide containing the transferrin receptor binding mutation described herein.

[0005] In some embodiments, antibodies that specifically bind to TREM2 include: (a) A CDR-H1 sequence containing the sequence iG-FTFT-α6-FYMS (sequence number 28) (where α6 is D or N), ii. VIRN-β5-β6-N-β8-YT-β 11 -β 12 -YNPSVKG(Sequence ID 29)(In the sequence, β5 is K or R, β6 is A or P, β8 is G or A, β 11 is A or T, and β 12 CDR-H2 sequence containing the sequence (which is G or D), iii. A CDR-H3 sequence containing the sequence γ1-RL-γ4-YGFDY (Sequence ID 30) (wherein γ1 is A or T and γ4 is T or S), iv.QSSKSLLHS-δ 10 -GKTYLN(sequence number 31)(in sequence, δ 10 CDR-L1 sequence containing the sequence (is N or T), The CDR-L2 sequence containing the sequence of v.WMSTRAS (sequence number 8), and vi. A variable region containing the CDR-L3 sequence which includes the sequence QQFLE-φ6-PFT (sequence number 32) (where φ6 is Y or F), (b) A first Fc polypeptide modified to specifically bind to the transferrin receptor, and (c) Second Fc polypeptide.

[0006] In some embodiments, the first Fc polypeptide and the second Fc polypeptide associate to form an Fc dimer.

[0007] In some embodiments, the CDR-H1 sequence is selected from sequence numbers 4 or 12. In some embodiments, the CDR-H2 sequence is selected from sequence numbers 5, 13, or 25. In some embodiments, the CDR-H3 sequence is selected from sequence numbers 6, 14, or 17. In some embodiments, the CDR-L1 sequence is selected from sequence numbers 7 or 23. In some embodiments, the CDR-L3 sequence is selected from sequence numbers 9 or 18.

[0008] In some embodiments, the variable region includes: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.

[0009] In some embodiments, the variable region has a V sequence having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26 H and includes.

[0010] In certain embodiments, the V H sequence has at least 90% sequence identity to SEQ ID NO: 15. In certain embodiments, the V H sequence has at least 95% sequence identity to SEQ ID NO: 15. In certain embodiments, the V H sequence includes SEQ ID NO: 15.

[0011] In certain embodiments, the V H sequence has at least 90% sequence identity to SEQ ID NO: 24. In certain embodiments, the V H sequence has at least 95% sequence identity to SEQ ID NO: 24. In certain embodiments, the V H sequence includes SEQ ID NO: 24.

[0012] In some embodiments of this model, the variable region has at least 85% sequence identity with respect to any one of sequence numbers 3, 11, 16, 20, 22, and 27. L Includes arrays.

[0013] In a particular embodiment, V L The sequence has at least 90% sequence identity with respect to sequence number 16. In a particular embodiment, V L The sequence has at least 95% sequence identity with respect to sequence number 16. In a particular embodiment, V L The array includes sequence number 16.

[0014] In a particular embodiment, V L The sequence has at least 90% sequence identity with respect to sequence number 22. In a particular embodiment, V L The sequence has at least 95% sequence identity with respect to sequence number 22. In a particular embodiment, V L The array includes sequence number 22.

[0015] In a particular embodiment, V L The sequence has at least 90% sequence identity with respect to sequence number 27. In a particular embodiment, V L The sequence has at least 95% sequence identity with respect to sequence number 27. In a particular embodiment, V L The array includes sequence number 27.

[0016] In some embodiments of this model, the variable region includes: (a) V containing sequence number 15 H V containing the sequence and sequence number 16 L array, or (b) V containing sequence number 19 H V including the sequence and sequence number 20 L array, or (c) V containing sequence number 21 H V including the sequence and sequence number 20 L array, or (d) V containing sequence number 19 H V including the sequence and sequence number 22 L array, or (e) V containing sequence number 21 H V including the sequence and sequence number 22 L array, or (f) V containing sequence number 24 H V including the sequence and sequence number 20 L array, or (g) V containing sequence number 26 H V including the sequence and sequence number 20 L array, or (h) V containing sequence number 24 H V including the sequence and sequence number 22 L array, or (i) V containing sequence number 26 H V including the sequence and sequence number 22 L array, or (j) V containing sequence number 2 H V containing the sequence and sequence number 3 L array, or (k) V containing sequence number 10 H V containing the sequence and sequence number 11 L array, or (l) V containing sequence number 24 H V including the sequence and sequence number 27 L array.

[0017] In some embodiments of this model, the first Fc polypeptide comprises, according to EU numbering, Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, or Val at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and Phe at position 421. In some embodiments, the first Fc polypeptide binds to the apical domain of the transferrin receptor. In certain embodiments, the antibody exhibits improved uptake into the brain compared to an antibody having a wild-type Fc dimer.

[0018] In a particular embodiment, according to EU numbering, the first Fc polypeptide has the T366W substitution, and the second Fc polypeptide has the T366S, L368A, and Y407V substitutions.

[0019] In other embodiments, according to EU numbering, the first Fc polypeptide has T366S, L368A, and Y407V substitutions, and the second Fc polypeptide has T366W substitution.

[0020] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide include modifications that reduce effector function. In certain embodiments, the modifications that reduce effector function include substitutions that are Ala at position 234 and Ala at position 235, according to EU numbering.

[0021] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide include amino acid modifications to the native Fc sequence that extend the serum half-life. In certain embodiments, the amino acid modification includes substitutions that are Leu at position 428 and Ser at position 434, according to EU numbering.

[0022] In some embodiments of this model, the first Fc polypeptide comprises the sequence of SEQ ID NO: 41 or SEQ ID NO: 64, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. In a particular embodiment, the antibody comprises (i) V containing SEQ ID NO: 24. H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 41, and V containing SEQ ID NO: 24. H (iii) a second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and V containing SEQ ID NO: 22 LThe antibody comprises two light chains, each containing the respective amino acid sequence. In a particular embodiment, the antibody comprises two light chains, each containing (i) a first heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 64, (ii) a second heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL containing SEQ ID NO: 22. In a particular embodiment, the antibody comprises a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 42, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 53, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54. In a particular embodiment, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 65, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LCs) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0023] In some embodiments of this model, the first Fc polypeptide comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 66, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. In a particular embodiment, the antibody comprises (i) V containing SEQ ID NO: 24. H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 44, and V containing SEQ ID NO: 24. H (iii) a second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and V containing SEQ ID NO: 22 LThe antibody comprises two light chains, each containing the following. In a particular embodiment, the antibody comprises two light chains, each containing (i) a first heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 66, (ii) a second heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL containing SEQ ID NO: 22. In a particular embodiment, the antibody comprises a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 45, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 53, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54. In a particular embodiment, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 67, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LCs) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0024] In some embodiments of this model, the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. In a particular embodiment, the antibody comprises (i) V containing SEQ ID NO: 24. H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 47, and V containing SEQ ID NO: 24. H (iii) V containing the second Fc polypeptide containing SEQ ID NO: 39, a second heavy chain (HC), and (iii) V containing SEQ ID NO: 22. LThe antibody comprises two light chains, each containing the respective amino acid sequence. In a particular embodiment, the antibody comprises two light chains, each containing (i) a first heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 68, (ii) a second heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL containing SEQ ID NO: 22. In a particular embodiment, the antibody comprises a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 48, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 53, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54. In a particular embodiment, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 69, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LCs) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0025] In some embodiments of this model, the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 61 or SEQ ID NO: 84. In a particular embodiment, the antibody comprises (i) V containing SEQ ID NO: 24. H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 47, and V containing SEQ ID NO: 24. H (iii) V containing the second Fc polypeptide containing SEQ ID NO: 61, a second heavy chain (HC), and (iii) V containing SEQ ID NO: 22 LThe antibody comprises two light chains, each containing the following. In a particular embodiment, the antibody comprises two light chains, each containing (i) a first heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 68, (ii) a second heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 84, and (iii) a VL containing SEQ ID NO: 22. In a particular embodiment, the antibody comprises a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 48, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 52, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54. In a particular embodiment, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 69, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 72, and (iii) first and second light chains (LCs) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0026] In some embodiments of this model, the first Fc polypeptide comprises the sequence of SEQ ID NO: 50 or SEQ ID NO: 70, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. In a particular embodiment, the antibody comprises (i) V containing SEQ ID NO: 24. H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 50, and V containing SEQ ID NO: 24. H (iii) a second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and V containing SEQ ID NO: 22 LThe antibody comprises two light chains, each containing the respective amino acid sequence. In a particular embodiment, the antibody comprises two light chains, each containing (i) a first heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 70, (ii) a second heavy chain (HC) comprising a VH containing SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL containing SEQ ID NO: 22. In a particular embodiment, the antibody comprises a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 51, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 53, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54. In a particular embodiment, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence described in SEQ ID NO: 71, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LCs) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0027] In another aspect, the disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), wherein the antibody comprises (i) SEQ ID NO: 24 H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 41, and V containing SEQ ID NO: 24. H (iii) a second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and V containing SEQ ID NO: 22 L The antibody comprises two light chains, each containing the amino acid sequence described in (i) SEQ ID NO: 42, a first heavy chain (HC) comprising or consisting thereof, (ii) a second HC comprising or consisting thereof, the amino acid sequence described in SEQ ID NO: 53, and (iii) first and second light chains (LC) comprising or consisting thereof, the amino acid sequences described in SEQ ID NO: 54.

[0028] In another embodiment, the Disclosure provides an isolated antibody that specifically binds to human bone marrow cell-expressed trigger receptor 2 (TREM2), the antibody comprising two light chains, each comprising (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 64, (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL comprising SEQ ID NO: 22. In some embodiments, the antibody comprises a first heavy chain (HC) comprising or consisting of (i) the amino acid sequence described in SEQ ID NO: 65, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0029] In another aspect, the disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), wherein the antibody comprises (i) SEQ ID NO: 24 H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 44, and V containing SEQ ID NO: 24. H (iii) a second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and V containing SEQ ID NO: 22 L The antibody comprises two light chains, each containing the amino acid sequence described in (i) SEQ ID NO: 45, a first heavy chain (HC) containing or comprising the amino acid sequence described in SEQ ID NO: 53, and a second HC containing or comprising the amino acid sequence described in (iii) SEQ ID NO: 54.

[0030] In another embodiment, the Disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), the antibody comprising two light chains, each comprising (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 66, (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL comprising SEQ ID NO: 22. In some embodiments, the antibody comprises a first heavy chain (HC) comprising or consisting of (i) the amino acid sequence described in SEQ ID NO: 67, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0031] In another aspect, the disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), wherein the antibody comprises (i) SEQ ID NO: 24 H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 47, and V containing SEQ ID NO: 24. H (iii) V containing the second Fc polypeptide containing SEQ ID NO: 39, a second heavy chain (HC), and (iii) V containing SEQ ID NO: 22. L The antibody comprises two light chains, each containing the amino acid sequence described in (i) SEQ ID NO: 48, a first heavy chain (HC) containing or comprising the amino acid sequence described in SEQ ID NO: 53, and first and second light chains (LCs) containing or comprising the amino acid sequences described in SEQ ID NO: 54, respectively.

[0032] In another embodiment, the Disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), the antibody comprising two light chains, each comprising (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 68, (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL comprising SEQ ID NO: 22. In some embodiments, the antibody comprises a first heavy chain (HC) comprising or consisting of (i) the amino acid sequence described in SEQ ID NO: 69, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0033] In another aspect, the disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), wherein the antibody comprises (i) SEQ ID NO: 24 H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 47, and V containing SEQ ID NO: 24. H (iii) V containing the second Fc polypeptide containing SEQ ID NO: 61, a second heavy chain (HC), and (iii) V containing SEQ ID NO: 22 L It comprises two light chains, each containing the amino acid sequence described in (i) SEQ ID NO: 48, a first heavy chain (HC) containing or comprising the amino acid sequence described in SEQ ID NO: 52, and (iii) first and second light chains (LC) containing or comprising the amino acid sequences described in SEQ ID NO: 54, respectively.

[0034] In another embodiment, the Disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), the antibody comprising two light chains, each comprising (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 68, (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 61, and (iii) a VL comprising SEQ ID NO: 22. In some embodiments, the antibody comprises a first heavy chain (HC) comprising or consisting of (i) the amino acid sequence described in SEQ ID NO: 69, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 72, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0035] In another aspect, the disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), wherein the antibody comprises (i) SEQ ID NO: 24 H (ii) a first heavy chain (HC) comprising a first Fc polypeptide containing SEQ ID NO: 50, and V containing SEQ ID NO: 24. H (iii) a second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and V containing SEQ ID NO: 22 L The antibody comprises two light chains, each containing the amino acid sequence described in (i) SEQ ID NO: 51, a first heavy chain (HC) containing or comprising the amino acid sequence described in SEQ ID NO: 53, and a first and second light chain (LC) containing or comprising the amino acid sequences described in SEQ ID NO: 54, respectively.

[0036] In another embodiment, the Disclosure provides an isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), the antibody comprising two light chains, each comprising (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 70, (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63, and (iii) a VL comprising SEQ ID NO: 22. In some embodiments, the antibody comprises a first heavy chain (HC) comprising or consisting of (i) the amino acid sequence described in SEQ ID NO: 71, (ii) a second HC comprising or consisting of the amino acid sequence described in SEQ ID NO: 73, and (iii) first and second light chains (LC) comprising or consisting of the amino acid sequences described in SEQ ID NO: 54.

[0037] In some embodiments of any of the embodiments described herein, the antibody reduces the level of soluble TREM2 protein (sTREM2). In some embodiments, the antibody enhances TREM2 activity. In some embodiments, the antibody enhances phagocytosis or enhances the migration, differentiation, function, or survival of bone marrow cells, microglia, or macrophages. In some embodiments, the antibody enhances microglial function without increasing neuroinflammation. In some embodiments, the antibody enhances Syk phosphorylation. In some embodiments, the antibody enhances Syk phosphorylation in the presence of a TREM2 ligand. In some embodiments, the antibody exhibits cross-reactivity with cynomolgus monkey TREM2 protein.

[0038] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody as described herein and a pharmaceutically acceptable carrier.

[0039] In another aspect, the Disclosure provides a kit comprising an isolated antibody or a pharmaceutical composition as described herein, and instructions for use thereof.

[0040] In another embodiment, the Disclosure provides a method for treating a neurodegenerative disease in a subject, comprising administering an isolated antibody or a pharmaceutical composition described herein to the subject. In some embodiments, the neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with chromosome 17-linked parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / parkinsonian dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, Boxer dementia, diffuse neurofibrillary tangle disease with calcification, Down syndrome, familial Anglo-British dementia, familial Danish dementia, Gerstmann-Sträussler-Schaft dementia The group is comprised of Inker's disease, glial tauopathy, Guadeloupean parkinsonism with dementia, Guadeloupean PSP, Haller-Vorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-dominant dementia, Niemann-Pick disease type C, bulbopontigrine degeneration, Parkinson's disease, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and neurofibrillary senile dementia (tangle-only dementia).

[0041] In another aspect, the Disclosure provides a method for reducing sTREM2 levels in subjects with neurodegenerative diseases, comprising administering an isolated antibody or a pharmaceutical composition described herein to the subject.

[0042] In another aspect, the Disclosure provides a method for enhancing TREM2 activity in subjects with neurodegenerative diseases, comprising administering an isolated antibody or a pharmaceutical composition described herein to the subject.

[0043] In another aspect, the present disclosure provides isolated polynucleotides comprising a nucleotide sequence encoding an antibody described herein.

[0044] In another embodiment, the Disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding any one of SEQ ID NOs: 42, 45, 48, 51, 53, 54, and 61.

[0045] In another embodiment, the disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 42, 53, and 54.

[0046] In another embodiment, the disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 45, 53, and 54.

[0047] In another embodiment, the disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 48, 53, and 54.

[0048] In another embodiment, the disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 48, 52, and 54.

[0049] In another embodiment, the disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 51, 53, and 54.

[0050] In another aspect, the present disclosure provides a vector comprising a polynucleotide as described herein.

[0051] In another aspect, the present disclosure provides a host cell comprising a polynucleotide or a vector as described herein.

[0052] In another aspect, the present disclosure provides a method for expressing an antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), comprising culturing the host cells described herein under conditions suitable for the expression of the antibody. [Invention 1001] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (a) iG-FTFT-α 6 -FYMS(sequence number 28)(in the sequence, α 6 CDR-H1 sequence containing the sequence (which is D or N), ii. VIRN-β 5 -β 6 -N-β 8 -YT-β 11 -β 12 -YNPSVKG(Sequence ID 29)(in sequence, β 5 is K or R, and β 6 is A or P, and β 8 is G or A, and β 11 is A or T, and β 12 CDR-H2 sequence containing the sequence (which is G or D), iii.γ 1 -RL-γ 4 -YGFDY(sequence number 30)(γ in sequence) 1 is A or T, γ 4 CDR-H3 sequence containing the sequence (which is T or S), iv.QSSKSLLHS-δ 10 -GKTYLN(sequence number 31)(in sequence, δ 10 CDR-L1 sequence containing the sequence (is N or T), The CDR-L2 sequence containing the sequence of v.WMSTRAS (sequence number 8), and vi.QQFLE-φ 6 -PFT(sequence number 32)(in the sequence, φ 6 CDR-L3 sequence containing the sequence (is Y or F) Variable region, (b) A first Fc polypeptide modified to specifically bind to the transferrin receptor, and (c) Second Fc polypeptide The antibody, which includes the antibody. [Invention 1002] The antibody of the present invention 1001, wherein the CDR-H1 sequence is selected from SEQ ID NO: 4 or 12. [Invention 1003] The antibody of the present invention 1001 or 1002, wherein the CDR-H2 sequence is selected from SEQ ID NOs. 5, 13, or 25. [Invention 1004] An antibody according to any of the present invention 1001 to 1003, wherein the CDR-H3 sequence is selected from SEQ ID NOs. 6, 14, or 17. [Invention 1005] An antibody according to any of the present invention 1001 to 1004, wherein the CDR-L1 sequence is selected from SEQ ID NO: 7 or 23. [Invention 1006] An antibody according to any of the present invention 1001 to 1005, wherein the CDR-L3 sequence is selected from SEQ ID NO: 9 or 18. [Invention 1007] The aforementioned variable region is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 6, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 9, or (f) CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, CDR-H3 containing the amino acid sequence of SEQ ID NO: 14, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 9 An antibody according to any of the present invention 1001 to 1006, including the above. [Invention 1008] The variable region has at least 85% sequence identity with any one of sequence numbers 2, 10, 15, 19, 21, 24, and 26. H An antibody according to any of the present invention 1001 to 1007, comprising the sequence. [Invention 1009] The aforementioned V H The antibody of the present invention 1008, wherein the sequence has at least 90% sequence identity with respect to sequence number 15. [Invention 1010] The aforementioned V H The antibody of the present invention 1009, wherein the sequence has at least 95% sequence identity with SEQ ID NO: 15. [Invention 1011] The aforementioned V H The antibody of the present invention 1010, wherein the sequence includes sequence number 15. [Invention 1012] The aforementioned V H The antibody of the present invention 1008, wherein the sequence has at least 90% sequence identity with respect to sequence number 24. [Invention 1013] The aforementioned V H The antibody of the present invention 1012, wherein the sequence has at least 95% sequence identity with respect to sequence number 24. [Invention 1014] The aforementioned V H The antibody of the present invention 1013, wherein the sequence includes sequence number 24. [Invention 1015] The variable region has at least 85% sequence identity with any one of sequence numbers 3, 11, 16, 20, 22, and 27. L An antibody comprising any of the present invention 1001 to 1014, wherein the sequence is included. [Invention 1016] The aforementioned V L The antibody of the present invention 1015, wherein the sequence has at least 90% sequence identity with respect to sequence number 16. [Invention 1017] The aforementioned V L The antibody of the present invention 1016, wherein the sequence has at least 95% sequence identity with SEQ ID NO: 16. [Invention 1018] The aforementioned V L The antibody of the present invention 1017, wherein the sequence includes sequence number 16. [Invention 1019] The aforementioned V L The antibody of the present invention 1015, wherein the sequence has at least 90% sequence identity with respect to sequence number 22. [Invention 1020] The aforementioned V L The antibody of the present invention 1019, wherein the sequence has at least 95% sequence identity with respect to sequence number 22. [Invention 1021] The aforementioned V L The antibody of the present invention 1020, wherein the sequence includes sequence number 22. [Invention 1022] The aforementioned V L The antibody of the present invention 1015, wherein the sequence has at least 90% sequence identity with respect to sequence number 27. [Invention 1023] The aforementioned V L The antibody of the present invention 1022, wherein the sequence has at least 95% sequence identity with respect to sequence number 27. [Invention 1024] The aforementioned V L The antibody of the present invention 1023, wherein the sequence includes sequence number 27. [Invention 1025] The aforementioned variable region is (a) V containing sequence number 15 H V containing the sequence and sequence number 16 L array, or (b) V containing sequence number 19 H V including the sequence and sequence number 20 L array, or (c) V containing sequence number 21 H V including the sequence and sequence number 20 L array, or (d) V containing sequence number 19 H V including the sequence and sequence number 22 L array, or (e) V containing sequence number 21 H V including the sequence and sequence number 22 L array, or (f) V containing sequence number 24 H V including the sequence and sequence number 20 L array, or (g) V containing sequence number 26 H V including the sequence and sequence number 20 L array, or (h) V containing sequence number 24 H V including the sequence and sequence number 22 L array, or (i) V containing sequence number 26 H V including the sequence and sequence number 22 L array, or (j) V containing sequence number 2 H V containing the sequence and sequence number 3 L array, or (k) V containing sequence number 10 H V containing the sequence and sequence number 11 L array, or (l) V containing sequence number 24 H V including the sequence and sequence number 27 L array An antibody according to any of the present invention 1001 to 1007, including the above. [Invention 1026] An antibody according to any of the Invention 1001 to 1025, wherein the first Fc polypeptide comprises, according to EU numbering, Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, or Val at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and Phe at position 421. [Invention 1027] The antibody of the present invention 1026, wherein the first Fc polypeptide binds to the apical domain of the transferrin receptor. [Invention 1028] The antibody of the present invention 1026 or 1027, which exhibits improved uptake into the brain compared to antibodies having wild-type Fc dimers. [Invention 1029] An antibody according to any of the invention 1001 to 1028, wherein the first Fc polypeptide has a T366W substitution and the second Fc polypeptide has T366S, L368A, and Y407V substitutions, in accordance with EU numbering. [Invention 1030] An antibody according to any of Invention 1001 to 1028, wherein the first Fc polypeptide has T366S, L368A, and Y407V substitutions, and the second Fc polypeptide has a T366W substitution, in accordance with EU numbering. [Invention 1031] An antibody according to any of the present invention 1001 to 1030, wherein the first Fc polypeptide and / or the second Fc polypeptide includes a modification that reduces effector function. [Invention 1032] The antibody of the present invention 1031, wherein the modification that reduces the effector function includes substitutions of Ala at position 234 and Ala at position 235, according to EU numbering. [Invention 1033] The first Fc polypeptide and / or the second Fc polypeptide, Amino acid modifications to the natural Fc sequence that extend the serum half-life An antibody according to any of the present invention 1001 to 1032, including the above. [Invention 1034] The antibody of the present invention 1033, wherein the amino acid modification includes substitutions such as Leu at position 428 and Ser at position 434, according to EU numbering. [Invention 1035] An antibody according to any of the present invention 1001 to 1034, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 41 or SEQ ID NO: 64, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. [Invention 1036] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 41, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1035, which includes the antibody of the present invention. [Invention 1037] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 64, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1035, which includes the antibody of the present invention. [Invention 1038] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 42, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 An antibody according to the present invention 1035 or 1036, comprising: [Invention 1039] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 65, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1035 or 1037, comprising the above. [Invention 1040] An antibody according to any of the present invention 1001 to 1034, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 66, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. [Invention 1041] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 44, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1040, which includes the antibody of the present invention. [Invention 1042] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 66, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1040, which includes the antibody of the present invention. [Invention 1043] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 45, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 An antibody according to the present invention 1040 or 1041, comprising: [Invention 1044] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 67, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1040 or 1042, comprising the above. [Invention 1045] An antibody according to any of the present invention 1001 to 1034, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. [Invention 1046] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1045, which includes the antibody of the present invention. [Invention 1047] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1045, which includes the antibody of the present invention. [Invention 1048] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 An antibody according to the present invention 1045 or 1046, comprising: [Invention 1049] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1045 or 1047, comprising the above. [Invention 1050] An antibody according to any of the present invention 1001 to 1034, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 61 or SEQ ID NO: 84. [Invention 1051] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing SEQ ID NO: 61, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1050, which includes the present invention. [Invention 1052] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 84, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1050, which includes the present invention. [Invention 1053] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 52, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1050 or 1051, comprising the above. [Invention 1054] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 72, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1050 or 1052, comprising the above. [Invention 1055] An antibody according to any of the present invention 1001 to 1034, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 50 or SEQ ID NO: 70, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 63. [Invention 1056] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 50, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1055, which includes the antibody of the present invention. [Invention 1057] (i) V containing sequence number 24 H A first heavy chain (HC) comprising the first Fc polypeptide containing SEQ ID NO: 70, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody of the present invention 1055, which includes the antibody of the present invention. [Invention 1058] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 51, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1055 or 1056, comprising the above. [Invention 1059] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 71, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1055 or 1057, comprising the above. [Invention 1060] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 41, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1061] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 42, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1060, which includes the antibody of the present invention. [Invention 1062] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 64, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1063] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 65, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1062, which includes the antibody of the present invention. [Invention 1064] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H and a first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 44, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1065] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 45, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1064, which includes the antibody of the present invention. [Invention 1066] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H and a first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 66, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1067] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 67, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1066, which includes the antibody of the present invention. [Invention 1068] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H and a first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1069] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1068, which includes the antibody of the present invention. [Invention 1070] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H and a first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1071] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1070, which includes the antibody of the present invention. [Invention 1072] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H and a first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 61, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1073] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 52, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1072, which includes the antibody of the present invention. [Invention 1074] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H and a first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 61, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1075] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 72, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1074, which includes the antibody of the present invention. [Invention 1076] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 50, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 39, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1077] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 51, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1076, which includes the antibody of the present invention. [Invention 1078] An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 H A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 70, (ii) V containing sequence number 24 H A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 L Two light chains each containing The antibody, which includes the antibody. [Invention 1079] (i) A first heavy chain (HC) containing or consisting of the amino acid sequence described in Sequence ID No. 71, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody of the present invention 1078, which includes the antibody of the present invention. [Invention 1080] An antibody according to any of the Invention 1001-1079 that reduces the level of soluble TREM2 protein (sTREM2). [Invention 1081] An antibody according to any of the present invention 1001 to 1080 that enhances TREM2 activity. [Invention 1082] An antibody according to any of the Invention 1001-1081, which enhances phagocytosis or enhances the migration, differentiation, function, or survival of bone marrow cells, microglia, or macrophages. [Invention 1083] An antibody according to any of the invention 1001-1082 that enhances microglial function without increasing neuroinflammation. [Invention 1084] An antibody according to any of the present invention 1001-1083 that enhances Syk phosphorylation. [Invention 1085] The antibody of the present invention 1084, which enhances Syk phosphorylation in the presence of TREM2 ligand. [Invention 1086] An antibody according to any of the present invention 1001 to 1085 that exhibits cross-reactivity with the cynomolgus monkey TREM2 protein. [Invention 1087] A pharmaceutical composition comprising an isolated antibody according to any of invention 1001 to 1086 and a pharmaceutically acceptable carrier. [Invention 1088] An isolated antibody according to any of Invention 1001 to 1086 or a pharmaceutical composition according to Invention 1087, The instruction manual and A kit that includes this. [Invention 1089] A method for treating a neurodegenerative disease in a subject, comprising administering to the subject an isolated antibody according to any of the inventions 1001 to 1086 or a pharmaceutical composition according to the invention 1087. [Invention 1090] The aforementioned neurodegenerative diseases include Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with chromosome 17-linked parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / parkinsonian dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, Boxer dementia, diffuse neurofibrillary tangle disease with calcification, Down syndrome, familial Anglo-American dementia, familial Danish-American dementia, and Gerstmann-Sträussler-Scheinker disease. The method of the present invention 1089, selected from the group consisting of globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadeloupean PSP, Haller-Vorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-dominant dementia, Niemann-Pick disease type C, bulbopontigrine degeneration, Parkinson's disease, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and neurofibrillary senile dementia (tangle-only dementia). [Invention 1091] A method for reducing the level of sTREM2 in a subject having a neurodegenerative disease, comprising administering an isolated antibody according to any of the inventions 1001 to 1086 or a pharmaceutical composition according to the invention 1087 to the subject. [Invention 1092] A method for enhancing TREM2 activity in a subject with a neurodegenerative disease, comprising administering an isolated antibody according to any of the inventions 1001 to 1086 or a pharmaceutical composition according to the invention 1087 to the subject. [Invention 1093] An isolated polynucleotide containing a nucleotide sequence encoding any of the antibodies described in invention 1001 to 1086. [Invention 1094] An isolated polynucleotide containing a nucleotide sequence encoding one of sequence numbers 42, 45, 48, 51, 53, 54, and 61. [Invention 1095] Isolated polynucleotides containing nucleotide sequences encoding sequence numbers 42, 53, and 54. [Invention 1096] Isolated polynucleotides containing nucleotide sequences encoding SEQ ID NOs: 45, 53, and 54. [Invention 1097] Isolated polynucleotides containing nucleotide sequences encoding sequence numbers 48, 53, and 54. [Invention 1098] Isolated polynucleotides containing nucleotide sequences encoding sequence numbers 48, 52, and 54. [Invention 1099] Isolated polynucleotides containing nucleotide sequences encoding SEQ ID NOs: 51, 53, and 54. [Invention 1100] A vector comprising any of the polynucleotides 1093 to 1099 of the present invention. [Invention 1101] A host cell containing any polynucleotide of Invention 1093 to 1099 or a vector of Invention 1100. [Invention 1102] A method for expressing an antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), comprising culturing the host cells of the present invention 1101 under conditions suitable for the expression of the antibody. [Brief explanation of the drawing]

[0053] [Figure 1A] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1B] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1C]This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1D] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1E] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1F] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1G] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 1H] This shows the cell-binding curves for dose-configured humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells. [Figure 2] This shows the dose-response binding curves for representative ATV:TREM2 and corresponding anti-TREM2 antibodies ("anti-TREM2") with non-transferrin-bound Fc in HEK293 cells against human TREM2. [Figure 3] This shows dose-response binding curves of pSyk signaling activation by representative ATV:TREM2 and the corresponding anti-TREM2 antibody (TREM2 IgG) in TREM2-expressing HEK293 cells. [Figure 4A] This shows the dose-response curve of lipid clearance in iPSC microglia in response to treatment with a representative ATV:TREM2. [Figure 4B]This shows the dose-response curve of lipid clearance in iPSC microglia in response to treatment with a representative ATV:TREM2. [Figure 5A] This image shows a representative picture of lipid accumulation in iPSC-derived microglia cells treated with ATV:TREM2 after oleic acid stimulation. [Figure 5B] This shows the quantitative analysis of lipid accumulation in treated cells. [Figure 5C] This heatmap shows the regulation of triglyceride, acylcarnitine, and TCA cycle intermediate species levels in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 5D] The bar graph shows the changes in representative triglyceride species levels in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 5E] The bar graph shows the changes in representative acylcarnitine species levels in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 5F] This bar graph shows the changes in representative TCA cycle intermediate species levels in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 6A] This bar graph shows the changes in the levels of specific triglyceride species in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 6B] This bar graph shows the changes in the levels of specific ceramide species in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 6C] This bar graph shows the changes in the levels of specific acylcarnitine species in iPSC-derived microglia cells treated with ATV:TREM2 after myelin stimulation. [Figure 7A] This is a representative Western blot image of mTOR signaling pathway targets in iPSC-derived microglia treated with ATV:TREM2. [Figure 7B]This plot shows the changes in mTOR signaling pathway target levels in iPSC-derived microglia treated with ATV:TREM2. [Figure 7C] This plot shows the changes in mTOR signaling pathway target levels in iPSC-derived microglia treated with ATV:TREM2. [Figure 7D] This plot shows the changes in mTOR signaling pathway target levels in iPSC-derived microglia treated with ATV:TREM2. [Figure 7E] This plot shows the changes in mTOR signaling pathway target levels in iPSC-derived microglia treated with ATV:TREM2. [Figure 8] This bar graph shows the changes in progranulin (PGRN) levels in iPSC-derived microglia treated with ATV:TREM2. [Figure 9] This graph shows the changes in the levels of representative bis(monoacylglycero)phosphate (BMP) species in iPSC-derived microglia treated with ATV:TREM2. [Figure 10A] This is a representative kinetic graph of oxygen consumption in iPSC-derived microglia treated with ATV:TREM2. [Figure 10B] This bar graph shows the maximum respiratory capacity of iPSC-derived microglia treated with ATV:TREM2, both in the presence and absence of a CPT1 inhibitor. [Figure 11A] This is a dose-response curve of cell viability in human macrophage cells treated with anti-TREM2 antibody. [Figure 11B] Figure 11A shows a bar graph illustrating the EC50 of the dose-response curve. [Figure 11C] Figure 11A shows a bar graph illustrating the Emax of the dose-response curve. [Figure 12] This is a heatmap of relative cytokine release in human macrophage cells treated with anti-TREM2 antibody. [Figure 13A]A volcano plot showing the relative changes in triglyceride species in iPSC-derived microglia cells treated with anti-TREM2 antibody is shown. [Figure 13B] A volcano plot showing the relative changes in triglyceride species in iPSC-derived microglia cells treated with anti-TREM2 antibody is shown. [Figure 13C] A volcano plot showing the relative changes in triglyceride species in iPSC-derived microglia cells treated with anti-TREM2 antibody is shown. [Figure 13D] The bar graph shows the changes in the levels of representative triglyceride species in iPSC-derived microglia cells treated with anti-TREM2 antibody. [Figure 13E] The bar graph shows the changes in the levels of representative triglyceride species in iPSC-derived microglia cells treated with anti-TREM2 antibody. [Figure 14A] This is a plot of TREM2 levels against antibody concentration in cell lysates of iPSC-derived microglia cells treated with anti-TREM2 antibody. [Figure 14B] This is a plot of TREM2 levels against antibody concentration in the cell culture medium of iPSC-derived microglia cells treated with anti-TREM2 antibody. [Figure 15] This plot shows the pharmacokinetic profile of anti-TREM2 antibody administered to cynomolgus monkeys. [Figure 16] A and B are plots showing EdU+Iba+ cells (A) and relative Iba+ regions (B) per 1 mm² in the brain of TB36 / hTfR KI mice treated with either ATV:TREM2 or ATV:RSV. [Figure 17]Plots A and B show EdU+ Iba+ cells (A) and relative Iba+ regions (B) per 1 mm² in the brain of TB36 / hTfR KI mice treated with either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV. The graphs show the mean ± SEM and p-values: one-way ANOVA and Tukey's multiple comparison test; *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 18] Plots A and B show cytokine IP-10 levels (A) and cytokine MCP-5 levels (B) in the brain of TB36 / hTfR KI mice treated with either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV. The graphs show the mean ±SEM of the replicated experiments. The graphs show the mean ±SEM and p-values: one-way ANOVA and Tukey's multiple comparison test; **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 19] This plot shows glial marker CSF1R levels in the brains of TB36 / hTfR KI mice treated with either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV. The graph shows mean ± SEM and p-values: one-way ANOVA and Tukey's multiple comparison test; *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 20] This plot shows the plasma PK profiles of ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV in TB36 / hTfR KI mice. [Figure 21] This plot shows the brain PK profiles of ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV in TB36 / hTfR KI mice. [Modes for carrying out the invention]

[0054] Detailed explanation I. Introduction TREM2 is a transmembrane receptor expressed on the cell surface of microglia, dendritic cells, macrophages, and osteoclasts. Although not bound by any specific theory, it is thought that after ligand binding, TREM2 forms a signaling complex with the transmembrane adapter protein DNAX-activating protein 12 (DAP12), and is subsequently tyrosine-phosphorylated by the protein kinase SRC. The activated TREM2 / DAP12 signaling complex is thought to mediate intracellular signaling by recruiting and phosphorylating kinases such as Syk kinase. TREM2 / DAP12 signaling regulates phagocytosis, cell proliferation and survival, inflammatory cytokine secretion, and cell migration, including that of microglia and macrophages. TREM2 undergoes regulated intramembrane proteolysis, in which the membrane-bound full-length TREM2 is cleaved by the metalloproteinase ADAM10 into an sTREM2 portion that is shed from the cell and a membrane-retained C-terminal fragment that is further degraded by gamma-secretase. Altered levels of sTREM2 have been reported in patients with Alzheimer's disease or frontotemporal dementia who also have TREM2 mutations. Furthermore, TREM2 mutations are associated with functional changes such as impaired phagocytosis and reduced microglial function.

[0055] As detailed in the Examples section below, antibodies were constructed that specifically bind to human TREM2 and modulate one or more downstream functions of the TREM2 / DAP12 signaling complex. In certain embodiments, the antibody further comprises an Fc polypeptide containing a mutation that enables the Fc polypeptide to bind to a transferrin receptor (e.g., human-derived TfR). In some embodiments, the antibodies disclosed herein can bind to a transferrin receptor protein (e.g., one expressed on the surface of brain endothelial cells (BECs)) via the modified Fc polypeptide, thereby being able to cross the blood-brain barrier (BBB) ​​more effectively than antibodies lacking the TfR-binding Fc mutation. In certain embodiments, the antibodies disclosed herein include mutations in the Fc polypeptide that reduce or eliminate effector function, and mutations that increase the in vivo half-life, for example, by increasing the binding of antibody Fc to the Fc embryonic receptor (FcRn).

[0056] In some embodiments, anti-TREM2 antibodies enhance TREM2 activity (for example, by enhancing phagocytosis or by enhancing the differentiation, function, migration, or survival of bone marrow cells, microglia, or macrophages). Therefore, in another embodiment, a method is provided for enhancing TREM2 activity, for example, in subjects with neurodegenerative diseases.

[0057] In some embodiments, anti-TREM2 antibodies reduce sTREM2 shedding. Therefore, in another embodiment, a method is provided for reducing sTREM2 levels, for example, in subjects with neurodegenerative diseases.

[0058] II. Definition As used herein, unless otherwise clearly indicated by the context, the singular forms “a,” “an,” and “the” refer to multiple objects. Therefore, for example, a reference to “antibody” optionally includes two or more such molecules or combinations.

[0059] As used herein, the terms “about” and “approximately” indicate, when used to modify a quantity specified by a number or range, that a reasonable deviation from that number and a value known to those skilled in the art, e.g., ±20%, ±10%, or ±5%, is within the intended meaning of the stated value.

[0060] As used herein, the term “TREM2 protein” refers to the myeloid cell expression trigger receptor 2 protein encoded by the gene TREM2. As used herein, “TREM2 protein” refers to the natural (i.e., wild-type) TREM2 protein of any vertebrate, e.g., 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 the sequence identified in UniprotKB accession number Q9NZC2 (SEQ ID NO: 1).

[0061] As used herein, the term "anti-TREM2 antibody" refers to an antibody that specifically binds to the TREM2 protein (e.g., human TREM2).

[0062] As used herein, the term “antibody” refers to a protein having an immunoglobulin fold that specifically binds to an antigen via its variable region. This term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term “antibody” also includes, but is not limited to, antibody fragments that retain binding specificity via their variable region, including Fab, F(ab')2, Fv, scFv, and bivalent scFv. Antibodies may contain a light chain classified as either kappa or lambda. Antibodies may contain a heavy chain classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0063] As used herein, the term “anti-TREM2 antigen-binding moiety” refers to an antigen-binding segment or the whole that specifically binds to the TREM2 protein (e.g., human TREM2). The terms “antigen-binding moiety” and “antigen-binding fragment” are used interchangeably herein and refer to fragments of one or more antibodies that retain the ability to specifically bind to an antigen (e.g., the TREM2 protein) via their variable region. An example of an antigen-binding fragment is the Fab fragment (V L , V H (A monovalent fragment consisting of CL and CH1 domains), F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by disulfide bridges in the hinge region), single-stranded Fv(scFv), disulfide bond Fv(dsFv), complementarity-determining region (CDR), V L (Light chain variable region), and V H (Heavy chain variable region) is one example, but it is not limited to these.

[0064] The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that originates from germline variable (V) genes, diversity (D) genes, or binding (J) genes (but not from constant (Cμ and Cδ) gene segments) and gives the antibody specificity to bind to an antigen. Typically, an antibody variable region contains four conserved "framework" regions interspersed with three hypervariable "complementarity-determining regions."

[0065] The term "complementarity-determining region" or "CDR" refers to the three hypervariable regions within each chain that intersect the four framework regions constructed by the light chain variable region and the heavy chain variable region. CDRs are primarily responsible for antibody binding to the antigen's epitope. The CDRs on each chain are usually numbered sequentially from the N-terminus as CDR1, CDR2, and CDR3, and are usually identified by the chain on which a particular CDR is located. Therefore, V H CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain where it is found, while V L CDR1 or CDR-L1 is a CDR1 derived from the variable region of the antibody light chain in which it is found.

[0066] Different light chain or heavy chain "framework regions" or "FRs" are relatively conserved within a species. The antibody framework region, i.e., the combined framework regions of the constituent light and heavy chains, functions to arrange and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available reference literature. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database for human and mouse sequences.

[0067] The amino acid sequences of the CDR and framework region may be determined using various well-known definitions in the art, such as Kabat, Chothia, the International ImMunoGeneTics Database (IMGT), AbM, and observed antigen contact ("Contact"). In some embodiments, the CDR is determined according to the Contact definition. See MacCallum et al., J.Mol.Biol., 262:732-745 (1996). In some embodiments, the CDR is determined by a combination of the Kabat, Chothia, and / or Contact CDR definitions.

[0068] The term "epitope" refers to a region or area of ​​an antigen to which an antibody's CDR specifically binds, and may include several amino acids or a portion of several amino acids, e.g., five or six or more, e.g., twenty or more amino acids, or a portion of those amino acids. For example, if the target is a protein, the epitope may consist of a sequence of amino acids (e.g., a linear epitope) or amino acids from different parts of the protein that are adjacent by protein folding (e.g., a discontinuous epitope or a structural epitope). In some embodiments, the epitope is phosphorylated at one amino acid (e.g., at a serine or threonine residue).

[0069] As used herein, the phrase "recognizes an epitope," when used in reference to an anti-TREM2 antibody, means that the antibody CDR interacts with or specifically binds to the antigen (i.e., the TREM2 protein) at its epitope or at the portion of the antigen containing that epitope.

[0070] A "monoclonal antibody" refers to an antibody produced from a single clone or single cell line of cells, consisting of, or essentially being, an antibody molecule identical to their primary amino acid sequence.

[0071] A "polyclonal antibody" refers to an antibody obtained from a heterogeneous antibody population in which different antibodies within the population bind to different epitopes of the antigen.

[0072] A "chimeric antibody" refers to an antibody molecule in which the constant region or a portion thereof is altered, substituted, or exchanged, resulting in an antigen-binding site (i.e., a variable region, CDR, or a portion thereof) being linked to a constant region of a different or altered class, effector function, and / or species, or in which the variable region or a portion thereof is altered, substituted, or exchanged by a variable region having a different or altered antigen specificity (e.g., a CDR and framework region derived from a different species). In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region derived from one source or species (e.g., mouse) and a constant region derived from another source or species (e.g., human). Methods for producing chimeric antibodies are described in the art.

[0073] A "humanized antibody" is a chimeric immunoglobulin derived from a non-human source (e.g., mouse) that contains minimal sequences derived from a non-human immunoglobulin outside the CDR. Generally, a humanized antibody contains at least one (e.g., two) antigen-binding variable domains, the CDR region substantially corresponds to the CDR region of a non-human immunoglobulin, and the framework region substantially corresponds to the framework region of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of the human immunoglobulin sequence. Methods for antibody humanization are known in the art.

[0074] A “human antibody” or “fully human antibody” is typically an antibody that has human heavy and light chain sequences derived from human germline genes. In some embodiments, the antibody is produced by human cells, by non-human animals utilizing the human antibody repertoire (e.g., transgenic mice genetically engineered to express human antibody sequences), or by a phage display platform.

[0075] The term "specifically binds" refers to a molecule (e.g., an antibody or its antigen-binding moiety) that binds to an epitope or target in a sample with higher affinity, higher binding strength, and / or longer duration than it binds to another epitope or non-target compound (e.g., a structurally different antigen). In some embodiments, an antibody (or its antigen-binding moiety) that specifically binds to an epitope or target is an antibody (or its antigen-binding moiety) that binds to the epitope or target with at least 5 times higher affinity than other epitopes or non-target compounds, e.g., at least 5 times, 10 times, 100 times, 1,000 times, or 10,000 times or more affinity. As used herein, the terms "specifically binds to," "specifically binds to," or "specific to" a particular epitope or target mean, for example, 10 times higher affinity to the epitope or target to which it binds. -4 M or less, for example, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Equilibrium dissociation constant K for M D This can be represented by molecules having [a certain characteristic]. Those skilled in the art will understand that an antibody that specifically binds to a target derived from a certain species (e.g., the TREM2 protein) may also specifically bind to an ortholog of that target (e.g., the TREM2 protein).

[0076] The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules, for example, between an antibody (or its antigen-binding moiety) and an antigen. Therefore, unless otherwise indicated or the context makes clear, the term may refer to a 1:1 interaction between an antibody (or its antigen-binding moiety) and an antigen. Binding affinity is determined by the equilibrium dissociation constant (K). D This can be quantified by measuring the coupling rate constant (k a ,time -1 M -1 The dissociation rate constant (k) divided by ) d ,time -1 ) refers to K D The kinetics of complex formation and dissociation can be determined by measuring them using, for example, surface plasmon resonance (SPR) (e.g., the Biacore® system), binding equilibrium exclusion methods such as KinExA®, and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, "binding affinity" refers not only to formal binding affinity that reflects a 1:1 interaction between the antibody (or its antigen-binding portion) and the antigen, but also to K, which may reflect strong binding. D This includes the apparent affinity from which the value is calculated.

[0077] As used herein, the term "cross-reactivity" refers to the ability of an antibody to bind to an antigen other than the antigen that produced the antibody. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen originating from a different species than the antigen that produced the antibody. As a non-limiting example, the anti-TREM2 antibodies described herein, produced against human TREM2 peptides, may exhibit cross-reactivity with TREM2 peptides or proteins originating from different species (e.g., monkeys or mice).

[0078] As used herein, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is described in SEQ ID NO: 62. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee: accession number XP_003310238.1, rhesus macaque: NP_001244232.1, dog: NP_001003111.1, cattle: NP_001193506.1, mouse: NP_035768.1, rat: NP_073203.1, and chicken: NP_990587.1). The term “transferrin receptor” also includes exemplary reference sequences encoded by genes located at the transferrin receptor protein 1 chromosomal locus, such as allele variants of the human sequence. The full-length transferrin receptor protein includes a short N-terminal intracellular domain, a transmembrane domain, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is described in SEQ ID NO: 55.

[0079] As used herein, the terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides. In the context of IgG antibodies, the CH3 domain polypeptide refers to the amino acid segment around positions 341 to 447, as numbered according to the EU numbering scheme, and the CH2 domain polypeptide refers to the amino acid segment around positions 231 to 340, as numbered according to the EU numbering scheme. CH2 and CH3 domain polypeptides can also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, and according to the IMGT Scientific chart numbering (IMGT website), the CH2 domain numbers are 1 to 110 and the CH3 domain numbers are 1 to 107. The CH2 and CH3 domains are part of the Fc region of immunoglobulins. In the context of IgG antibodies, the Fc region refers to the amino acid segment around positions 231 to 447, as numbered according to the EU numbering scheme. As used herein, the term “Fc region” may also include at least a portion of the hinge region of an antibody. An example partial hinge region sequence is shown in Sequence ID No. 57.

[0080] When used in the context of identifying a given amino acid residue in a polypeptide sequence, the terms "corresponds to," "determined by reference to," or "numbered by reference to" refer to the position of a residue in a particular reference sequence when a given amino acid sequence is compared to the reference sequence in a maximally aligned manner. Therefore, for example, if optimally aligned to SEQ ID NO: 38, and the amino acids and residues in SEQ ID NO: 38 align, then the amino acid residues in the polypeptide "correspond" to the amino acids in the amino acid region 111-217 of SEQ ID NO: 38. A polypeptide aligned to a reference sequence does not need to be the same length as the reference sequence.

[0081] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a natural immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. The Fc polypeptide contains a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may contain at least a portion of the hinge region, but does not contain a variable region.

[0082] A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., substitution, deletion, or insertion, compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0083] The term "isolated," when used in reference to nucleic acids or proteins (e.g., antibodies), indicates that the nucleic acid or protein essentially does not contain other cellular components that it was associated with in its native 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, 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.

[0084] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and mimics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Natural α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of natural α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. "Amino acid analogs" refer to compounds that have the same basic chemical structure as natural amino acids, i.e., compounds with an α-carbon bonded to a hydrogen, carboxyl group, amino group, and R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as natural amino acids. "Amino acid mimes" refer to chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar way to natural amino acids.In this specification, amino acids may be referred to by either the commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0085] The terms “polypeptide” and “peptide” are used interchangeably herein and refer to polymers of amino acid residues in a single chain. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as natural and non-natural amino acid polymers. Amino acid polymers may consist entirely of L-amino acids, entirely of D-amino acids, or as mixtures of L-amino acids and D-amino acids.

[0086] As used herein, the term “protein” refers to one polypeptide, a dimer (i.e., two polypeptides), or a polymer (i.e., three or more polypeptides) of a single-chain polypeptide. The single-chain polypeptides of a protein may be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

[0087] The terms “polynucleotide” and “nucleic acid” are interchangeable to refer to a chain of nucleotides of any length, including DNA and RNA. A nucleotide can be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or their analogues, or any substrate that can be incorporated into a chain by DNA polymerase or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogues. Examples of polynucleotides as intended herein include single-stranded DNA and double-stranded DNA, single-stranded RNA and double-stranded RNA, and hybrid molecules having mixtures of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0088] The terms "conservative substitution" and "conservative mutation" refer to changes that result in the substitution of one amino acid with another amino acid that can be classified as having similar characteristics. Examples of classifications of these defined conservative amino acid groups include the "charged / polar group" containing Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); the "aromatic group" containing Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" containing 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). Subgroups can also be identified within each group. For example, a group of charged or polar amino acids can be subdivided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His; a "negatively charged subgroup" consisting of Glu and Asp; and a "polar subgroup" consisting of Asn and Gln. In another example, a group of aromatic or cyclic amino acids can be subdivided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp; and a "phenyl subgroup" consisting of Phe and Tyr. In yet another example, an aliphatic group can be subdivided into subgroups, for example, an "aliphatic nonpolar subgroup" consisting of Val, Leu, Gly, and Ala; and an "aliphatic micropolar subgroup" consisting of Met, Ser, Thr, and Cys.Examples of the classification of conservative mutations include amino acid substitutions of amino acids within the aforementioned subgroups, such as, but not limited to, substitutions that can maintain a positive charge, such as replacing Arg with Lys or vice versa; replacing Asp with Glu or vice versa, such as maintaining a negative charge; replacing Thr with Ser or vice versa, such as maintaining a free -OH group; and replacing Asn with Gln or vice versa, such as maintaining a free -NH2 group. In some embodiments, hydrophobic amino acids are substituted for native hydrophobic amino acids, for example, in the active site, in order to maintain hydrophobicity.

[0089] In relation to two or more polypeptide sequences, the terms “identical” or “percent “identical” refer to two or more sequences or subsequences that, when compared and aligned to the greatest extent possible across a comparison window, or across a specified region measured by a sequence comparison algorithm, or by manual alignment and visual inspection, have amino acid residues that are identical or identical to a certain percentage, 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, across a specified region.

[0090] In polypeptide sequence comparison, a single amino acid sequence typically functions as a reference sequence, which is then compared to the candidate sequence. To obtain the best alignment, alignment can be performed using various methods available to those skilled in the art, such as visual alignment or publicly available software using known algorithms. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to obtain the best alignment can be determined by those skilled in the art. For the purpose of this application, polypeptide sequence comparison uses the standard protein BLAST of the BLASTP algorithm to align two protein sequences using default parameters.

[0091] The terms “subject,” “individual,” and “patient,” as used interchangeably herein, refer to mammals including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the subject, individual, or patient is a human.

[0092] As used herein, terms such as “to treat” and “treatment” generally mean obtaining a desired pharmacological and / or physiological effect. “To treat” or “treatment” can refer to any indication of success in treating or improving a neurodegenerative disease (e.g., Alzheimer’s disease or another neurodegenerative disease described herein), including any objective and subjective parameters such as relief, remission, improvement in patient survival, increased survival time or survival rate, reduction of symptoms or making the disease more tolerable for the patient, slowing the rate of degeneration or decline, or improving the patient’s physical or mental well-being. Treatment or improvement of symptoms may be based on objective or subjective parameters. The effects of treatment can be compared to an untreated individual or a pool of individuals, or to the same patient at different points in time before or between treatments.

[0093] The term "pharmaceutically acceptable excipient" refers to, but is not limited to, non-active medicinal ingredients that are biologically or pharmacologically compatible for use in humans or animals, such as buffers, carriers, or preservatives.

[0094] As used herein, “therapeutic dose” or “therapeutic effective dose” of a drug (e.g., an antibody as described herein) is the amount of drug that treats, reduces, alleviates, or reduces the severity of the symptoms of a disease in a subject. A “therapeutic dose” of a drug (e.g., an antibody as described herein) can improve patient survival, increase survival time or survival rate, reduce symptoms, make injury, disease or condition (e.g., neurodegenerative disease) more tolerable, slow the rate of degeneration or decline, or improve the patient’s physical or mental well-being.

[0095] The term "administer" refers to a method of delivering a drug, compound, or composition to a desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the antibody described herein is administered intravenously.

[0096] The terms “control” or “control value” refer to a reference value or baseline value. An appropriate control can be determined by those skilled in the art. In some cases, the control value may be determined against the baseline of the same subject or within the same experiment; for example, a measured value of sTREM2 taken before treatment with an anti-TREM2 antibody may be the control value against the measured sTREM2 level after treatment in the same subject. In other cases, the control value may be determined against a control subject (e.g., a healthy control or a disease control) or against the mean value in a population of control subjects (e.g., a population of 10, 20, 50, 100, 200, 500, or more control subjects); for example, a measured sTREM2 level of a subject at baseline or after treatment may be compared to the value of a healthy control.

[0097] III. Anti-TREM2 antibody In one embodiment, an antibody that specifically binds to the TREM2 protein is provided. In some embodiments, the antibody specifically binds to the human TREM2 protein. In some embodiments, the anti-TREM2 antibody is selective for TREM2 compared to other TREM-like receptors (e.g., TREM1).

[0098] In some embodiments, the anti-TREM2 antibody is an antibody comprising one or more complementarity-determining region (CDR) sequences, heavy chain variable region sequences, and / or light chain variable region sequences disclosed herein. In some embodiments, the anti-TREM2 antibody comprises one or more CDR sequences, heavy chain variable region sequences, and / or light chain variable region sequences disclosed herein, and further comprises one or more functional properties disclosed herein, for example, an antibody that enhances TREM2 activity (e.g., enhances phagocytosis or enhances the migration, differentiation, function, or survival of cells such as bone marrow cells, microglia, or macrophages), or an antibody that reduces the level of sTREM2. In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide comprising one or more modifications described herein.

[0099] 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.

[0100] Anti-TREM2 sequence In some embodiments, the heavy chain sequence or a portion thereof, and / or the light chain sequence or a portion thereof, are derived from the anti-TREM2 antibodies described herein (e.g., clone CL0020306, clone CL0020188, or clone CL0020307). The CDR, heavy chain variable region, and light chain variable region amino acid sequences of these clones are listed in Table 8.

[0101] In some embodiments, the anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of: (a) A heavy chain CDR1 (CDR-H1) sequence having at least 90% sequence identity with respect to one of the amino acid sequences of SEQ ID NOs: 4 and 12, or having up to two amino acid substitutions with respect to one of the amino acid sequences of SEQ ID NOs: 4 and 12, (b) A heavy chain CDR2 (CDR-H2) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs. 5, 13, and 25, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs. 5, 13, and 25, (c) A heavy chain CDR3 (CDR-H3) sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs. 6, 14, and 17, or having up to two amino acid substitutions to any one of the amino acid sequences of SEQ ID NOs. 6, 14, and 17. (d) A light chain CDR1 (CDR-L1) sequence having at least 90% sequence identity with respect to either one of the amino acid sequences of SEQ ID NOs. 7 and 23, or having up to two amino acid substitutions with respect to either one of the amino acid sequences of SEQ ID NOs. 7 and 23, (e) A light chain CDR2 (CDR-L2) sequence having at least 90% sequence identity with any one amino acid sequence of SEQ ID NO: 8, or having up to two amino acid substitutions with any one amino acid sequence of SEQ ID NO: 8, and (f) A light chain CDR3 (CDR-L3) sequence having at least 90% sequence identity with respect to either one of the amino acid sequences of SEQ ID NOs. 9 and 18, or having up to two amino acid substitutions with respect to either one of the amino acid sequences of SEQ ID NOs. 9 and 18.

[0102] In some embodiments, the anti-TREM2 antibody comprises two, three, four, five, or all six of (a) to (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, a CDR having up to two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR having up to two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

[0103] In some embodiments, the anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of: (a) A CDR-H1 sequence containing one of the amino acid sequences of SEQ ID NOs: 4 and 12, (b) A CDR-H2 sequence containing one of the amino acid sequences of SEQ ID NOs. 5, 13, and 25, (c) A CDR-H3 sequence containing one of the amino acid sequences of SEQ ID NOs. 6, 14, and 17, (d) A CDR-L1 sequence containing one of the amino acid sequences of SEQ ID NOs. 7 and 23, (e) A CDR-L2 sequence containing any one of the amino acid sequences of SEQ ID NO: 8, and (f) A CDR-L3 sequence containing one of the amino acid sequences of SEQ ID NOs. 9 and 18.

[0104] In some embodiments, the anti-TREM2 antibody comprises two, three, four, five, or all six of (a) to (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).

[0105] In some embodiments, the anti-TREM2 antibody includes: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (e) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 6, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 9, or (f) CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, CDR-H3 containing the amino acid sequence of SEQ ID NO: 14, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 9, or (g) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 9.

[0106] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26.

[0107] In some embodiments, the anti-TREM2 antibody includes a light chain variable region containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27.

[0108] In some embodiments, the anti-TREM2 antibody includes 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 with any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26, 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 with any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27. In some embodiments, anti-TREM2 includes a heavy chain variable region comprising any one amino acid sequence of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26, and a light chain variable region comprising any one amino acid sequence of SEQ ID NOs: 3, 11, 16, 20, 22, and 27.

[0109] In some embodiments, the anti-TREM2 antibody includes: (a) V having at least 85% sequence identity with respect to Sequence ID No. 2 H V has at least 85% sequence identity with respect to the sequence and sequence number 3. L array, or (b) V having at least 85% sequence identity with respect to sequence number 10 H V has at least 85% sequence identity with respect to sequence number 11. L array, or (c) V having at least 85% sequence identity with respect to sequence number 15 H V has at least 85% sequence identity with respect to the sequence and sequence number 16. L array, or (d) V having at least 85% sequence identity with sequence number 19 H V has at least 85% sequence identity with respect to sequence number 20. L array, or (e) V having at least 85% sequence identity with respect to sequence number 21 H V has at least 85% sequence identity with respect to sequence number 20. L array, or (f) V having at least 85% sequence identity with sequence number 19 H V has at least 85% sequence identity with respect to sequence and sequence number 22. L array, or (g) V having at least 85% sequence identity with sequence number 21 H V has at least 85% sequence identity with respect to sequence and sequence number 22. L array, or (h) V having at least 85% sequence identity with sequence number 24 H V has at least 85% sequence identity with respect to sequence number 20. L array, or (i) V having at least 85% sequence identity with sequence number 26 H V has at least 85% sequence identity with respect to sequence number 20. L array, or (j) V having at least 85% sequence identity with sequence number 24 H V has at least 85% sequence identity with respect to sequence and sequence number 22. L array, or (k) V having at least 85% sequence identity with sequence number 26 H V has at least 85% sequence identity with respect to sequence and sequence number 22. L array, or (l) V having at least 85% sequence identity with sequence number 24 H V has at least 85% sequence identity with respect to sequence and sequence number 27. L array.

[0110] In some embodiments, an anti-TREM2 antibody comprises one or more sequences encompassed by a consensus sequence disclosed herein. As a non-limiting example, a consensus sequence can be identified by aligning a heavy-chain or light-chain sequence (e.g., a CDR) against an antibody derived from the same (or similar) germline. In some embodiments, a consensus sequence may be generated from an antibody containing sequences of the same (or similar) length and / or at least one very similar CDR (e.g., very similar CDR3). In some embodiments, such sequences in those antibodies may be aligned and compared to identify conserved amino acids or motifs (i.e., locations where changes in the sequence may alter protein function) and / or regions where mutations reside in the sequence (i.e., locations where mutations in the sequence are unlikely to significantly affect protein function). Alternatively, consensus sequences may be identified by aligning a heavy-chain or light-chain sequence (e.g., a CDR) with an antibody that binds to the same or similar (e.g., overlapping) epitopes to determine conserved amino acids or motifs (i.e., locations where changes in the sequence may alter protein function) and regions where mutations are present in the sequence alignment (i.e., locations where mutations in the sequence are unlikely to significantly affect protein function). In some embodiments, one or more consensus sequences may be identified with antibodies that recognize the same or similar epitopes as the anti-TREM2 antibodies disclosed herein. Exemplary consensus sequences include SEQ ID NOs. 28–32. In the consensus sequences of SEQ ID NOs. 28–32, capital letters represent amino acid residues that are fully conserved between the aligned sequences (e.g., the aligned CDR sequences), while "X" or Greek letters (e.g., "α", "β", "γ", "δ", "ε", or "φ") represent amino acid residues that are not fully conserved between the aligned sequences. When selecting an amino acid to be inserted at a position indicated by "X" or a Greek letter, it will be understood that in some embodiments, the amino acid is selected from amino acids found at the corresponding position in the aligned sequence.

[0111] Clones CL0020188, CL0020306, CL0020307, ​​and variants of CL0020188 In some embodiments, the anti-TREM2 antibody includes: (a) A CDR-H1 sequence containing the sequence GFTFT-α6-FYMS (sequence number 28) (where α6 is D or N), (b) VIRN-β5-β6-N-β8-YT-β 11 -β 12 -YNPSVKG(Sequence ID 29)(In the sequence, β5 is K or R, β6 is A or P, β8 is G or A, β 11 is A or T, and β 12 CDR-H2 sequence containing the sequence (which is G or D), (c) CDR-H3 sequence containing the sequence γ1-RL-γ4-YGFDY (Sequence ID 30) (wherein γ1 is A or T and γ4 is T or S), (d) QSSKSLLHS-δ 10 -GKTYLN(sequence number 31)(in sequence, δ 10 CDR-L1 sequence containing the sequence (is N or T), (e) CDR-L2 sequence containing the sequence of WMSTRAS (sequence number 8), and (f) A CDR-L3 sequence containing the sequence QQFLE-φ6-PFT (sequence number 32) (where φ6 is Y or F).

[0112] In some embodiments, the anti-TREM2 antibody includes a CDR-H1 sequence selected from SEQ ID NOs: 4 and 12. In some embodiments, the anti-TREM2 antibody includes a CDR-H2 sequence selected from SEQ ID NOs: 5, 13, and 25. In some embodiments, the anti-TREM2 antibody includes a CDR-H3 sequence selected from SEQ ID NOs: 6, 14, and 17. In some embodiments, the anti-TREM2 antibody includes a CDR-L1 sequence selected from SEQ ID NOs: 7 and 23. In some embodiments, the anti-TREM2 antibody includes a CDR-L3 sequence selected from SEQ ID NOs: 9 and 18.

[0113] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing 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%) to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26.

[0114] In some embodiments, the anti-TREM2 antibody includes a light chain variable region containing 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%) to any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27.

[0115] Clone CL0020188 In some embodiments, the anti-TREM2 antibody includes a CDR-H1 sequence containing the amino acid sequence of SEQ ID NO: 4, a CDR-H2 sequence containing the amino acid sequence of SEQ ID NO: 5, a CDR-H3 sequence containing the amino acid sequence of SEQ ID NO: 17, a CDR-L1 sequence containing the amino acid sequence of SEQ ID NO: 7, a CDR-L2 sequence containing the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence containing the amino acid sequence of SEQ ID NO: 18.

[0116] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0117] In some embodiments, the anti-TREM2 antibody includes a light chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0118] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and a light chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16.

[0119] In some embodiments, the anti-TREM2 antibody includes heavy chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 4, 5, and 17, respectively, and includes a heavy chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 15 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody includes light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 7, 8, and 18, respectively, and includes a light chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 16 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0120] In some embodiments, the anti-TREM2 antibody competes for binding with the antibodies described herein (for example, antibodies comprising heavy chain CDR1-3 and light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs. 4, 5, 17, 7, 8, and 18, respectively, or antibodies comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16).

[0121] In some embodiments, the anti-TREM2 antibody includes a CDR-H1 sequence containing the amino acid sequence of SEQ ID NO: 4, a CDR-H2 sequence containing the amino acid sequence of SEQ ID NO: 25, a CDR-H3 sequence containing the amino acid sequence of SEQ ID NO: 17, a CDR-L1 sequence containing the amino acid sequence of SEQ ID NO: 23, a CDR-L2 sequence containing the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence containing the amino acid sequence of SEQ ID NO: 18.

[0122] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97%) with respect to SEQ ID NO: 24.

[0123] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a light chain variable region containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 22 (e.g., at least 90%, 95%, or 97% sequence identity).

[0124] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97% sequence identity), and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 22 (e.g., at least 90%, 95%, or 97% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 24, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22.

[0125] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes heavy chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 4, 25, and 17, respectively, and includes a heavy chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 23, 8, and 18, respectively, and includes a light chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 22 (e.g., at least 90%, 95%, or 97% sequence identity).

[0126] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety competes for binding with the antibodies described herein (for example, antibodies comprising heavy chain CDR1-3 and light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs. 4, 25, 17, 23, 8, and 18, respectively, or antibodies comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NOs. 24 and a light chain variable region containing the amino acid sequence of SEQ ID NOs. 22).

[0127] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a CDR-H1 sequence containing the amino acid sequence of SEQ ID NO: 4, a CDR-H2 sequence containing the amino acid sequence of SEQ ID NO: 25, a CDR-H3 sequence containing the amino acid sequence of SEQ ID NO: 17, a CDR-L1 sequence containing the amino acid sequence of SEQ ID NO: 7, a CDR-L2 sequence containing the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence containing the amino acid sequence of SEQ ID NO: 9.

[0128] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97%) with respect to SEQ ID NO: 24.

[0129] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a light chain variable region containing an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97%) with respect to SEQ ID NO: 27.

[0130] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97% sequence identity), and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 27 (e.g., at least 90%, 95%, or 97% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 24, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 27.

[0131] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes heavy chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 4, 25, and 17, respectively, and includes a heavy chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and includes a light chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 27 (e.g., at least 90%, 95%, or 97% sequence identity).

[0132] In some embodiments, the anti-TREM2 antibody or antigen-binding moiety is an antibody that competes for binding with the antibodies described herein (for example, an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 containing 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 containing the amino acid sequence of SEQ ID NO: 24 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 27).

[0133] Clone CL0020306 In some embodiments, the anti-TREM2 antibody includes a CDR-H1 sequence containing the amino acid sequence of SEQ ID NO: 4, a CDR-H2 sequence containing the amino acid sequence of SEQ ID NO: 5, a CDR-H3 sequence containing the amino acid sequence of SEQ ID NO: 6, a CDR-L1 sequence containing the amino acid sequence of SEQ ID NO: 7, a CDR-L2 sequence containing the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence containing the amino acid sequence of SEQ ID NO: 9.

[0134] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2.

[0135] In some embodiments, the anti-TREM2 antibody includes a light chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0136] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), and a light chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 3.

[0137] In some embodiments, the anti-TREM2 antibody includes heavy chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and includes a heavy chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 2 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody includes light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and includes a light chain variable region having at least 85% sequence identity with respect to SEQ ID NO: 3 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0138] In some embodiments, the anti-TREM2 antibody competes for binding with the antibodies described herein (for example, antibodies comprising heavy chain CDR1-3 and light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively, or antibodies comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 3).

[0139] Clone CL0020307 In some embodiments, the anti-TREM2 antibody includes a CDR-H1 sequence containing the amino acid sequence of SEQ ID NO: 12, a CDR-H2 sequence containing the amino acid sequence of SEQ ID NO: 13, a CDR-H3 sequence containing the amino acid sequence of SEQ ID NO: 14, a CDR-L1 sequence containing the amino acid sequence of SEQ ID NO: 7, a CDR-L2 sequence containing the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence containing the amino acid sequence of SEQ ID NO: 9.

[0140] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 10 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding moiety includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10.

[0141] In some embodiments, the anti-TREM2 antibody includes a light chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 11.

[0142] In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and a light chain variable region containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the anti-TREM2 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11.

[0143] In some embodiments, the anti-TREM2 antibody includes heavy chain CDR1-3 containing the amino acid sequences of SEQ ID NOs. 12, 13, and 14, respectively, and includes a heavy chain variable region having at least 85% sequence identity with respect to SEQ ID NO. 10 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody includes light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs. 7, 8, and 9, respectively, and includes a light chain variable region having at least 85% sequence identity with respect to SEQ ID NO. 11 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0144] In some embodiments, the anti-TREM2 antibody is an antibody that competes for binding with the antibodies described herein (for example, an antibody comprising heavy chain CDR1-3 and light chain CDR1-3 containing the amino acid sequences of SEQ ID NOs. 12, 13, 14, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11).

[0145] Binding properties of anti-TREM2 antibodies In some embodiments, the antibodies described herein that specifically bind to the TREM2 protein bind to TREM2 expressed on cells (e.g., primary cells or cell lines that endogenously express TREM2, such as human macrophages, or primary cells or cell lines that have been engineered to express TREM2, such as those described in the Examples section below). In some embodiments, the antibodies described herein that specifically bind to the TREM2 protein bind to purified or recombinant TREM2 protein or a portion thereof, or to a chimeric protein containing TREM2 or a portion thereof (e.g., an Fc fusion protein containing TREM2 or an Fc fusion protein containing the external domain of TREM2).

[0146] In some embodiments, antibodies that specifically bind to the human TREM2 protein exhibit cross-reactivity with one or more other TREM2 proteins of different species. In some embodiments, antibodies that specifically bind to the human TREM2 protein exhibit cross-reactivity with cynomolgus monkey ("cyno") TREM2 protein. In some embodiments, antibodies that specifically bind to the human TREM2 protein exhibit cross-reactivity with mouse TREM2 protein. In some embodiments, anti-TREM2 antibodies exhibit cross-reactivity with human TREM2, cyno TREM2, and mouse TREM2.

[0147] 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)), binding equilibrium exclusion (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, binding equilibrium exclusion is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, a BioLayer interference assay is used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0148] Epitope recognized by anti-TREM2 antibody In some embodiments, the anti-TREM2 antibody recognizes the same or substantially the same human TREM2 epitope as the epitope recognized by the antibody clone described herein. As used herein, the term “substantially the same” means, when used in relation to the epitope recognized by the antibody clone described herein, that the anti-TREM2 antibody recognizes an epitope that is identical to, within, or substantially identical to, the epitope recognized by the antibody clone described herein (e.g., having at least 90% sequence identity to, or having one, two, or three amino acid substitutions, e.g., conservative substitutions to, or substantially overlapping to, the epitope (e.g., overlapping to, at least 50%, 60%, 70%, 80%, 90%, or 95%) the epitope recognized by the antibody clone described herein.

[0149] In some embodiments, the anti-TREM2 antibody recognizes the same or substantially the same human TREM2 epitopes recognized by antibody clones selected from the group consisting of clones CL0020306, CL0020188, CL0020307, ​​and their variants.

[0150] In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope within the stalk region of TREM2. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that includes, is located in, or consists of residues 129-172 or 131-169 of SEQ ID NO: 1. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 that includes, is located in, or consists of residues 129-148 (e.g., 143-148 of SEQ ID NO: 1). In some embodiments, the anti-TREM2 antibody is an agonist that activates TREM2 / DAP12 signaling (e.g., by inducing phosphorylation of a kinase such as Syk) and binds to human TREM2 at an epitope within the stalk region of TREM2. In some embodiments, an anti-TREM2 antibody binds to human TREM2 at an epitope within the stalk region of TREM2, inhibiting cleavage of TREM2 by a protease (e.g., ADAM17).

[0151] Functional properties of anti-TREM2 antibodies In some embodiments, an anti-TREM2 antibody (e.g., an antibody having one or more disclosed CDR sequences, heavy chain variable region sequences, and / or light chain variable region sequences) functions in one or more TREM2 activities disclosed herein. For example, in some embodiments, an anti-TREM2 antibody modulates the level of sTREM2 protein (e.g., the level of sTREM2 shed from the cell surface to an extracellular sample), modulates the recruitment or phosphorylation of kinases (e.g., Syk kinase) that interact with the TREM2 / DAP12 signaling complex, and / or modulates one or more downstream activities of the signaling complex, such as phagocytosis, cell proliferation, cell survival, cell differentiation, cytokine secretion, or cell migration.

[0152] In some embodiments, anti-TREM2 antibodies enhance one or more ligand-induced TREM2 activities (e.g., those described herein). In some embodiments, the ligand is a lipid ligand. Examples of TREM2 lipid ligands include 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), 2-arachidonoylglycerol (2-AG), 7-ketocholesterol (7-KC), 24(S)-hydroxycholesterol (24OHC), 25(S)-hydroxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), acylcarnitine (AC), alkylacylglycerophosphocholine (PAF), and α-galactosyl Luceramide (KRN7000), bis(monoacylglycero)phosphate (BMP), cardiolipin (CL), ceramide, ceramide-1-phosphate (C1P), cholesteryl ester (CE), cholesterol phosphate (CP), diacylglycerol 34:1 (DG34:1), diacylglycerol 38:4 (DG38:4), diacylglycerol pyrophosphate (DGPP), dihydroceramide (DhCer), dihydrosphingomyelin (DhSM), ether phosphatidylcholine (PCe), Cholesterol (FC), galactosylceramide (GalCer), galactosylsphingosine (GalSo), ganglioside GM1, ganglioside GM3, glucosylsphingosine (GlcSo), Hanks equilibrium salt solution (HBSS), Kdo2-lipid A (KLA), lactosylceramide (LacCer), lysoalkylacylglycerophosphocholine (LPAF), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LP E) Lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysosphingomyelin (LSM), lysophosphatidylserine (LPS), N-acylphosphatidylethanolamine (NAPE), N-acylserine (NSer), oxidized phosphatidylcholine (oxPC), 9-hydroxystearic acid palmitate (PAHSA), phosphatidylethanolamine (PE), phosphatidylethanol (PEtOH), phosphatidic acid (PA),Examples include, but are not limited to, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphinganine, sphinganine-1-phosphate (Sa1P), sphingomyelin (SM), sphingosine, sphingosine-1-phosphate (So1P), and sulfatides.

[0153] Adjusting sTREM2 shedding In some embodiments, the anti-TREM2 antibody alters the level of sTREM2 protein in the sample, for example, the level of sTREM2 shedding from the cell surface to the extracellular sample. In some embodiments, the anti-TREM2 antibody decreases the level of sTREM2.

[0154] In some embodiments, an anti-TREM2 antibody reduces the level of sTREM2 if the amount of sTREM2 in the treated sample decreases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control value. In some embodiments, an anti-TREM2 antibody reduces the level of sTREM2 if the amount of sTREM2 in the treated sample decreases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control value. In some embodiments, the control value is the amount of sTREM2 in an untreated sample (e.g., supernatant from TREM2-expressing cells not treated with an anti-TREM2 antibody, or a sample from a subject not treated with an anti-TREM2 antibody) or a sample treated with a suitable non-TREM2-binding antibody.

[0155] In some embodiments, sTREM2 shedding is measured using a sample containing a body fluid, such as blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample contains cerebrospinal fluid. In some embodiments, the sample contains supernatant from a cell culture (e.g., supernatant from a primary cell or cell line that intrinsically expresses TREM2, such as human macrophages, or from a primary cell or cell line that has been engineered to express TREM2, as described in the Examples section below).

[0156] In some embodiments, the level of sTREM2 in a sample is measured using an immunoassay. Immunosounds are known in the art and are not limited to, but include enzyme immunoassays (EIA) such as enzyme multiplex immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), and electrochemiluminescence immunoassay (ECL). In some embodiments, the sTREM2 level is measured using an ELISA assay. In some embodiments, the sTREM2 level is measured using an ELISA assay as described in the Examples section below.

[0157] Kinase recruitment or regulation of phosphorylation In some embodiments, anti-TREM2 antibodies induce phosphorylation of kinases that interact with the TREM2 / DAP12 signaling complex (e.g., Syk, ZAP70, PI3K, Erk, AKT, or GSK3b). In some embodiments, anti-TREM2 antibodies induce phosphorylation of kinases that interact with the TREM2 / DAP12 signaling complex without blocking the binding of native TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance phosphorylation of kinases that interact with the TREM2 / DAP12 signaling complex induced by TREM2 ligands (e.g., lipid ligands). In some embodiments, anti-TREM2 antibodies induce or enhance the phosphorylation of Syk. In some embodiments, an anti-TREM2 antibody induces or enhances Syk phosphorylation if the level of Syk phosphorylation in a sample treated with the 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%, or at least 90% compared to a control value. In some embodiments, an anti-TREM2 antibody induces Syk phosphorylation if the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control value. In some embodiments, the control value is the level of Syk phosphorylation in untreated samples (e.g., samples containing TREM2-expressing cells not treated with anti-TREM2 antibody, or samples from subjects not treated with anti-TREM2 antibody), samples treated with TREM2 ligand but not with anti-TREM2 antibody, or samples treated with appropriate non-TREM2-binding antibody.

[0158] In some embodiments, immunoassays are used to detect and / or quantify phosphorylation (e.g., Syk phosphorylation) in a sample. In some embodiments, the immunoassay is enzyme immunoassay (EIA), enzyme multiplex immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemiluminescence immunoassay (ECL). In some embodiments, phosphorylation is detected and / or quantified using an immunoassay utilizing an amplified luminescent proximity homogenous assay (AlphaLISA®, PerkinElmer Inc.).

[0159] In some embodiments, phosphorylation is measured using a sample comprising one or more cells, for example, one or more TREM2-expressing cells (e.g., primary cells or cell lines that endogenously express TREM2, such as human macrophages or iPSC-derived microglia, or primary cells or cell lines that have been engineered to express TREM2, for example, as described in the Examples section below). In some embodiments, the sample comprises body fluids, for example, blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises tissues (e.g., lungs, brain, kidneys, spleen, nerve tissue, or skeletal muscle) or cells from such tissues. In some embodiments, the sample comprises endogenous body fluids, tissues, or cells (e.g., from human or non-human subjects).

[0160] Regulation of phagocytosis 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 native TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance phagocytosis induced by TREM2 ligands (e.g., lipid ligands). In some embodiments, anti-TREM2 antibodies enhance phagocytosis if the level of phagocytosis in a sample treated with the 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%, or at least 90% compared to a control value. In some embodiments, anti-TREM2 antibodies enhance phagocytosis if the level of phagocytosis in a sample treated with the anti-TREM2 antibody increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control value. In some embodiments, the control value is the level of phagocytosis in untreated samples, samples treated with TREM2 ligand but not with anti-TREM2 antibody, or samples treated with appropriate non-TREM2-binding antibody.

[0161] In some embodiments, phagocytosis is measured using a phagocytic assay with a labeled substrate. Phagocytic assays are known in the art. In some embodiments, the phagocytic assay is performed on a sample containing cells that endogenously express TREM2, such as human macrophages or microglia. In some embodiments, the phagocytic assay is performed on a sample containing cells that have been engineered to express TREM2. In some embodiments, phagocytosis is measured using a human macrophage phagocytic assay described in the Examples section below.

[0162] Regulation of cell differentiation, function, migration, and survival In some embodiments, anti-TREM2 antibodies enhance cell migration, cell survival, cell function, or cell differentiation (e.g., 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 microglia). In some embodiments, anti-TREM2 antibodies enhance cell survival of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance cell function of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance the cell differentiation of one or more cell types (e.g., myeloid cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance the migration, survival, function, and / or differentiation of myeloid cells. In some embodiments, anti-TREM2 antibodies enhance the migration, survival, function, and / or differentiation of macrophages. In some embodiments, anti-TREM2 antibodies enhance the migration, survival, function, and / or differentiation of microglia. In some embodiments, anti-TREM2 antibodies enhance microglial activation. In some embodiments, anti-TREM2 antibodies enhance the migration, survival, function, and / or differentiation of disease-associated microglia. In some embodiments, anti-TREM2 antibodies enhance cell migration, cell survival, cell function, or cell differentiation without blocking the binding of native 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).

[0163] In some embodiments, an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of activity (e.g., migration, survival, function, or differentiation) in a sample treated with the 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%, or at least 90% compared to a control value. In some embodiments, an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of activity (e.g., migration, survival, function, or differentiation) in a sample treated with the anti-TREM2 antibody increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control value. In some embodiments, the control value is the level of activity (e.g., migration, survival, function, or differentiation) in untreated samples (e.g., samples not treated with anti-TREM2 antibody), samples treated with TREM2 ligand but not with anti-TREM2 antibody, or samples treated with appropriate non-TREM2 binding antibody.

[0164] In some embodiments, cell migration is measured using a chemotaxis assay, which is known in the art. In some embodiments, the cell migration assay (e.g., a chemotaxis assay) is performed on a sample containing cells that endogenously express TREM2, such as human macrophages. In some embodiments, the cell migration assay (e.g., a chemotaxis assay) is performed on a sample containing 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.

[0165] In some embodiments, cell viability is measured using a cell viability assay, which is known in the art. In some embodiments, the cell viability assay (e.g., a cell viability assay) is performed on a sample containing cells that endogenously express TREM2, such as human macrophages. In some embodiments, the cell viability assay (e.g., a cell viability assay) is performed on a sample containing cells that have been engineered to express TREM2. In some embodiments, cell viability is measured using a human macrophage viability assay, as described in the Examples section below.

[0166] In some embodiments, cell function is measured using a functional assay appropriate for the cell. For example, in some embodiments, macrophage cell function is evaluated using a phagocytic assay, as described, for example, in the Examples section below.

[0167] In some embodiments, cell differentiation is measured by evaluating the differentiation ability of cells that endogenously express TREM2. For example, in some embodiments, cell differentiation is measured by evaluating the ability of macrophages to differentiate from monocytes, as described, for example, in the Examples section.

[0168] In some embodiments, microglial activation is measured in vivo. In some embodiments, microglial activation is measured using TSPO-PET imaging. TSPO-PET imaging is known in the art.

[0169] In some embodiments, anti-TREM2 antibodies enhance microglial function without increasing neuroinflammation. The level of neuroinflammation 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 immunoassay (EIA), multiplex enzyme immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemiluminescence immunoassay (ECL).

[0170] IV. Fc polypeptide mutations in proteins containing an anti-TREM2 antigen binding moiety In some embodiments, the anti-TREM2 antibody comprises two Fc polypeptides, one or both of which may each contain independently selected modifications (e.g., mutations), or may be a wild-type Fc polypeptide, e.g., human IgG1Fc polypeptide. Non-limiting examples of mutations that can be introduced into one or both Fc polypeptides include, for example, mutations that enable the binding of the Fc polypeptide (or antibody containing it) to BBB receptors, e.g., transferrin receptor (TfR) proteins (e.g., human or cynomolgus monkey TfRs, such as those that may be expressed on brain endothelial cells), mutations that increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or mutations that result in knob and hole heterodimerization of the Fc polypeptide.

[0171] Transferrin receptor binding mutation In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide that includes modifications (e.g., amino acid substitutions) that enable the Fc polypeptide to bind to the TfR protein. Briefly, binding to the TfR protein (e.g., its apical domain) expressed on brain endothelial cells, for example, may, in some embodiments, enable the modified Fc polypeptide of this disclosure or an antibody containing it to cross the blood-brain barrier via receptor-mediated transcytosis. In certain embodiments, receptor-mediated transcytosis may enhance or improve the ability of the protein containing the Fc polypeptide to be present in the brain (i.e., on the luminal side of the blood-brain barrier), thereby enabling improved binding to TREM2 in the CNS and other functions, such as clearance, neutralization, or target immunodepletion.

[0172] Exemplary TfR-binding amino acid modifications to Fc (e.g., CH2 and / or CH3 moieties, fragments, or domains), as well as Fc polypeptides and moieties thereof containing amino acid modifications, are described in PCT Patent Publication WO2018 / 152326A1. These amino acid modifications, TfR-binding Fc polypeptide sequences and TfR-binding Fc polypeptides, as well as techniques for producing and testing them, are incorporated herein by reference. One or two Fc polypeptides of the Fc dimers of this disclosure can be manipulated to include modifications that enable binding to TfR. In certain embodiments, one Fc polypeptide of an Fc dimer includes modifications that enable binding to TfR, while the other Fc polypeptide does not.

[0173] In some embodiments, the modified Fc polypeptide includes the YxTEWSS (SEQ ID NO: 58) motif. In some embodiments, the modified Fc polypeptide includes the TxxExxxxF (SEQ ID NO: 59) motif. In some embodiments, the modified Fc polypeptide includes both the YxTEWSS (SEQ ID NO: 58) and TxxExxxxF (SEQ ID NO: 59) motifs.

[0174] In some embodiments, the modified Fc polypeptide contains wild-type amino acid residues at positions 380, 389, 390, and 415 according to EU numbering, where the wild-type amino acid residues are found at the corresponding positions in SEQ ID NO: 38.

[0175] In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide having the following amino acids according to EU numbering: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, or Val at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and Phe at position 421.

[0176] In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide having the following amino acids according to EU numbering: Trp at position 380, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 390, Thr at position 413, Glu at position 415, Glu at position 416, and Phe at position 421.

[0177] In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide having the following amino acids according to EU numbering: Glu at position 380, Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Asn at position 390, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421.

[0178] In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide having the following amino acids according to EU numbering: Glu at position 380, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Val at position 389, Asn at position 390, Thr at position 413, Glu at position 415, Glu at position 416, and Phe at position 421.

[0179] In some embodiments, the anti-TREM2 antibody comprises an Fc polypeptide having the following amino acids according to EU numbering: Glu at position 380, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Asn at position 390, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421.

[0180] In some embodiments, the modified Fc polypeptide contains a sequence having at least 90% identity to the amino acid sequence described in any one of SEQ ID NOs: 40, 43, 46, and 49. In some embodiments, the modified Fc polypeptide contains or consists of the amino acid sequence described in any one of SEQ ID NOs: 41, 44, 47, and 50.

[0181] Examples of adding modified Fc polypeptides are shown in Table 8.

[0182] Mutations that promote heterodimerization of Fc polypeptides In some embodiments, the Fc polypeptide present in the anti-TREM2 antibodies disclosed herein includes knob and hole mutations that promote heterodimer formation and inhibit homodimer formation. Generally, this modification involves introducing a projection ("knob") at the interface of the first polypeptide and a corresponding void ("hole") at the interface of the second polypeptide, with the projection positioned within the void such that heterodimer formation is promoted and homodimer formation is inhibited. The projection is constructed by substituting a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary depressions of the same or similar size as the projection are created on the interface of the second polypeptide by substituting a larger amino acid side chain with a smaller side chain (e.g., alanine or threonine). In some embodiments, such additional mutations are located in the Fc polypeptide at positions that do not adversely affect (e.g., do not inhibit) the binding of the Fc polypeptide to a BBB receptor, e.g., TfR.

[0183] In an exemplary embodiment of the knob-hole approach for dimerization, one Fc polypeptide present in the protein described herein has a T366W knob mutation, while the other Fc polypeptide has a Y407V mutation, typically accompanied by T366S and L368A hole mutations.

[0184] In some embodiments, one or both Fc polypeptides may be manipulated to include other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the CH3-CH3 interface, which is naturally charged, or hydrophobic patch modifications.

[0185] In some embodiments, modifications may be introduced to enhance the serum half-life. For example, in some embodiments, one or both Fc polypeptides present in the anti-TREM2 protein of this disclosure may contain tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, when numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides may have M252Y, S254T, and T256E substitutions. Alternatively, one or both Fc polypeptides may have M428L and / or N434S substitutions according to EU numbering. Alternatively, one or both Fc polypeptides may have N434S or N434A substitutions.

[0186] Fc effect pedal function In some embodiments, one or both Fc polypeptides of the anti-TREM2 proteins disclosed herein may include modifications that reduce effector function, i.e., modifications that reduce their ability to induce certain biological functions when bound to Fc receptors expressed on effector cells that mediate effector function. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector function may vary depending on the antibody class. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity when bound to appropriate Fc receptors present on immune system cells, while native human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function when bound to appropriate Fc receptors present on immune system cells.

[0187] In some embodiments, one or both Fc polypeptides may include modifications that adjust the effector function.

[0188] In some embodiments, one or both Fc polypeptides may contain modifications that reduce or eliminate effector function. Exemplary Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in the CH2 domain, e.g., substitutions at positions 234 and 235 according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides may contain alanine residues at positions 234 and 235. Therefore, one or both Fc polypeptides may have L234A and L235A (LALA) substitutions.

[0189] Additional Fc polypeptide mutations that modulate effector function may include, but are not limited to, mutations in which proline at position 329 is replaced with glycine, arginine, serine, or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and the tryptophan residues Trp87 and Trp110 of FcγRIII. Additional exemplary substitutions include S228P, E233P, L235E, N297A, N297D, N297G, and P331S, according to the EU numbering scheme. Multiple substitutions may exist, for example, according to the EU numbering scheme, including L234A and L235A of the human IgG1 Fc region, L234A, L235A, and P329G of the human IgG1 Fc region, L234A, L235A, and P329S of the human IgG1 Fc region, S228P and L235E of the human IgG4 Fc region, L234A and G237A of the human IgG1 Fc region, L234A, L235A, and G237A of the human IgG1 Fc region, V234A and G237A of the human IgG2 Fc region, L235A, G237A, and E318A of the human IgG4 Fc region, and S228P and L236E of the human IgG4 Fc region. In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, for example, substitutions at positions 298, 333, and / or 334 according to the EU numbering scheme.

[0190] Mutations that prolong the FcRn binding site and serum half-life In certain embodiments, the Fc polypeptide present in the anti-TREM2 protein of this disclosure (e.g., a modified Fc polypeptide) may include an FcRn binding site. In some embodiments, the FcRn binding site is located in the Fc polypeptide or a fragment thereof.

[0191] In some embodiments, the FcRn binding site includes a native FcRn binding site. In some embodiments, the FcRn binding site does not involve amino acid changes to the amino acid sequence of the native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, for example, a human IgG binding site. In some embodiments, the FcRn binding site includes modifications that alter the FcRn binding.

[0192] In some embodiments, the FcRn binding site has one or more mutated, e.g., substituted amino acid residues, which either prolong the serum half-life or substantially reduce it (i.e., the reduction in serum half-life is 25% or less when assayed under the same conditions compared to an equivalent Fc polypeptide with a wild-type residue at the mutated position). In some embodiments, the FcRn binding site has one or more substituted amino acid residues at positions 251-256, 428, and 433-436 according to the EU numbering scheme.

[0193] In some embodiments, one or more residues at or near the FcRn binding site are mutated relative to the native human IgG sequence to extend the serum half-life of the polypeptide. In some embodiments, the mutation is introduced at one, two, or three of positions 252, 254, and 256. In some embodiments, the mutations are M252Y, S254T, and T256E. In some embodiments, the Fc polypeptide further comprises the mutations M252Y, S254T, and T256E. In certain embodiments, one or both Fc polypeptides present in the anti-TREM2 protein of this disclosure may contain tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 when numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides may have the M252Y, S254T, and T256E substitutions.

[0194] In some embodiments, the mutation is M428L and / or N434S. In some embodiments, the Fc polypeptide further comprises the mutation N434S, with or without M428L. In some embodiments, the Fc polypeptide contains mutations at one, two, or all three of the positions T307, E380, and N434 according to the EU numbering scheme. In some embodiments, the mutation is T307Q and N434A. In some embodiments, the Fc polypeptide comprises mutations T307A, E380A, and N434A. In some embodiments, the Fc polypeptide contains mutations at positions T250 and M428 according to the EU numbering scheme. In some embodiments, the Fc polypeptide contains mutations T250Q and / or M428L. In some embodiments, the Fc polypeptide contains mutations at positions M428 and N434 according to the EU numbering scheme. In some embodiments, the Fc polypeptide contains mutations M428L and N434S. In some embodiments, the antibody of the Disclosure may comprise two Fc polypeptides, each of which comprises an M428L and / or N434S substitution. In some embodiments, the Fc polypeptide comprises an N434S or N434A mutation. In some embodiments, the antibody of the Disclosure may comprise two Fc polypeptides, each of which comprises an N434S or N434A substitution.

[0195] V. Antibody Preparation In some embodiments, antibodies are prepared by immunizing an animal (e.g., mouse, rabbit, or rat) with an antigen or antigen mixture to induce an antibody response. In some embodiments, the antigen or antigen mixture is administered in combination with an adjuvant (e.g., Freund's adjuvant). After the initial immunization, one or more subsequent immunizations with antigens may be administered to enhance antibody production. After immunization, antigen-specific B cells are recovered, for example, from the spleen and / or lymphoid tissue. To produce monoclonal antibodies, the B cells are fused with myeloma cells and subsequently screened for antigen specificity. Methods for preparing antibodies are also described in the Examples section below.

[0196] The genes encoding the heavy and light chains of a target antibody can be cloned from cells. For example, the gene encoding a monoclonal antibody can be cloned from a hybridoma to produce a recombinant monoclonal antibody. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be constructed from hybridomas or plasma cells. Alternatively, phage or yeast display techniques can be used to identify antibodies and Fab fragments that specifically bind to a selected antigen. Antibodies can also be bispecific, meaning they can recognize two different antigens. Antibodies can also be heteroconjugates, such as two covalently linked antibodies or immunotoxins.

[0197] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is mammalian cell expression, e.g., hybridoma or CHO cell expression systems. Numerous such systems are widely available from commercial suppliers. H and V L In embodiments that include both regions, V H and V L The region may be expressed using a single vector, for example, in a dicistronic expression unit or under the control of different promoters. In other embodiments, V H and V L The region may be expressed using a separate vector. H or V L The region may optionally contain methionine at its N-terminus.

[0198] In some embodiments, the antibody is a chimeric antibody. Methods for producing chimeric antibodies are known in the art. For example, a chimeric antibody can be produced in which the antigen-binding region (heavy chain variable region and light chain variable region) of one species, such as mouse, is fused with the effector region (constant domain) of another species, such as human. As another example, a "class-switched" chimeric antibody can be produced in which the effector region of the antibody is replaced with the effector region of a different immunoglobulin class or subclass.

[0199] In some embodiments, the antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity. A humanized antibody typically includes one or more variable regions (e.g., CDRs) or a portion thereof that are non-human (e.g., derived from mouse variable region sequences), and optionally several framework regions or a portion thereof that are non-human, and further includes one or more constant regions derived from a human antibody sequence. Methods for humanizing non-human antibodies are known in the art. Humanized antibodies or human antibodies can be expressed using other organisms, such as transgenic mice or other mammals. Other methods for humanizing antibodies include, for example, variable domain resurfacing, CDR grafting, specificity-determining residue (SDR) grafting, guided selection, and framework shuffling.

[0200] As an alternative to humanization, fully human antibodies can be produced. As a non-limiting example, it is possible to produce transgenic animals (e.g., mice) that, upon immunization, can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been explained that homozygous deletion of the antibody heavy chain binding region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Introducing a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen exposure. Alternatively, human antibodies can be produced by hybridoma-based methods, such as using primary human B cells to create cell lines that produce human monoclonal antibodies.

[0201] Human antibodies can also be produced using phage display or yeast display techniques. In phage display, a repertoire of variable heavy and variable light chain genes is amplified and expressed in a phage display vector. In some embodiments, the antibody library is a native repertoire amplified from human sources. In some embodiments, the antibody library is a synthetic library created by cloning and recombining heavy and light chain sequences to produce a large pool of antibodies with varying antigen specificities. Phages typically present antibody fragments (e.g., Fab fragments or scFv fragments), which are then screened for binding to the antigen of interest.

[0202] In some embodiments, antibody fragments (Fab, Fab', F(ab')2, scFv, V H , or V HHVarious techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be recovered directly from E. coli cells and chemically coupled to form F(ab')2 fragments. By another approach, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for producing antibody fragments will be obvious to those skilled in the art.

[0203] In some embodiments, the antibody or antibody fragment is conjugated (pegylated) to another molecule, such as polyethylene glycol, or to serum albumin, to extend its half-life in vivo.

[0204] VI. Nucleic acids, vectors, and host cells In some embodiments, the anti-TREM2 antibodies disclosed herein are prepared using recombinant methods. Accordingly, in some embodiments, the disclosure provides isolated nucleic acids comprising nucleic acid sequences encoding any of the anti-TREM2 antibodies described herein (e.g., one or more of the CDR, heavy chain variable region, and light chain variable region described herein); vectors comprising such nucleic acids; and host cells into which nucleic acids used for replicating and / or expressing antibodies are introduced.

[0205] In some embodiments, the polynucleotide (e.g., isolated polynucleotide) comprises a nucleotide sequence encoding an antibody described herein (e.g., one described in the preceding section under the heading "Anti-TREM2 Antibody Sequence"). In some embodiments, the polynucleotide comprises a nucleotide sequence encoding one or more amino acid sequences (e.g., a CDR sequence, a heavy chain sequence, or a light chain sequence) disclosed in Table 8 below. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to the sequences disclosed in Table 8 below (e.g., a CDR sequence, a heavy chain sequence, or a light chain sequence). In some embodiments, the polynucleotide described herein is operably linked to a heterologous nucleic acid, e.g., a heterologous promoter.

[0206] Suitable vectors containing polynucleotides encoding the antibodies or fragments thereof of this disclosure include cloning vectors and expression vectors. The cloning vector selected may vary depending on the host cell to which it is intended to be used, but useful cloning vectors generally have the ability to self-replicate, may have a single target of a particular restriction endonuclease, and / or may possess a marker gene that can be used for selecting the clone containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage 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.

[0207] Expression vectors are generally replicable polynucleotide constructs containing the nucleic acids of this disclosure. Expression vectors can replicate in host cells either as episomes or as integral parts of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, and retroviruses), and any other vectors.

[0208] Suitable host cells for cloning or expressing the polynucleotides or vectors described herein include prokaryotic cells or eukaryotic cells. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is a eukaryote, such as Chinese hamster ovary (CHO) cells or lymphoid cells. In some embodiments, the host cell is a human cell, such as a human embryonic kidney (HEK) cell.

[0209] In another embodiment, a method for producing the anti-TREM2 antibody described herein is provided. In some embodiments, the method comprises culturing the host cells described herein (e.g., host cells expressing the polynucleotide or vector described herein) under conditions suitable for antibody expression. In some embodiments, the antibody is subsequently recovered from the host cells (or host cell culture medium).

[0210] VII. Treatment methods using anti-TREM2 antibodies In another embodiment, a therapeutic method is provided that uses an anti-TREM2 antibody disclosed herein (e.g., an anti-TREM2 antibody described in Section III above). In some embodiments, a method for treating neurodegenerative diseases is provided. In some embodiments, a method for modulating one or more TREM2 activities (e.g., in a subject having a neurodegenerative disease) is provided.

[0211] In some embodiments, methods for treating neurodegenerative diseases are provided. In some embodiments, the neurodegenerative diseases include Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with chromosome 17-linked parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / parkinsonian dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, Boxer dementia, diffuse neurofibrillary tangle disease with calcification, Down syndrome, familial Anglo-British dementia, familial Danish-British dementia, and Gerstmann-Sträussler-Scheinka dementia. The neurodegenerative disease is selected from the group consisting of Alzheimer's disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadeloupean PSP, Haller-Vorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-dominant dementia, Niemann-Pick disease type C, bulbopontigrine degeneration, Parkinson's disease, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and neurofibrillary senile dementia. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neurodegenerative disease is Nasu-Hakola disease. In some embodiments, the neurodegenerative disease is frontotemporal dementia. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the method involves administering to a target an isolated antibody or its antigen-binding fragment (e.g., the anti-TREM2 antibody described herein) that specifically binds to the human TREM2 protein, or a pharmaceutical composition containing the anti-TREM2 antibody described herein.

[0212] In some embodiments, the anti-TREM2 antibody (or its antigen-binding moiety or pharmaceutical composition) described herein is used to treat neurodegenerative diseases characterized by TREM2 mutations. In some embodiments, the neurodegenerative disease characterized by TREM2 mutations is Alzheimer's disease, for example, Alzheimer's disease characterized by the R47H mutation in TREM2.

[0213] In some embodiments, methods are provided for modulating one or more TREM2 activities in subjects (e.g., subjects with neurodegenerative diseases). In some embodiments, the methods include modulating sTREM2 levels, modulating the recruitment or phosphorylation of kinases (e.g., Syk kinase) that interact with the TREM2 / DAP12 signaling complex, modulating phagocytosis (e.g., phagocytosis of cell debris, amyloid-beta particles, etc.), modulating cell migration (e.g., migration of myeloid cells, macrophages, microglia, and disease-associated microglia), and / or modulating cell differentiation (e.g., myeloid cells, macrophages, microglia, and disease-associated microglia). In some embodiments, methods are provided for enhancing one or more TREM2 activities in subjects with neurodegenerative diseases. In some embodiments, methods are provided for decreasing sTREM2 levels in subjects with neurodegenerative diseases. In some embodiments, a method for modulating one or more TREM2 activities in a subject includes administering to the subject an isolated antibody or its antigen-binding moiety that specifically binds to the human TREM2 protein (e.g., the anti-TREM2 antibody described herein), or a pharmaceutical composition containing the anti-TREM2 antibody described herein.

[0214] In some embodiments, the subject being treated is a human, such as an adult human or a pediatric human.

[0215] In some embodiments, methods are provided for reducing plaque accumulation in subjects having neurodegenerative diseases. In some embodiments, the method comprises administering an antibody or pharmaceutical composition described herein to a subject. In some embodiments, the subject has Alzheimer's disease. In some embodiments, the subject is an animal model of the neurodegenerative disease (e.g., a 5XFAD or APP / PS1 mouse model). In some embodiments, plaque accumulation is measured by amyloid plaque imaging and / or tau imaging, for example, using positron emission tomography (PET) scanning. In some embodiments, administration of an anti-TREM2 antibody reduces plaque accumulation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a baseline value (e.g., the level of plaque accumulation in the subject before administration of the anti-TREM2 antibody).

[0216] In some embodiments, the anti-TREM2 antibody is administered to the subject in a therapeutically effective dose or amount. However, the dose may vary depending on several factors, including the chosen route of administration, the composition of the drug, the patient's response, the severity of the condition, the subject's body weight, and the prescribing physician's judgment. The dose may be increased or decreased over time as needed by the individual patient. In certain cases, the patient is initially given a low dose, which is then increased to an effective dose that the patient can tolerate. Determining the effective dose is well within the capabilities of those skilled in the art.

[0217] The routes of administration of the anti-TREM2 antibody described herein may be oral, intraperitoneal, percutaneous, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intrafocal, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or ear delivery, or any other method known in the art. In some embodiments, the antibody is administered orally, intravenously, or intraperitoneally.

[0218] In some embodiments, the anti-TREM2 antibody (and optionally another therapeutic agent) is administered to the subject over an extended period, for example, for at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or 350 days or longer.

[0219] VIII. Pharmaceutical Compositions and Kits In another embodiment, pharmaceutical compositions and kits are provided that include antibodies that specifically bind to the human TREM2 protein. In some embodiments, the pharmaceutical compositions and kits are for use in the treatment of neurodegenerative diseases. In some embodiments, the pharmaceutical compositions and kits are for use in the modulation (e.g., enhancement or inhibition) of one or more TREM2 activities, such as Syk phosphorylation. In some embodiments, the pharmaceutical compositions and kits are for use in the modulation (e.g., reduction) of sTREM2 levels.

[0220] Pharmaceutical composition 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 the antibody or antigen-binding fragment described in Section III above.

[0221] 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. The 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 activator. A variety of pharmaceutically acceptable excipients are well known in the art.

[0222] In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration. A pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the activator(s). Examples of physiologically acceptable compounds include carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of activators; or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are well known in the art.

[0223] The pharmaceutical compositions described herein may be prepared in ways known to those skilled in the art, for example, by conventional mixing, dissolution, granulation, sugar-coated tablet production, emulsification, encapsulation, encapsulation, or freeze-drying processes. The following methods and excipients are merely illustrative and not limiting.

[0224] For oral administration, anti-TREM2 antibodies can be formulated by combining them with pharmaceutically acceptable carriers known in the art. Such carriers allow the compound to be formulated for oral intake by patients being treated as tablets, pills, sugar-coated tablets, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, and the like. Pharmaceutical preparations for oral use can be obtained by mixing the compound with a solid excipient, optionally grinding the resulting mixture, processing the granular mixture, and, if desired, adding appropriate excipients to obtain tablets or sugar-coated tablet cores. Suitable excipients include fillers such as sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, may be added.

[0225] Anti-TREM2 antibodies can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the compound(s) can be formulated into a preparation by dissolving, suspending, or emulsifying the compound(s) in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and, if necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the compound(s) can be formulated in an aqueous solution, such as Hanks' solution, Ringer's solution, or a physiologically compatible buffer such as physiological saline buffer. The injectable formulation can be provided in unit dosage forms, such as ampoules or multi-dose containers, with preservatives added. The composition(s) can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0226] Pharmaceutical compositions intended for in vivo administration are typically sterilized. Sterilization can be achieved by methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0227] The dosage and desired drug concentration of the pharmaceutical compositions of this disclosure may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is well within the technical scope of those skilled in the art. Suitable dosages are also described in Section VII above.

[0228] kit In some embodiments, a kit containing an anti-TREM2 antibody is provided. In some embodiments, the anti-TREM2 antibody is the antibody or its antigen-binding fragment described in Section III above.

[0229] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises the anti-TREM2 antibody described herein and further comprises one or more additional therapeutic agents for use in the treatment of 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 use in the treatment of cognitive or behavioral symptoms of neurodegenerative diseases. In some embodiments, the therapeutic agent is a neuroprotective agent (e.g., carbidopa / levodopa, anticholinergics, dopamine agonists, monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, glutamatergics, histone deacetylase (HDAC) inhibitors, cannabinoids, caspase inhibitors, melatonin, anti-inflammatory agents, hormones (e.g., estrogen or progesterone), or vitamins).

[0230] 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).

[0231] In some embodiments, the kit further includes explanatory materials (e.g., instructions for using the kit for the treatment method described herein, i.e., a protocol) for carrying out the methods described herein. The explanatory materials typically consist of written or printed materials, but are not limited thereto. Any medium on which such instructions can be stored and communicated to end users is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses to internet sites providing such explanatory materials. [Examples]

[0232] IX. Examples This disclosure will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit this disclosure.

[0233] Example 1. Preparation and initial characterization of anti-TREM2 antibodies Recombinant expression and purification of human TREM2 ECD fused with mouse Fc. The external domain (residues 19-172) of human TREM2 (UniProtKB ID-Q9NZC2) was subcloned into a pRK vector, incorporating a secretory signal derived from mouse IgG kappa chain V-III and amino acids 1-20 (UniProtKB ID-P01661) in the N-terminal region, and a mouse Fc tag (with GGGGS (SEQ ID NO: 34) between TREM2 ECD and Fc) in the C-terminal region.

[0234] Purified plasmids were transfected into Expi293F® cells (Thermo Fisher) using the Expi293F® Expression System Kit according to the manufacturer's instructions. To inhibit N-linked glycan maturation and reduce glycosylation heterogeneity, kifunensin (Sigma), a high mannosidase I inhibitor, was added to the culture at a concentration of 1 μg / mL immediately after transfection. The transfected cells were incubated in an orbital shaker (Infors HT Multitron) at 37°C and 125 rpm in a humid atmosphere of 6% CO2. Sixteen hours after transfection, ExpiFectamine® 293 Transfection Enhancer 1 and 2 were added to the cells, and the supernatant was collected 96 hours after transfection. The clarified supernatant was supplemented with an EDTA-free protease inhibitor (Roche) and stored at -80°C.

[0235] To isolate rhTREM2-Fc, the clarified culture medium supernatant was loaded onto a HiTrap MabSelect SuRe Protein A affinity column (GE Healthcare Life Sciences) and washed with 200 mM arginine and 137 mM succinate buffer (pH 5.0). The fusion protein was eluted in 100 mM QB citrate buffer and 50 mM NaCl at pH 3.0. Immediately after elution, the pH was neutralized by adding 1 M Tris-HCl buffer at pH 8.0 to the protein solution. 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 retained in 20 mM Tris-HCl, 100 mM NaCl, and 50 mM arginine at pH 8.0, which also served as the protein preservation buffer. All chromatography steps were performed using either the AKTA pure system or the AKTA Avant system (GE Healthcare Life Sciences).

[0236] Recombinant expression and purification of His-tagged TREM2 ECD The external domain (residues 19-172) of TREM2 (UniProtKB-Q9NZC2) was subcloned into a pRK vector, incorporating a secretory signal derived from mouse Ig kappa chain V-III and amino acids 1-20 (UniProtKB ID-P01661) in the N-terminal region, and a 6X-His tag (SEQ ID NO: 35) in the C-terminal region. Insertion was confirmed by sequencing, and maxiprep plasmid purification was performed.

[0237] Purified plasmids were transfected into Expi293F® cells (Thermo Fisher) using the Expi293F® Expression System Kit according to the manufacturer's instructions. The transfected cells were incubated in an orbital shaker (Infors HT Multitron) in a humid atmosphere of 6% CO2 at 37°C and 125 rpm. ExpiFectamine® 293 Transfection Enhancer 1 and 2 were added to the cells 16 hours after transfection, and the supernatant was collected 96 hours after transfection.

[0238] The collected medium was supplemented with 1 M imidazole at pH 8.0 to a final concentration of 10 mM, 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) was washed with MQ water and equilibrated with load buffer (20 mM Tris, 150 mM NaCl, and 10 mM imidazole at pH 8.0). Affinity purification was performed using the gravity flow method. The collected medium was loaded onto the resin, and nonspecifically bound proteins were washed with load buffer supplemented with 50 mM and 100 mM imidazole. The bound His-tagged TREM2 external domain was eluted with 20 mM Tris, 150 mM NaCl, and 200 mM imidazole at pH 8.0. The eluted protein was concentrated using an Amicon 10kDa concentrator, and the concentrated protein was further purified by gel filtration chromatography using an AKTA Avant system (GE Healthcare Life Sciences). This protein was loaded onto a HiLoad Superdex 200 16 / 600 (GE Healthcare Life Sciences) column equilibrated with 1×PBS, eluted using 1×PBS as the electrophoresis buffer, and fractionated. The eluted fractions were analyzed by electrophoresis on polyacrylamide (PAGE) gels under denaturation and natural conditions. The eluted fractions were further characterized by analytical size exclusion chromatography and intact protein mass spectrometry. Using the results from PAGE and analytical characterization, highly glycosylated protein fractions were pooled and separated and stored at -80°C.

[0239] Antibody production Rodents (mice and rats) were immunized using a standard protocol with BWZ cells expressing rhTREM2-Fc immunogen or full-length Trem2 receptor. Titers were measured throughout the immunization process using serum collected at various time points. Antigen-specific immune responses were detected using flow cytometry with BWZ live cells expressing rhTREM2-Fc immunogen and full-length TREM2. Candidate antibody selection criteria included the specificity of rodent antibody production and binding to TREM2 as detected by flow cytometry. Antibody-secreting cells were isolated from animal immune tissues, including the spleen, lymph nodes, and bone marrow.

[0240] Single-cell suspensions were analyzed to determine the binding properties of secreted antibodies. Antibody-secreting cells were loaded into microfluidic devices and isolated in nanoliter reaction chambers, allowing detection of secreted antibodies using fluorescence and bright-field imaging-based microscopy assays (see, e.g., U.S. Patent No. 9,188,593). Binding assays were performed, including detection of antibodies bound to antigen-coated microbeads, detection of soluble fluorescently labeled antigens bound to antibodies immobilized on beads, and detection of antibodies bound to cell surface-expressed antigens. Cell surface-expressed antigens included both recombinant and native antigens presented on the cell surface.

[0241] Using image analysis, chambers exhibiting a positive fluorescence signal indicating the presence of single cells producing antibodies with desired properties were identified. The contents of these chambers were collected and lysed in a 384-well plate (see, e.g., U.S. Patent No. 10,087,408). Single-cell lysates were then subjected to RT-PCR to amplify the heavy chain and light chain variable region sequences. The resulting amplicons were then sequenced to determine the cDNA sequences of the paired heavy chain and light chain variable regions from the selected single cells. The resulting sequences were manually examined and analyzed to determine sequence diversity and somatic hypermutation. Based on the screening data and sequence diversity, sequences were selected for expression. The expressed antibodies were tested to confirm antigen-binding specificity.

[0242] Example 2. Sequence optimization and humanization of anti-TREM2 antibody. Exemplary anti-TREM2 antibodies were sequence-optimized, humanized, and then characterized for their binding kinetics and binding specificity.

[0243] Sequence optimization was performed by searching for residues susceptible to chemical modification (e.g., asparagine deamidation motif (NG), aspartate isomerization motif (DS)) and residues potentially oxidized (tryptophan (W) and methionine (M)) within the CDR sequence, and by amino acid substitution with conserved and germline residues to eliminate such sequence tendencies. Subsequently, humanized and sequence-optimized anti-TREM2 antibody variants were analyzed for binding kinetics using dose-controlled cell binding to Biacore and HEK293-H6 cells (see Example 5 for a representative protocol).

[0244] Example 3. Preparation of an anti-TREM2 antibody ("ATV:TREM2") containing a modified Fc polypeptide. Fd(V) of humanized affinity mature anti-TREM2 antibody HThe +CH1) region (SEQ ID NOs. 22 and 24) was cloned into an expression vector containing a sequence encoding an Fc polypeptide manipulated to bind to the human transferrin receptor (TfR) (CH3C.35.23.1.1, CH3C.35.23.3, CH3C.35.23.3 cisLALA, or CH3C.35.24) or a sequence encoding an Fc polypeptide that binds to the cynomolgus monkey transferrin receptor (CH3C.35.21). The Fc polypeptide encoding sequence contained a "knob" (T366W) mutation that prevents homodimerization and promotes heterodimerization with an Fc polypeptide containing a "hole" (T366S / L368A / Y407V) mutation. The Fd region was also cloned into a corresponding "hole" vector containing a sequence encoding an Fc polypeptide that has the hole mutation but lacks the TfR-binding mutation. The coding sequences (both Fd-knob-Fc and Fd-hole-Fc constructs) also contained the "LALA" (L234A;L235A) mutation in the hinge region that reduces effector function (Wines et al., J.Immunol. 164:5313-5318 (2000)) and the "LS" (M428L;N434S) mutation in the FcCH3 region that increases binding to FcRn (see, for example, Zalevsky et al., Nat.Biotech. 28(2):157-159 (2010)). The final encoded heavy chain sequences expressed by the vector are listed in Table 1.

[0245] (Table 1) ATV:TREM2 array TIFF0007870250000001.tif41161

[0246] The corresponding knob and hole vectors were cotransfected into ExpiCHO or Expi293 cells with the corresponding light chain vector (SEQ ID NO: 54) in a knob:hole:light chain ratio of 1:1:2. The expressed proteins were purified by protein A chromatography followed by preparative size exclusion chromatography (SEC) to isolate the purified anti-TREM2 protein.

[0247] The binding of anti-TREM2 protein to the human transferrin receptor was determined as follows: Anti-human Fab was immobilized on a CM5 chip and the anti-TREM2 protein was captured. Full-length human TfR or human TfR apical domain was sequentially diluted (e.g., to concentrations of 1–1,000 nM) and flowed onto the chip (with an association time of 180 seconds), and then dissociated. Fitting was performed using a 1:1 binding model.

[0248] Example 4. Characterization of anti-TREM2 antibodies The following sections describe various assays performed to evaluate the binding and functional properties of the prepared anti-TREM2 antibody.

[0249] Affinity measurement using BiaASA kinetics The affinity of anti-TREM2 antibodies to human and cynomolgus monkey TREM2 ECD was measured using surface plasmon resonance (Biacore® 8K instrument). Anti-TREM2 antibodies were captured on a Biacore Series S CM5 sensor chip (GE Healthcare Life Sciences, catalog number 29149604) using the Human Fab Capture Kit (GE Healthcare Life Sciences, catalog number 28958325). Three-fold serial dilutions of recombinant human TREM2 or recombinant cynomolgus monkey TREM2 were injected at a flow rate of 30 μL / min. Antibody binding was monitored for 300 seconds, followed by monitoring of antibody dissociation for 600 seconds or more in HBS-EP+ electrophoresis buffer (GE Healthcare Life Sciences, catalog number BR100669). Binding response was corrected by subtracting the RU value from an empty flow cell. Kinetic analysis was performed using k on and k off The 1:1 Languir model was used for simultaneous fitting. D The join value is k on and k off It was calculated from that.

[0250] Evaluation of TREM2 binding in TREM2-expressing HEK cells The binding properties of the anti-TREM2 antibody were evaluated in HEK293 cells expressing human TREM2 as follows.

[0251] HEK293 cell lines stably expressing human TREM2 / DAP12 were generated by transfecting cells with vectors expressing wild-type human TREM2 and DAP12, and with vectors expressing DAP12 alone. Stable-expressing clones were selected, and cell surface TREM2 expression was evaluated by flow cytometry. Surface TREM2 expression was detected using an APC conjugate 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 clone was named "HEK293-DAP12#1".

[0252] HEK293 (HEK293-H6) and HEK293 (B5) that highly express human TREM2 and GFP were harvested with 0.05% trypsin and incubated at 37°C for 2 hours. After incubation, the cells were centrifuged and washed twice with FACS buffer (PBS + 0.5% BSA). The mixed cells were then placed in FACS buffer containing human Trustin FcX solution (Biolegend, catalog no. 422302) at a rate of 10 per cell line. 6The cells were resuspended at a density of / mL. The mixed cell lines were seeded at 200,000 cells per well in a 96-well round-bottom plate and incubated at room temperature for 20 minutes. After incubation, the cells were centrifuged and incubated with dose-defined anti-TREM2 antibody on ice for 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. The cells were then incubated with secondary antibody (Alexa Fluor 647 AffiniPure F(ab')2 Fragment Goat Anti-human IgG(H+L), Jackson ImmunoResearch Laboratories, catalog no. 109-606-088, 1:800 dilution) on ice for 30 minutes. After incubation, the cells were washed three times with FACS buffer, resuspended in 100 μL of FACS buffer, and analyzed by flow cytometry (BD FACSCanto II, San Jose, CA), obtaining 30,000 events per sample. The average fluorescence intensity per cell was calculated using FlowJo software and used to create dose-response coupling curves.

[0253] Activation of TREM2-dependent pSyk signaling Activation of TREM2-dependent pSyk signaling was measured in human macrophage cells or HEK293-H6 cells using Perkin-Elmer's commercially available AlphaLisa assay.

[0254] In all experiments involving the use of lipid vesicles containing 70% DOPC and 30% POPS, the lipid vesicles were prepared within two weeks of the experiment as follows: 7 mg of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and 3 mg of POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) were combined in chloroform in a glass vial and dried under a flow of N2 gas for 1-2 hours, or until completely dry. The lipid mixture was resuspended in 1 mL of HBSS (to a final lipid concentration of approximately 10 mg / mL) and vortexed for 2-3 minutes. Subsequently, the lipid suspension was extruded using an Avanti mini-extruder consisting of a single 100 nm pore size membrane to form small monolayer vesicles at 10 mg / mL.

[0255] 1. Administration of antibodies to cells The day before the assay, human macrophage cells or HEK293-H6 cells were seeded at 100,000 cells / well or 40,000 cells / well in poly-D-lysine-coated 96-well plates, respectively. Antibodies were diluted in PBS in 10 sequential dilutions, with 3-fold dilutions between dilutions. For antagonist dose-response curves, lipid vesicles containing 70% DOPC and 30% POPS at a final concentration of 1 mg / mL were also included in the antibody / PBS mixture. After washing cells three times with HBSS using a Biotek 405 / 406 plate washer, 50 μL of antibody / PBS (with or without vesicles) solution per well was added using a Hamilton Nimbus liquid handler. The cell plates were then transferred to a 37°C incubator for 5 minutes. The plate was flicked to remove the liposome / antibody solution, and 40 μL of lysis buffer (Cell Signaling Technologies, CST) containing 1 μM PMSF was added using a liquid handler. The lysate was then frozen at -80°C or immediately assayed using the AlphaLisa assay.

[0256] Human macrophage cells were prepared for the assay as follows: Human monocytes were isolated from fresh blood according to the RosetteSep Human Monocyte Concentration Cocktail Protocol (Stemcell Technologies, reference #15068). The isolated monocytes were washed with washing buffer (PBS + 2% FBS) and resuspended in 10 mL of ACK lysis buffer (ThermoFisher Scientific, catalog no. A10492) to lyse the erythrocytes. Cell lysis was stopped by adding 20 mL of washing buffer, the sample was centrifuged, and washed again with culture medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% glutamac, 1% non-essential amino acids, and 1% penicillin-streptomycin). The human monocytes were then differentiated into macrophage cells in a 250 mL flask in the presence of 50 ng / mL of human recombinant M-CSF (Gibco, catalog no. PHC9501). Fresh human M-CSF was added on day 3, followed by the collection of human macrophages on day 5, which were then used in the assay.

[0257] 2. AlphaLisa Assay Cell lysates were assayed for pSyk using the standard protocol for the Perkin Elmer pSyk AlphaLisa kit. Briefly, 10 μL of lysate / well was transferred to an opaque white 384-well Optiplate (Perkin Elmer). Next, 5 μL of Acceptor Mix (containing the working solution for the acceptor beads) was added to each well, followed by sealing the plate with foil and incubating at room temperature for 1 hour. Subsequently, 5 μL of Donor Mix (containing the working solution for the donor beads) was added to each well under dimmed light conditions. The plate was sealed again and incubated at room temperature for 1 hour. Finally, the plate was read using the AlphaLisa setting on a Perkin Elmer EnVision plate reader.

[0258] Human macrophage cell survival assay Human monocytes were isolated according to the RosetteSep Human Monocyte Concentration Cocktail Protocol (Stemcell Technologies, catalog number 15068). The isolated monocytes were washed with washing buffer (PBS + 2% FBS) and resuspended in 10 mL of ACK lysis solution (ThermoFisher Scientific, catalog number A10492) to lyse the erythrocytes. Dissolution was stopped by adding 20 mL of washing buffer. The cell suspension was centrifuged and washed once with culture medium (RPMI1640 + 10% FBS + penicillin / streptomycin). The cells were then 10 6 The cells were resuspended in culture medium at a density of μL / mL and used in the survival assay described below.

[0259] The day before the assay, 96-well plates were pre-coated with anti-TREM2 antibody or isotype control in dose-setting stages (45 μL / well, 12 stages in total) and incubated overnight at 4°C. After overnight incubation, the pre-coated plates were washed twice with PBS, and then human monocytes (10) were incubated in the presence of a low concentration of human M-CSF (5 ng / mL, Gibco, catalog number PHC9501). 5 Cells were loaded into plates (per well). After 5 days at 37°C, the culture medium was aspirated, and 100 μL PBS + 100 μL Celltiter-glo medium (Promega, catalog no. G7571) was added to each well. After 10 minutes of incubation, the cell medium was transferred to a multi-well plate suitable for use with a luminometer, and luminescence related to cell viability was recorded.

[0260] Lipid storage assay Prior to the assay, human pluripotent stem cells (iPSCs) were first differentiated into hematopoietic progenitor cells (HPCs) using a commercially available kit (StemCell Technologies' STEMdiff Hematopoietic Kit). The HPCs were transferred to plates containing primary human astrocytes and co-cultured for 14–21 days. After identifying that the suspension cells in the co-culture were mainly (>80%) mature microglia, the microglia were used in the assay.

[0261] Cells (iPSC-derived human microglia, 30,000 cells / well) were seeded in PDL-coated 96-well plates containing complete serum medium. After 24 hours at 37°C, the medium was replaced with complete serum medium containing oleic albumin (final concentration 10 μM or 33 μM, Sigma O3008) or purified unlabeled myelin (final concentration 50 μg / mL, purified from wild-type C57Bl / 6 mouse brain (Jackson Laboratories) using the method described in Safaiyan et al. (2016, Nature Neuroscience 19(8):995-998)). After lipid treatment at 37°C for 24 hours, the medium was replaced with medium containing anti-TREM2 antibody. For each experiment, the concentration of anti-TREM2 antibody used was 100 nM. For dose-response curves, the medium containing 100 nM anti-TREM2 antibody was serially diluted 3-fold in a total of 10 steps. RSV was used as a control. The cells were incubated at 37°C for a further 48 hours, after which they were either imaged using Bodipy staining or extracted for lipidomics, as described below.

[0262] For Bodipy imaging, the supernatant was removed, and cells were incubated at 37°C for 30 minutes in live cell imaging buffer (Life Technologies, catalog no. A14291DJ) containing 1 mg / mL of Bodipy 493 / 503 solution (Thermo-Fisher D3922) in 1:2500 DMSO and 1 drop / mL of Nucblue (ThermoFisher, catalog no. R37605). After the incubation period, the staining solution was removed, and cells were either imaged alive or fixed with 4% paraformaldehyde. Cells were imaged using an Opera Phoenix high-content confocal imager with the Alexa 488 channel and DAPI irradiation settings for Bodipy. Lipid spots were analyzed using the spot detection algorithm of the instrument's Harmony software.

[0263] For lipidomic analysis, the cells were washed once with PBS while kept on ice. A 70 μL volume of 9:1 methanol:aqueous solution containing a 1:100 internal standard was added to the cells in a 96-well plate. The plate was shaken in a shaker at 1200 rpm at 4°C for 20 minutes, and then centrifuged at 300 × g for 5 minutes. A 50 μL sample of the supernatant was transferred to an LC-MS vial and kept at -80°C until instrumental analysis.

[0264] Lipid levels were analyzed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled with electrospray mass spectrometry (QTRAP 6500 + Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected into a BEH C18 1.7 μm, 2.1 × 100 mm column (Waters Corporation, Milford, Massachusetts, USA) at a flow rate of 0.25 mL / min at 55°C. In cationization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium formate + 0.1% formic acid, and mobile phase B consisted of 90:10 isopropyl alcohol / acetonitrile (v / v) containing 10 mM ammonium formate + 0.1% formic acid. In the anionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium acetate, and mobile phase B consisted of 90:10 isopropyl alcohol / acetonitrile (v / v) containing 10 mM ammonium acetate. The gradient was programmed as follows: from 45%B to 99%B at 0.0–8.0 mins, at 99%B at 8.0–9.0 mins, to 45%B at 9.0–9.1 mins, and to 45%B at 9.1–10.0 mins. Electrospray ionization was performed in either cation or anion mode using the following settings: curtain gas: 30, collision gas: moderate, ion spray voltage: 5500 (positive mode) or 4500 (negative mode), temperature: 250°C (positive mode) or 600°C (negative mode), ion source gas 1:50, ion source gas 2:60. Analyst 1.6.3 (Sciex) was used in multiple reaction monitoring mode (MRM) to acquire data using the following parameters: residence time (msec) and collision energy (CE), declustering potential (DP): 80, entrance potential (EP): 10 (positive mode) or -10 (negative mode), and collision cell exit potential (CXP): 12.5 (positive mode) or -12.5 (negative mode). Lipids were quantified using a mixture of non-endogenous internal standards.Lipids were identified based on their retention time and the MRM properties of commercially available reference materials (Avanti Polar Lipids, Birmingham, AL, USA).

[0265] Activation of TREM2-dependent mTOR signaling Human microglia derived from wild-type iPSCs were cultured and treated for 96 hours with either an anti-TREM2 antibody (final concentration 100 nM) and either DMSO or a commercially available mTOR inhibitor (Selleckchem, catalog no. AZD8055, final concentration 20 nM). Subsequently, the treated cells were lysed, and cell lysates were prepared for Western blotting to investigate the phosphorylation of major signaling targets in the mTOR pathway. The primary antibodies for Western blotting were obtained from Cell Signaling Technologies: (1) phospho-mTOR (Ser2448), product number 5536T; (2) mTOR (7C10), product number 2983T; (3) phospho-AKT (Ser473), product number 9271T; (4) phospho-GSK-3beta (Ser9), product number 5558T; (5) phospho-S6 ribosomal protein (S235 / 236), product number 4858T; (6) phospho-4E-BP1 (Thr37 / 46), product number 2855T; (7) beta-actin, product number 58169S.

[0266] Example 5. Results Table 2 and Figures 1A-1H show the results of the analysis of the binding characteristics of variants of the humanized and sequence-optimized antibody CL0020188. The NG motifs in the CL0020188 CDR-H2 sequence (SEQ ID NO: 5) and CDR-L1 sequence (SEQ ID NO: 7) were modified and grafted onto the human framework region for analysis. Table 2 shows the K20020188 binding characteristics measured by Biacore in HEK293-H6 cells. D The EC was measured by a binding assay with set values ​​and doses. 50 The values ​​are shown. Figures 1A-1H contain representative dose-response curves for binding to TREM2 expressed by HEK293-H6 cells for humanized and sequence-optimized variants. Variants are represented by filled black circles (●), and isotype controls are represented by white circles (○).

[0267] (Table 2) Binding characteristics of the sequence-optimized and humanized CL0020188 variant TIFF0007870250000002.tif72163

[0268] As shown in Table 2, when the humanized and sequence-optimized CL00201088 clone was measured with Biacore, the parental antibody (K D It showed similar affinity values ​​to hTREM2 compared to the parent antibody (EC2000 = 9.5 nM). This is consistent with the cell binding results of HEK293-H6 cells shown in Table 1, and the corresponding dose-response curves are shown in Figures 1A-1H. 50 Compared to the parent antibody (=0.44nM), the humanized and sequence-optimized clone showed comparable and sub-nanomolemic affinity for TREM2 expressed in HEK293-H6 cells. Overall, the results demonstrate comparable binding kinetics between the parent antibody and the humanized and sequence-optimized variant.

[0269] The results for the ATV:TREM2 variant described in Example 3 are summarized in Table 3 below. Exemplary cell binding curves based on binding to human TREM2-expressing HEK cells and analysis by FACS are shown in Figure 2.

[0270] (Table 3) Overview of ATV:TREM2 characteristics TIFF0007870250000003.tif52163

[0271] Antibodies were evaluated for their ability to regulate TREM2-dependent pSyk signaling in HEK-H6 cells, promote human macrophage cell survival, and regulate lipid accumulation in iPSC-derived human microglia cells (hereinafter referred to as "iPSC microglia" or "iMG"). Figure 3 shows the results for the ATV:TREM2 variant (ATV:TREM2#3) and the corresponding anti-TREM2 antibody. ATV:TREM2 was able to activate pSyk signaling in TREM2-expressing HEK293-H6 cells to a significantly greater extent than the corresponding TREM2 antibody, indicating that its efficacy can be enhanced by adding ATV to the molecule (Figure 3). Furthermore, ATV:TREM2 induced macrophage survival and EC 50 The concentration was 4.1 + 0.3 nM. Finally, the anti-TREM2 antibody demonstrated the ability to reduce lipid accumulation in myelin-treated iMG (Figures 4A and 4B), and showed IC12 inhibition of lipid storage. 50 The value was 0.20 nM (97.7 + 0.3% maximum inhibition).

[0272] Additional studies were conducted to investigate the ability of ATV:TREM2 to reduce lipid accumulation. Figures 5A–5F and 6A–6C show that a representative ATV:TREM2 variant (ATV:TREM2#3) reduces lipid accumulation while increasing fatty acid oxidation intermediates, suggesting a potential role of ATV:TREM2 in enhancing mitochondrial function. Cells (iMGs) incubated with ATV:TREM2 after treatment with oleic acid lipid stimulation (33 μM) were able to reduce lipid accumulation, as shown by Bodipy staining (Figures 5A and 5B). LC-MS analysis of iMGs incubated with ATV:TREM2 for 48 hours after 24 hours of myelin treatment showed that ATV:TREM2 reduces triglyceride (TG) species while simultaneously increasing beta-oxidation intermediates (acylcarnitine) and TCA cycle intermediates (Figures 5C–5F). Figure 5C provides a heatmap showing all TG, acylcarnitine, and TCA cycle intermediate species that exhibited change ratios greater than 1.5 (p<0.05). Meanwhile, Figures 5D-5F show representative species changes in vehicle and myelin-stimulated iMGs incubated with ATV:TREM2 or isotype controls after stimulation. Figures 6A-6C show changes in specific TG, acylcarnitine, and TCA cycle intermediate species in iMGs incubated with ATV:TREM2 or isotype controls after myelin stimulation. As shown in Figures 6A-6C, ATV:TREM2 reduces all TG and ceramide species while increasing certain short-chain acylcarnitine species, indicating that ATV:TREM2 may enhance mitochondrial function.

[0273] Microglial cell proliferation is associated with the activation and coordination of mTOR signaling. Therefore, we explored the role of ATV:TREM2 downstream of mTOR pathway signaling. The phosphorylation status of mTOR signaling pathway targets was analyzed by Western blotting in wild-type iPSC microglia incubated with a representative ATV:TREM2 variant (ATV:TREM2#3) in the presence and absence of an mTOR inhibitor. Figure 7A shows representative Western blot images of mTOR signaling pathway targets. Quantification of Western blot data is shown in Figures 7B-7E (phosphorylation levels normalized to beta-actin loading control). The phosphorylation levels of each sample treated with ATV:TREM2 were compared with samples treated with isotype antibody control for each independent experiment (n=6). The results showed that mTOR serine was elevated in samples treated with ATV:TREM2 compared to the isotype control. 2488 AKT serine 473 , serine of ribosomal protein S6 (RPS6) 235 / 236 As evidenced by the increased phosphorylation level of serine 9 in GSK3b, ATV:TREM2 activates mTOR pathway signaling (Figure 7B-7E; statistical summary: "ns" (p>0.05); * (p<0.01); ** (p<0.001)). RPS6 is a signal target downstream of the mTORC1 complex, and GSK3b is a signal target downstream of the mTORC2 complex. For all data generated in Figures 5A to 7E, the isotype control ("ISO") of ATV:TREM2#3 contains the sequences shown in Table 5.

[0274] Example 6. The role of ATV:TREM2 in lysosomal dysfunction. The role of ATV:TREM2 in lysosomal function was investigated. To assess its impact on lysosomal function, levels of progranulin (PGRN) and bis(monoacylglycero)phosphate (BMP) were measured in iPSC-derived microglia cells ("iMG") treated with an ATV:TREM2 variant.

[0275] To evaluate the effect of ATV:TREM2 on PGRN levels, iMG cells were seeded in 96-well plates at a density of 30,000 cells per well and incubated with ATV:TREM2#3 (100 nM) for 72 hours. Cell supernatant and cell lysates were then collected, and progranulin (PGRN) levels were analyzed using a colorimetric sandwich ELISA.

[0276] To measure PGRN levels, a Thermo Scientific 384-well Maxisorp plate was coated with 4 μg / mL of capture antibody (R&D anti-PGRN antibody, DuoSet ELISA kit, catalog number DY2420) diluted in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The sample wells were blocked with PBS containing 3% BSA for 90 minutes. Cell samples were diluted 1:10 with PBS containing 3% BSA and added to each sample well on the plate, followed by incubation at room temperature for 90 minutes. Then, detection antibody (R&D PGRN antibody, DuoSet ELISA kit, catalog number DY2420) diluted to 125 ng / mL was added to each sample well, and the plate was incubated at room temperature for 90 minutes. Finally, HRP-conjugated streptavidin (R&D SA-HRP, DuoSet ELISA kit, catalog number DY2420) diluted 1:200 was added to each sample well. The plate was incubated at room temperature for 20 minutes. After washing the sample wells with PBS, a chromogenic reagent (TMB substrate) was added and allowed to react for 5 minutes, after which the reaction was stopped with 4N H2SO4. Absorbance was measured using a BioTek Synergy Neo2 plate reader, and PGRN levels were determined by interpolation from a standard curve fitted to a 4-parameter logistic curve. As shown in Figure 8, incubation with ATV:TREM2 resulted in elevated PGRN levels in both the supernatant and cell lysates compared to the isotype control.

[0277] To evaluate the impact on BMP levels, iMG cells were stimulated with myelin or vehicle for 24 hours, followed by incubation with ATV:TREM2#3 (100 nM) for 48 hours. Cellular lipids were extracted by adding methanol containing an internal standard mixture, and BMP abundance was measured by liquid chromatography-mass spectrometry (LC-MS / MS) using a Q-trap 6500 (SCIEX) similar to that described in International PCT Publication WO2020 / 112889. BMP species were quantified using the internal standard BMP (14:0_14:0) and identified based on their retention time and MRM characteristics. Quantification was performed using MultiQuant 3.02 (Sciex) after correction for isotopic overlap. BMP species were normalized to the median of all measured lipid species content. Protein concentrations were measured using a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Representative BMP results are shown in Figure 9. As shown in Figure 9, iMGs stimulated with myelin and then treated with ATV:TREM2 showed reduced levels of BMP species, suggesting potential relief from myelin-induced lysosomal stimulation.

[0278] As shown in Figures 8 and 9, incubation of iMG with ATV:TREM2 increased PGRN levels and corrected myelin-induced BMP levels, demonstrating the role of ATV:TREM2 in regulating lysosomal effects. In Figures 8 and 9, the isotype control (ISO) of ATV:TREM2#3 contains the sequence shown in Table 5.

[0279] Example 7. The role of ATV:TREM2 in mitochondrial respiration. To evaluate the impact on mitochondrial respiration, oxygen consumption was measured in iPSC-derived microglia cells ("iMG") treated with ATV:TREM2 variants or isotype controls using a Seahorse XFe96 analyzer (Agilent) and the materials and protocol of the Seahorse XF Palmitate Oxidation Stress Kit (Agilent 103693). Prior to the Seahorse experiment, cells were cultured for 72 hours on XF96 microplates (Agilent, catalog no. 102416) with ATV:TREM2 #3 (100 nM) or isotype controls. Sixteen hours prior to the Seahorse experiment, cells were exposed to substrate-restricted medium consisting of Seahorse XF RPMI (Agilent 103576) supplemented with 0.5 mM glucose (Agilent 103577), 1 mM glutamine (Agilent 103579), 0.5 mM L-carnitine (part of the Seahorse XF Palmitate Oxidation Stress Kit), and 1% Hyclone FBS. Immediately before the experiment, cells were administered either palmitate-BSA conjugate (166 μM) or a BSA control. Mitochondrial respiratory capacity of cells was evaluated using sequential injections of (1) etomoxyl (4 μM) (carnitine palmitoyltransferase 1 (CPT1) inhibitor) or vehicle, (2) oligomycin (1.5 μM) (ATP synthase complex V inhibitor), (3) carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, mitochondrial uncoupler, 1 μM), and (4) rotenone / antimycin (0.5 mM each, complex I and complex III inhibitors, respectively). Oxygen consumption rate during the experiment was measured using a Seahorse analyzer (Agilent). A summary of the experimental conditions for evaluating mitochondrial respiration is shown in Table 4.

[0280] (Table 4) Experimental conditions TIFF0007870250000004.tif60161

[0281] A representative kinetic graph of oxygen consumption is shown in Figure 10A, and a bar graph showing the cellular maximum respiratory capacity is shown in Figure 10B. As shown in the figures, ATV:TREM2 increases maximum respiration to a similar extent as the fatty acid substrate palmitic acid (PAL). This effect was reduced in the presence of a CPT1 inhibitor. The results indicate that ATV:TREM2 increases maximum mitochondrial respiration, and that this effect appears to be conferred by an increase in fatty acid oxidation capacity.

[0282] Example 8. ATV:TREM2 characteristic comparison The properties of ATV:TREM2#1 and ATV:TREM2#3 were compared with those of the TREM2-binding reference antibody described in WO2019 / 028292. The heavy and light chain sequences of reference antibody #1 ("Ref.Ab.#1") are represented by SEQ ID NOs. 74 and 75, respectively. The heavy and light chain sequences of reference antibody #2 ("Ref.Ab.#2") are represented by SEQ ID NOs. 76 and 75, respectively. The heavy and light chain sequences of the isotype controls for each anti-TREM2 antibody are shown in Table 5.

[0283] (Table 5) Isotype control sequences TIFF0007870250000005.tif33161

[0284] ATV:TREM2 is more potent in vitro than the reference ab and induces less inflammation (less cytokine release). The anti-TREM2 antibody was evaluated using the human macrophage cell survival assay described in Example 4. Figure 11A shows the dose-response curve of cell viability with the anti-TREM2 antibody, where "ISO" refers to the isotype control of ATV:TREM2#3 (Table 5). The corresponding potency (EC50) and maximum response (Emax) values ​​determined from the dose-response curve are shown in Figures 11B and 11C. Each mark represents a single value obtained from a human cell donor (n=3). The results shown in Figures 11A-11C indicate that ATV:TREM2#3 is more potent in promoting human macrophage survival in vitro than reference antibodies #1 and #2.

[0285] In another experiment, human macrophage cells were treated with a 100 nM surface-immobilized anti-TREM2 antibody for 5 days. The cell culture medium from each cell set was then collected and analyzed for cytokine release using Luminex xMAP technology and a commercially available bead-based multiplex assay kit (Human Cytokine 42-Plex Discovery Assay®, Eve Technologies Corp.). A heatmap of relative cytokine release levels is shown in Figure 12 (using Z-scores for plotting). The results in Figure 12 indicate that ATV:TREM2#3 induces less inflammation in human macrophage cells than reference antibodies #1 and #2.

[0286] In summary, ATV:TREM2#3, reference antibody #1, and reference antibody #2 can promote the survival and proliferation of human macrophages (Figure 11A). However, ATV:TREM2#3 showed a stronger efficacy against cell survival and reduced the overall cytokine signature compared to reference antibodies #1 and #2 (Figures 11B, 11C, and 12).

[0287] ATV:TREM2 can reduce post-stimulation triglyceride species levels. Anti-TREM2 antibodies were evaluated by a lipid storage assay (using 10 μM oleic acid stimulation) as described in Example 4. The results for ATV:TREM2#3, reference antibody#1, and reference antibody#2 are shown in Figures 13A-13E.

[0288] Figures 13A-13C show volcano plots with a p<0.05 cutoff and a change factor greater than 1.5 for triglyceride species in iPSC-derived microglia cells ("iMG") quantified by LC-MS. The data were normalized against the isotype control of each antibody. As shown in Figures 13A-13C, ATV:TREM2#3 can modulate triglyceride (TG) species after oleic acid administration, but the reference antibody did not significantly alter the level of change in TG species after oleic acid stimulation. Figures 13D and 13E show bar graphs of representative TG species measurements, with data normalized against the isotype control of ATV:TREM2#3.

[0289] ATV:TREM2 can regulate TREM2 levels in vitro. To evaluate how anti-TREM2 antibodies affect TREM2 levels in iPSC-derived microglia cells ("iMGs"), iMGs were incubated with ATV:TREM2#3 or reference antibody#1 at various concentrations for 72 hours, followed by measurement of TREM2 levels in iMG cell lysates and cell culture media. TREM2 was measured as follows: Briefly, MSD small-spot streptavidin plates (Meso Scale Discovery) were coated with biotinylated goat anti-hTREM2 polyclonal antibody (R&D Systems, BAF1828) at room temperature for 1 hour. The plates were then blocked with MSD Block A buffer (Meso Scale Discovery) at room temperature for 1 hour. Samples and standards were prepared / diluted with assay buffer (25% MSD Block A buffer in TBST), blocked, and then 30 μL of sample and standard was loaded into the plates. After incubation at room temperature for 1 hour, the plates were washed with TBST and conjugated to the primary antibody (ATV:TREM2#3) at room temperature for 1 hour. Diluted sulfo-tagged goat anti-human IgG (Southern Biotech, 2049-01) was then added to the plates and incubated at room temperature for 1 hour. After washing with TBST, the MSD plates were colorimetrically treated with 2× MSD Read Buffer T, followed by detection using an MSD Sector plate reader. MSD values ​​were converted to absolute quantification of TREM2 by fitting a standard curve using Meso Scale Discovery software (Discovery Workbench).

[0290] Figures 14A and 14B show plots of TREM2 levels against antibody concentration in iMG cell lysates and cell culture media after incubation with anti-TREM2 antibodies. TREM2 levels for each antibody were normalized against their intrinsic isotype control. In Figures 14A and 14B, "ISO" represents the isotype control for ATV:TREM2#3. As shown in Figure 14A, total TREM2 levels increased with increasing antibody levels in iMG cell lysates treated with ATV:TREM2#3 and reference antibody #1. In contrast, soluble TREM2 levels decreased with increasing antibody levels in the cell culture media of antibody-treated cells.

[0291] ATV:TREM2 exhibits an excellent pharmacokinetic profile in non-human primates. The pharmacokinetic (PK) characteristics of anti-TREM2 antibodies were investigated in non-human primates. Briefly, 30 mg / kg of the test substance was administered intravenously as a single dose to young adult / adult male cynomolgus monkeys aged 36–53 months (Table 6, n=5 per cohort). CSF samples were collected at 24, 168, and 336 hours post-administration.

[0292] The results are shown in Table 6 and Figure 15. Table 6 shows the PK values ​​of anti-TREM2 antibodies in cerebrospinal fluid (CSF) of non-human primates. The PK values ​​of ATV:TREM2 variants indicate that these antibodies have higher exposure in CSF compared to TREM2 Ab (lack of transferrin receptor binding ability; heavy and light chain sequences represented by SEQ ID NOs. 83 and 54, respectively) and reference antibody #1.

[0293] (Table 6) CSF pharmacokinetic profiles of anti-TREM2 antibodies in non-human primates TIFF0007870250000006.tif52162

[0294] Figure 15 shows the PK profile of the anti-TREM2 antibody in the CSF of administered non-human primates. As shown in Figure 15 and Table 6, ATV:TREM2 shows at least a twofold increase in CSF PK compared to reference antibody #1.

[0295] Example 9. Characteristics of ATV:TREM2 in humanized TREM2 mice To investigate the in vivo effects of ATV:TREM2, BAC transgenic mice expressing human TREM2 were created. These mice were crossed with human transferrin receptor knock-in mice as described in U.S. Patent No. 10,143,187 to enable the characterization of the ATV:TREM2 molecule as described herein.

[0296] Creation of a human TREM2 BAC transgenic model To investigate the in vivo effects of ATV:TREM2, several transgenic mouse lines expressing human TREM2 were generated using modified BAC DNA CTD-2210D2 (ThermoFisher Scientific; catalog number 96012). In the original BAC DNA CTD-2210D2, the human TREM2 coding region and its regulatory elements are flanked by two other TREM-like genes, TREML1 and TREML2. To avoid interference from these TREM-like genes, their expression was stopped by deleting exon 1 from TREML1 and exon 3 from TREML2. This modified BAC CTD-2210D2 DNA construct was injected into the pronucleus of fertilized mouse oocytes of C57BL / 6J mice. Two independent founder lines (called TB36 and TB45) were obtained, demonstrating germline transmission.

[0297] To characterize and compare these two transgenic lines, hemizygous transgenic animals and wild-type non-transgenic littermates were used for analysis. The copy number of human TREM2 was determined using qPCR analysis of tail genomic DNA. Human TREM2 mRNA and protein levels in the brain, liver, lung, and spleen were measured by qRT-PCR and anMSD assays. Human TREML1 and TREML2 mRNA were analyzed in brain-selected microglia by qRT-PCR. Surface TREM2 expression in bone marrow-derived macrophages (BMDMs) was quantified by FACS. Human TREM2 function was evaluated by an in vitro BMDM survival assay.

[0298] The absence of human TREML1 and TREML2 in either TB36 or TB45 indicates the success of the deletion of these genes. qPCR analysis revealed that TB36 and TB45 each contained two and one copies of the human TREM2 transgene, corresponding to higher human TREM2 expression in TB36 compared to TB45 in brain and peripheral tissues. In vitro survival assays showed that the human TREM2 agonist antibody induced a stronger response in the TB36 lineage than in the TB45 lineage.

[0299] Based on these ex vivo and in vitro characterizations, TB36 was selected for the following breeding and in vivo studies. Hemizygous TB36 mice were backcrossed three more times with C57BL / 6J, and then crossed with hTfR KI mice (described in U.S. Patent No. 10,143,187) to produce human TREM2 BAC hemizygous; hTfR KI hemizygous mice for in vivo studies.

[0300] Pharmacokinetic and pharmacodynamic responses mediated by ATV:TREM2 in TB36 / hTfR KI mice To determine whether ATV:TREM2 can induce a microglial response in vivo, a single dose of ATV:TREM2#3 or the corresponding isotype control (ATV:RSV) (100 mg / kg) was administered intravenously to TB36 / hTfR KI mice on day 0, and the mice were sacrificed on day 1 or day 4 post-administration for ex vivo analysis. At the time of sacrifice, the animals were anesthetized by intraperitoneal injection of 2.5% Avertin. Terminal blood was collected from cardiac puncture into an EDTA tube, slowly inverted (10 times), and then centrifuged at 15,350 g for 7 minutes at 4°C. Plasma (upper layer) was transferred to a 1.5 ml Eppendorf tube and stored at -80°C until measurement. After blood collection, CSF samples were collected using glass capillary tubes pre-extracted from the large vat, then transferred to 0.5 mL Protein LoBind Eppendorf tubes and centrifuged at 12,700 rpm for 7 minutes at 4°C. The supernatant of the CSF samples was rapidly frozen on dry ice and stored at -80°C until measurement. The animals were then perfused with cold PBS, the brains were dissected, and the two hemispheres were separated. The right hemisphere was fixed by immersion in 4% paraformaldehyde at 4°C for 24 hours, then transferred to phosphate-buffered saline (PBS) solution containing 0.1% sodium azide and stored until ready for 30% sucrose treatment and sectioning. The left hemisphere was cut into two sections and rapidly frozen in two tubes each for PK measurement and synergistic / cytokine analysis of other targets.

[0301] The plasma and brain levels of human IgG were evaluated on days 1 and 4 after administration. The results are shown in Table 7 below.

[0302] (Table 7) Plasma and brain pharmacokinetic profiles in TB36 / hTfR-KI mice TIFF0007870250000007.tif43161

[0303] The effect of ATV:TREM2 on microglial proliferation Four doses of 5-ethinyl-2'-deoxyuridine (EdU, 80 mg / kg), a thymidine analog that can be incorporated into newly synthesized DNA, were administered intraperitoneally to mice on days 0, 1, 2, and 3, following treatment with ATV:TREM2#3 or ATV:RSV on day 0.

[0304] To detect proliferating microglia (EdU+Iba1+), brain sections taken 4 days after administration were treated with the Click-iT EdU imaging kit (ThermoFisher Scientific, C10637) and then immunostained for Iba1. EdU-Iba1 staining showed that ATV:TREM2 dramatically increased the number of newly formed microglia (EdU+Iba1+; Figure 16A) and total microglial coverage area (Iba1+ area; Figure 16B) in the brain compared to isotype controls, thus demonstrating that ATV:TREM2 significantly increases microglial proliferation compared to controls.

[0305] ATV:TREM2 is dramatically more potent than non-ATV TREM2 antibodies in promoting microglial proliferation in mouse models. To determine the minimum effective dose of ATV:TREM2 and compare it with non-ATV TREM2 antibodies, a single dose of ATV:TREM2#3 (1, 3, 10, 30 mg / kg) or the corresponding TREM2 reference antibody ("TREM2 Ab"; 30 mg / kg), reference antibody #2 (30 mg / kg), or isotype control (ATV:RSV; 30 mg / kg) was administered intravenously to TB36 / hTfR KI mice on day 0. To measure microglial proliferation, EdU was administered via IP to mice on days 0, 1, 2, and 3 after antibody treatment. Mice were sacrificed on days 1 and 4 for analysis. EdU-Iba1 double staining showed a dose-dependent increase in neonatal microglia in animals administered 1 mg / kg to 10 mg / kg of ATV:TREM2 on day 4, with 10 mg / kg of ATV:TREM2 showing the maximum effect on microglial proliferation (Figures 17A and 17B). Furthermore, the effect of 1 mg / kg of ATV:TREM2 on microglial proliferation was slightly higher than that of either anti-TREM2 or reference antibody #2, despite a 30-fold higher dose (not statistically significant).

[0306] ATV:TREM2 transiently increases brain cytokine levels in TB36 / hTfR KI mice. To determine the effect of ATV:TREM2 on cytokine (e.g., chemokine) levels, cytokine levels were measured in terminal plasma and brain lysates (prepared with Cell Signaling lysis buffer #9803) of TB36 / hTfR KI mice using Mouse Cytokine Array / Chemokine Array 44-Plex (MD44). Treatment with ATV:TREM2#3 did not alter cytokine levels measured in plasma, but several cytokine levels measured in the brain (e.g., IP-10 and MCP-5) increased sharply 24 hours post-administration, and these cytokine levels returned to baseline levels 96 hours post-administration. 3 mg / kg of ATV:TREM2#3 had the greatest effect on brain cytokine levels (Figures 18A and 18B).

[0307] ATV:TREM2 increases brain CSF1R levels in TB36 / hTfR KI mice. To determine the effect of ATV:TREM2 on the glial marker CSF1R levels, CSF1R protein levels were measured in brain lysates (prepared with Cell Signaling lysis buffer #9803) from TB36 / hTfR KI mice using a commercially available ELISA kit (Abcam ab240681). It was found that increasing the dose of ATV:TREM2#3 increased CSF1R protein levels on post-treatment day 1 and post-treatment day 4 (Figure 19).

[0308] ATV:TREM2 plasma PK profile A proportional increase in plasma PK was observed with ATV:TREM2#3 doses ranging from 1 mg / kg to 100 mg / kg. There were no significant differences in 24-hour plasma concentrations among ATV:TREM2#3, reference antibody #2, and TREM2 Ab at a dose of 30 mg / kg. Reference antibody #2 appeared to have lower clearance compared to ATV:TREM2#3 and TREM2 Ab at the same dose (Figure 20).

[0309] ATV:TREM2 Brain PK Profile At 24 hours, the brain concentration of 10 mg / kg ATV:TREM2#3 was similar to that of 30 mg / kg TREM2 Ab, and the brain concentrations of 1–3 mg / kg ATV:TREM2#3 were similar to those of 30 mg / kg reference antibody #2. At doses of 3 mg / kg–10 mg / kg of ATV:TREM2#3, an increase exceeding a proportional increase was observed, while a proportional increase was observed at other doses. At 96 hours, the brain concentration of 10 mg / kg ATV:TREM2#3 was similar to that of 30 mg / kg TREM2 Ab and reference antibody #2 (Figure 21). Overall, brain uptake of ATV:TREM2#3 was more effective than that of TREM2 Ab and reference antibody #2.

[0310] (Table 8) Abbreviated sequence list TIFF0007870250000008.tif210159TIFF0007870250000009.tif126165TIFF0007870250000010.tif211165TIFF0007870250000011.tif146165TIFF0007870250000012.tif202165TIFF0007870250000013.tif192165TIFF0007870250000014.tif220165TIFF0007870250000015.tif218165TIFF0007870250000016.tif222165TIFF0007870250000017.tif171165TIFF0007870250000018.tif191165TIFF0007870250000019.tif218165TIFF0007870250000020.tif219165TIFF0007870250000021.tif218165TIFF0007870250000022.tif192165

Claims

1. An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (a) i. CDR-H1 sequence containing the sequence of sequence number 4, ii. CDR-H2 sequence containing sequence number 5 or 25, iii. CDR-H3 sequence containing sequence number 17, iv. CDR-L1 sequence containing sequence number 7 or 23, v. The CDR-L2 sequence containing the sequence of sequence number 8, and vi. CDR-L3 sequence containing sequence number 18 Variable region, (b) A first Fc polypeptide modified to specifically bind to the transferrin receptor, and (c) Second Fc polypeptide The antibody, which includes the antibody.

2. The aforementioned variable region is (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, or (d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 23, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18 The antibody according to claim 1, comprising:

3. The variable region has at least 90% sequence identity with respect to any one of sequence numbers 15, 19, 21, 24, and 26. Ｈ An antibody according to claim 1 or 2, comprising a sequence.

4. The aforementioned V Ｈ The antibody according to claim 3, wherein the sequence has at least 90% or at least 95% sequence identity with respect to sequence number 15.

5. The aforementioned V Ｈ The antibody according to claim 4, wherein the sequence includes sequence number 15.

6. The aforementioned V Ｈ The antibody according to claim 3, wherein the sequence has at least 90% or at least 95% sequence identity with respect to sequence number 24.

7. The aforementioned V Ｈ The antibody according to claim 6, wherein the sequence includes sequence number 24.

8. The variable region has at least 90% sequence identity with respect to any one of sequence numbers 16, 20, and 22. Ｌ An antibody according to any one of claims 1 to 7, comprising a sequence.

9. The aforementioned V Ｌ The antibody according to claim 8, wherein the sequence has at least 90% or at least 95% sequence identity with respect to sequence number 16.

10. The aforementioned V Ｌ The antibody according to claim 9, wherein the sequence includes sequence number 16.

11. Said V Ｌ The antibody according to claim 8, wherein the sequence has at least 90% or at least 95% sequence identity to SEQ ID NO:

22.

12. The aforementioned V Ｌ The antibody according to claim 11, wherein the sequence includes sequence number 22.

13. The aforementioned variable region is (a) V containing Sequence ID No. 15 Ｈ V containing the sequence and sequence number 16 Ｌ array, or (b) V containing Sequence ID 19 Ｈ V including the array and sequence number 20 Ｌ array, or (c) V containing Sequence ID 21 Ｈ V including the array and sequence number 20 Ｌ array, or (d) V containing Sequence ID 19 Ｈ V including the sequence and sequence number 22 Ｌ array, or (e) V containing sequence number 21 Ｈ V including the sequence and sequence number 22 Ｌ array, or (f) V containing sequence number 24 Ｈ V including the array and sequence number 20 Ｌ array, or (g) V containing sequence number 26 Ｈ V including the array and sequence number 20 Ｌ array, or (h) V containing sequence number 24 Ｈ V including the sequence and sequence number 22 Ｌ array, or (i) V containing sequence number 26 Ｈ V including the sequence and sequence number 22 Ｌ array An antibody according to any one of claims 1 to 12, comprising:

14. The antibody according to any one of claims 1 to 13, wherein the first Fc polypeptide comprises, according to EU numbering, Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, or Val at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and Phe at position 421.

15. (a) The first Fc polypeptide binds to the apical domain of the transferrin receptor; and / or (b) Compared to antibodies with wild-type Fc dimers, uptake into the brain is improved. The antibody according to claim 14.

16. (a) In accordance with EU numbering, the first Fc polypeptide has the T366W substitution and the second Fc polypeptide has the T366S, L368A, and Y407V substitutions; or (b) According to EU numbering, the first Fc polypeptide has T366S, L368A, and Y407V substitutions, and the second Fc polypeptide has T366W substitution, The antibody according to any one of claims 1 to 15.

17. The first Fc polypeptide and / or the second Fc polypeptide include modifications that reduce the effector function. The antibody according to any one of claims 1 to 16.

18. The antibody according to claim 17, wherein the modification that reduces the effector function includes substitutions of Ala at position 234 and Ala at position 235, according to EU numbering.

19. The first Fc polypeptide and / or the second Fc polypeptide, Amino acid modifications to the natural Fc sequence that extend the serum half-life. An antibody according to any one of claims 1 to 18, comprising:

20. The antibody according to claim 19, wherein the amino acid modification includes substitutions that, according to EU numbering, are Leu at position 428 and Ser at position 434.

21. The antibody according to any one of claims 1 to 20, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 41 or SEQ ID NO: 64, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO:

63.

22. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 41, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing Sequence ID No. 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 42, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 21, comprising:

23. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 64, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 65, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 21, comprising:

24. The antibody according to any one of claims 1 to 20, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 66, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO:

63.

25. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 44, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing Sequence ID No. 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 45, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 24, comprising:

26. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 66, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 67, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 24, comprising:

27. The antibody according to any one of claims 1 to 20, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO:

63.

28. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing Sequence ID No. 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 27, comprising:

29. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 27, comprising:

30. The antibody according to any one of claims 1 to 20, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 61 or SEQ ID NO:

84.

31. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing Sequence ID No. 61, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 52, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 30, comprising:

32. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 84, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 72, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 30, comprising:

33. The antibody according to any one of claims 1 to 20, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 50 or SEQ ID NO: 70, and the second Fc polypeptide comprises the sequence of SEQ ID NO: 39 or SEQ ID NO:

63.

34. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 50, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing Sequence ID No. 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 51, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 33, comprising:

35. (a) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising the first Fc polypeptide containing sequence number 70, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising the second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; and / or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 71, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 33, comprising:

36. (a) An isolated antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 41, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (b) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 64, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (c) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 44, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (d)(i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 66, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (e) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (f)(i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (g) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 47, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 61, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (h)(i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 68, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 61, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (i) (i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 50, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing SEQ ID NO: 39, and (iii) V containing sequence number 22 Ｌ Two light chains each containing; or (j)(i) V containing sequence number 24 Ｈ A first heavy chain (HC) comprising a first Fc polypeptide containing sequence number 70, (ii) V containing sequence number 24 Ｈ A second heavy chain (HC) comprising a second Fc polypeptide containing sequence number 63, and (iii) V containing sequence number 22 Ｌ Two light chains each containing The antibody, which includes the antibody.

37. (a) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 42, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (b) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 65, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (c) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 45, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (d) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 67, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (e) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (f) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (g) (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 48, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 52, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (h)(i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 69, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 72, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 51, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 53, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54; or (j)(i) A first heavy chain (HC) comprising or consisting of the amino acid sequence described in Sequence ID No. 71, (ii) A second HC comprising or consisting of the amino acid sequence described in Sequence ID No. 73, and (iii) First and second light chains (LCs) each containing or consisting of the amino acid sequences described in Sequence ID No. 54 The antibody according to claim 36, comprising:

38. (a) Reduce the level of soluble TREM2 protein (sTREM2), and / or (b) Enhances TREM2 activity, and / or (c) enhances phagocytosis or enhances the migration, differentiation, function, or survival of bone marrow cells, microglia, or macrophages, and / or (d) enhance microglial function without increasing neuroinflammation, and / or (e) Enhances Syk phosphorylation, and / or (f) Shows cross-reactivity with cynomolgus monkey TREM2 protein, The antibody according to any one of claims 1 to 37.

39. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

40. An isolated antibody according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39, The instruction manual and A kit that includes this.

41. Use of an antibody according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39 in the manufacture of a drug for treating neurodegenerative diseases in a subject, reducing the level of sTREM2 in a subject having a neurodegenerative disease, or enhancing TREM2 activity in a subject having a neurodegenerative disease.

42. The aforementioned neurodegenerative diseases include Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with chromosome 17-linked parkinsonism, argyrophilic grain dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis / parkinsonian dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, Boxer dementia, diffuse neurofibrillary tangle disease with calcification, Down syndrome, familial Anglo-American dementia, familial Danish-American dementia, and Gerstmann-Sträussler-Scheinker disease. The use according to claim 41, selected from the group consisting of globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadeloupean PSP, Haller-Vorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle-dominant dementia, Niemann-Pick disease type C, bulbopontigrine degeneration, Parkinson's disease, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and neurofibrillary senile dementia (tangle-only dementia).

43. (a) A nucleotide sequence encoding the antibody according to any one of claims 1 to 38; or (b) A nucleotide sequence encoding any one of sequence numbers 42, 45, 48, 51, 53, or 54; or (c) A nucleotide sequence encoding SEQ ID NO: 42, a nucleotide sequence encoding SEQ ID NO: 53, and a nucleotide sequence encoding SEQ ID NO: 54; or (d) A nucleotide sequence encoding SEQ ID NO: 45, a nucleotide sequence encoding SEQ ID NO: 53, and a nucleotide sequence encoding SEQ ID NO: 54; or (e) a nucleotide sequence encoding SEQ ID NO: 48, a nucleotide sequence encoding SEQ ID NO: 53, and a nucleotide sequence encoding SEQ ID NO: 54; or (f) A nucleotide sequence encoding SEQ ID NO: 48, a nucleotide sequence encoding SEQ ID NO: 52, and a nucleotide sequence encoding SEQ ID NO: 54; or (g) Nucleotide sequence encoding SEQ ID NO: 51, Nucleotide sequence encoding SEQ ID NO: 53, and Nucleotide sequence encoding SEQ ID NO: 54 Isolated polynucleotides containing [the specified element].

44. A vector or host cell comprising the polynucleotide described in claim 43, or a host cell comprising the vector.

45. A method for expressing an antibody that specifically binds to human bone marrow cell expression trigger receptor 2 (TREM2), comprising culturing host cells containing the following under conditions suitable for the expression of the antibody: (a) A nucleotide sequence encoding the antibody according to any one of claims 1 to 38; (b) A nucleotide sequence encoding SEQ ID NO: 42, a nucleotide sequence encoding SEQ ID NO: 53, and a nucleotide sequence encoding SEQ ID NO: 54; (c) The nucleotide sequence that encodes Sequence ID No. 45, the nucleotide sequence that encodes Sequence ID No. 53, and the nucleotide sequence that encodes Sequence ID No. 54; (d) A nucleotide sequence encoding SEQ ID NO: 48, a nucleotide sequence encoding SEQ ID NO: 53, and a nucleotide sequence encoding SEQ ID NO: 54; (e) a nucleotide sequence encoding SEQ ID NO: 48, a nucleotide sequence encoding SEQ ID NO: 52, and a nucleotide sequence encoding SEQ ID NO: 54; or (f) A nucleotide sequence encoding SEQ ID NO: 51, a nucleotide sequence encoding SEQ ID NO: 53, and a nucleotide sequence encoding SEQ ID NO: 54.

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