TW202128771A - Anti-trem2 antibodies and methods of use thereof - Google Patents

Abstract

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

Description

Anti-TREM2 antibodies and methods of use

Trigger receptor-2 (TREM2) expressed on bone marrow cells is a transmembrane receptor expressed on microglial cells and is believed to play a role in regulating phagocytosis, cell survival, and the production of proinflammatory cytokines . Already in neurodegenerative diseases including Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia TREM2 mutations were identified in. In addition, it has been reported that the level of soluble TREM2 (sTREM2) changes in the cerebrospinal fluid of patients suffering from Alzheimer's disease or frontotemporal dementia and having a TREM2 mutation.

There is still a need for therapeutic agents that regulate the activity of TREM2 or the level of sTREM2 in the industry.

In one aspect, antibodies that specifically bind to human trigger receptor 2 (TREM2) expressed on bone marrow cells are provided. In some embodiments, the antibodies comprise modified Fc polypeptides that can bind to transferrin receptor protein. In any of the herein disclosed one embodiment, the antibody may comprise a sequence of an exemplary CDR, V _H and / or V _L according to any of the herein provided, and may further contain, as described herein comprising the transferrin Receptor binding mutant modified Fc polypeptide.

In some embodiments, the antibody that specifically binds to TREM2 includes: (a) a variable region, which includes: i. CDR-H1 sequence, which includes the sequence of GFTFT- α ₆ -FYMS (SEQ ID NO: 28), Wherein α ₆ is D or N; ii. CDR-H2 sequence, which comprises the sequence of VIRN- β ₅ - β ₆ -N- β ₈ -YT- β ₁₁ - β ₁₂ -YNPSVKG (SEQ ID NO: 29), wherein β ₅ is K or R; β ₆ is A or P; β ₈ is G or A; β ₁₁ is A or T; and β ₁₂ is G or D; iii. CDR-H3 sequence, which includes γ ₁ -RL- The _{sequence of γ 4} -YGFDY (SEQ ID NO: 30), wherein γ ₁ is A or T; and γ ₄ is T or S; iv. CDR-L1 sequence, which includes QSSKSLLHS- δ ₁₀ -GKTYLN (SEQ ID NO: 31), wherein δ ₁₀ is N or T; v. CDR-L2 sequence, which includes the sequence WMSTRAS (SEQ ID NO: 8); and vi. CDR-L3 sequence, which includes QQFLE- ϕ _{6-PFT (SEQ ID NO: 8);} ID NO: 32), wherein ϕ ₆ is Y or F; (b) the first Fc polypeptide modified to specifically bind to the transferrin receptor; and (c) the second Fc polypeptide.

In some embodiments, the first Fc polypeptide associates with the second Fc polypeptide to form an Fc dimer.

In some embodiments, the CDR-H1 sequence is selected from SEQ ID NO: 4 or 12. In some embodiments, the CDR-H2 sequence is selected from SEQ ID NO: 5, 13, or 25. In some embodiments, the CDR-H3 sequence is selected from SEQ ID NO: 6, 14 or 17. In some embodiments, the CDR-L1 sequence is selected from SEQ ID NO: 7 or 23. In some embodiments, the CDR-L3 sequence is selected from SEQ ID NO: 9 or 18.

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

_{In some embodiments, the variable region comprises a VH} sequence having at least 85% sequence identity with any of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26.

In certain embodiments, the _VH sequence has at least 90% sequence identity with SEQ ID NO:15. In certain embodiments, the _VH sequence has at least 95% sequence identity with SEQ ID NO:15. In certain embodiments, the _VH sequence comprises SEQ ID NO:15.

In certain embodiments, the _VH sequence has at least 90% sequence identity with SEQ ID NO:24. In certain embodiments, the _VH sequence has at least 95% sequence identity with SEQ ID NO:24. In certain embodiments, the _VH sequence comprises SEQ ID NO:24.

_{In some embodiments of this aspect, the variable region includes a VL} sequence having at least 85% sequence identity with any of SEQ ID NOs: 3, 11, 16, 20, 22, and 27.

In certain embodiments, V _L sequence of SEQ ID NO: 16 having a sequence identity of at least 90%. In certain embodiments, V _L sequence of SEQ ID NO: 16 having a sequence identity of at least 95%. In certain embodiments, V _L sequence comprises SEQ ID NO: 16.

In certain embodiments, V _L sequence of SEQ ID NO: 22 having a sequence identity of at least 90%. In certain embodiments, V _L sequence of SEQ ID NO: 22 having a sequence identity of at least 95%. In certain embodiments, V _L sequence comprises SEQ ID NO: 22.

In certain embodiments, V _L sequence of SEQ ID NO: 27 having a sequence identity of at least 90%. In certain embodiments, V _L sequence of SEQ ID NO: 27 having a sequence identity of at least 95%. In certain embodiments, V _L sequence comprises SEQ ID NO: 27.

In some embodiments of the aspects, the variable region comprising: (a) comprising SEQ ID NO: V _H and comprising a sequence of 15 SEQ ID NO: V 16 _L of sequence; or (b) comprises SEQ ID NO: 19 the V _H sequences and comprises SEQ ID NO: 20 of the V _L sequence; or (c) comprises SEQ ID NO: V 21 of the _H sequence and comprises SEQ ID NO: 20 of the V _L sequence; or (d) comprises SEQ ID NO : V _H sequence 19 of which comprise SEQ ID NO: 22 of the V _L sequence; or (e) comprises SEQ ID NO: V 21 of the _H sequence and comprises SEQ ID NO: 22 of the V _L sequence; or (f) comprising SEQ ID NO: V _H sequence 24 of which comprise SEQ ID NO: V _L sequence 20; or (g) comprises SEQ ID NO: V _H sequence 26 of which comprise SEQ ID NO: 20 of the V _L sequence; or (h) comprising SEQ ID NO: V _H sequence 24 of which comprise SEQ ID NO: 22 of the V _L sequence; or (i) comprises SEQ ID NO: V _H sequence 26 of which comprise SEQ ID NO: 22 of the V _L sequence; or ( j) comprising SEQ ID NO: V _H sequence two of which comprise SEQ ID NO: 3 of the V _L sequence; or (k) comprises SEQ ID NO: V _H sequence 10 of which comprise SEQ ID NO: V _L sequence 11 of; or (l) comprising SEQ ID NO: V 24 and the _H sequence comprises SEQ ID NO: V 27 _L of sequence.

In some embodiments of this aspect, according to EU numbering, the first Fc polypeptide comprises: 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 421 The Phe. In some embodiments, the first Fc polypeptide binds to the apical domain of the transferrin receptor. In a specific embodiment, the antibody has improved brain uptake compared to an antibody with a wild-type Fc dimer.

In certain embodiments, according to EU numbering, the first Fc polypeptide has T366W substitutions and the second Fc polypeptide has T366S, L368A, and Y407V substitutions.

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

In some embodiments, the first Fc polypeptide and/or the second Fc polypeptide comprise modifications that reduce effector function. In certain embodiments, according to EU numbering, the modification to reduce effector function includes the substitution of Ala at position 234 and Ala at position 235.

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

In some embodiments of this aspect, 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 certain embodiments, the antibody comprises: (i) a first heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 41 of a first Fc polypeptide; (ii) The second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising the V H comprising SEQ ID NO: : V _{L of} 22. In certain embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises a VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 64; (ii) A double heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising SEQ ID NO: 22 The VL. In a specific embodiment, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 42; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it. In certain embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 65; (ii) a second HC, which Contains or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 Or consist of it.

In some embodiments of this aspect, 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 certain embodiments, the antibody comprises: (i) a first heavy chain (HC) comprising a V _H comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 44; (ii) The second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising the V H comprising SEQ ID NO: : V _{L of} 22. In certain embodiments, the antibody comprises: (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 double heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising SEQ ID NO: 22 The VL. In a specific embodiment, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 45; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it. In certain embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 67; (ii) a second HC, which Contains or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 Or consist of it.

In some embodiments of this aspect, 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 certain embodiments, the antibody comprises: (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 47; (ii) The second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising the V H comprising SEQ ID NO: : V _{L of} 22. In certain embodiments, the antibody comprises: (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 double heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising SEQ ID NO: 22 The VL. In a specific embodiment, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it. In certain embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) a second HC, which Contains or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 Or consist of it.

In some embodiments of this aspect, 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 certain embodiments, the antibody comprises: (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 47; (ii) The second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 61; and (iii) two light chains, each comprising the V H comprising SEQ ID NO: : V _{L of} 22. In certain embodiments, the antibody comprises: (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 double heavy chain (HC) comprising the VH comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 84; and (iii) two light chains, each comprising SEQ ID NO: 22 The VL. In a specific embodiment, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 52 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it. In certain embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) a second HC, which Contains or consists of the amino acid sequence listed in SEQ ID NO: 72; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 Or consist of it.

In some embodiments of this aspect, 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 certain embodiments, the antibody comprises: (i) a first heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 50 of a first Fc polypeptide; (ii) The second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising the V H comprising SEQ ID NO: : V _{L of} 22. In certain embodiments, the antibody comprises: (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 double heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising SEQ ID NO: 22 The VL. In a specific embodiment, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 51; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it. In certain embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 71; (ii) a second HC, which Contains or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 Or consist of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which comprising comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 41 of a first Fc polypeptide; (ii) a second heavy chain (HC), which includes comprising SEQ ID NO: V _H 24 and comprising the SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 42; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which Contains the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 64; (ii) the second heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO : The second Fc polypeptide of 63; and (iii) two light chains, each of which includes a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 65; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 73 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which comprising comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 44 of a first Fc polypeptide; (ii) a second heavy chain (HC), which includes comprising SEQ ID NO: V _H 24 and comprising the SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 45; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which Contains the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 66; (ii) the second heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO : The second Fc polypeptide of 63; and (iii) two light chains, each of which includes a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 67; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 73 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which comprising comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 47 of a first Fc polypeptide; (ii) a second heavy chain (HC), which includes comprising SEQ ID NO: V _H 24 and comprising the SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which Contains the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 68; (ii) the second heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO : The second Fc polypeptide of 63; and (iii) two light chains, each of which includes a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 73 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which comprising comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 47 of a first Fc polypeptide; (ii) a second heavy chain (HC), which includes comprising SEQ ID NO: V _H 24 and comprising the SEQ ID NO: 61 of the second Fc polypeptide; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. In some embodiments, (i) the first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) the second HC, which comprises SEQ ID NO: The amino acid sequence listed in 52 or consists of it; and (iii) the first and second light chains (LC), each of which comprises or consists of the amino acid sequence listed in SEQ ID NO:54.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which Contains the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 68; (ii) the second heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO : The second Fc polypeptide of 61; and (iii) two light chains, each of which includes a VL comprising SEQ ID NO: 22. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 72 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which comprising comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 50 of a first Fc polypeptide; (ii) a second heavy chain (HC), which includes comprising SEQ ID NO: V _H 24 and comprising the SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 51; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 53 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) a first heavy chain (HC), which Contains the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 70; (ii) the second heavy chain (HC), which comprises the VH comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO : The second Fc polypeptide of 63; and (iii) two light chains, each of which includes a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises: (i) a first heavy chain (HC), which comprises or consists of the amino acid sequence listed in SEQ ID NO: 71; (ii) a second HC, which comprises The amino acid sequence listed in SEQ ID NO: 73 or consists of it; and (iii) the first and second light chains (LC), each of which includes the amino acid sequence listed in SEQ ID NO: 54 or Consists of it.

In some embodiments of any aspect 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, microglial cells, or macrophages. In some embodiments, the antibody enhances microglial cell function without increasing nerve inflammation. In some embodiments, the antibody enhances Syk phosphorylation. In some embodiments, the antibody enhances Syk phosphorylation in the presence of TREM2 ligand. In some embodiments, the antibody exhibits cross-reactivity with cynomolgus monkey TREM2 protein.

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

In another aspect, the present disclosure provides a kit comprising: the isolated antibody described herein or the pharmaceutical composition described herein; and instructions for use thereof.

In another aspect, the present disclosure provides a method of treating a neurodegenerative disease in an individual, which comprises administering to the individual the isolated antibody described herein or the pharmaceutical composition described herein. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, primary age-related tau disease, progressive supranuclear palsy (PSP), frontotemporal dementia Syndrome, frontotemporal dementia with parkinsonism linked to chromosome 17 (frontotemporal dementia with parkinsonism linked to chromosome 17), argyrophilic dementia, amyotrophic lateral sclerosis, Guam-type muscular atrophic spinal cord Lateral sclerosis/parkinsonism-dementia syndrome (amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, ALS-PDC), cortical basal nucleus degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease (Creutzfeldt-Jakob disease) ), boxer type dementia, diffuse neurofibrillary tangles with calcification, Down's syndrome, familial British dementia, familial Danish dementia dementia), Gerstmann-Straussler-Scheinker disease, globular glial tau disease, Guadeloupean parkinsonism with dementia , Guadeloupe PSP, Hallevorden-Spatz disease, hereditary diffuse white matter disease with spheroids (HDLS), Huntington's disease, inclusion bodies Myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangles dominant dementia, Niemann-Pick disease type C (Niemann-Pick disease type C), globus pallidus-pontine-black Quality deterioration, Parkinson's disease, Pick's disease, Parkinson's disease after encephalitis, prion protein cerebral amyloid angiopathy, progressive subcortical glioma disease, subacute Sclerosing panencephalitis and tangle-only dementia.

In another aspect, the present disclosure provides a method of reducing sTREM2 levels in an individual suffering from a neurodegenerative disease, which comprises administering to the individual the isolated antibody described herein or the pharmaceutical composition described herein.

In another aspect, the present disclosure provides a method of enhancing TREM2 activity in an individual suffering from a neurodegenerative disease, which comprises administering to the individual the isolated antibody described herein or the pharmaceutical composition described herein.

In another aspect, the present disclosure provides isolated polynucleotides comprising nucleotide sequences encoding the antibodies described herein.

In another aspect, the present disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding any one of SEQ ID NOs: 42, 45, 48, 51, 53, 54 and 61.

In another aspect, the present disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 42, 53, and 54.

In another aspect, the present disclosure provides an isolated polynucleotide comprising the nucleotide sequence encoding SEQ ID NO: 45, 53, and 54.

In another aspect, the present disclosure provides an isolated polynucleotide comprising the nucleotide sequence encoding SEQ ID NO: 48, 53, and 54.

In another aspect, the present disclosure provides isolated polynucleotides comprising nucleotide sequences encoding SEQ ID NOs: 48, 52, and 54.

In another aspect, the present disclosure provides an isolated polynucleotide comprising the nucleotide sequence encoding SEQ ID NO: 51, 53, and 54.

In another aspect, the present disclosure provides a vector comprising the polynucleotide described herein.

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

In another aspect, the present disclosure provides a method for expressing antibodies that specifically bind to human trigger receptor 2 (TREM2) expressed on bone marrow cells, which includes: culturing the antibodies described herein under conditions suitable for expressing the antibodies The host cell.

I. Introduction

TREM2 is a transmembrane receptor expressed on the cell surface of microglia, dendritic cells, macrophages and osteoclasts. Without being bound by a particular theory, it is believed that upon ligand binding, TREM2 forms a signaling complex with the transmembrane adaptor protein DNAX activated protein 12 (DAP12), which is then phosphorylated by the protein kinase SRC tyrosine. It is believed that the activated TREM2/DAP12 signaling complex mediates intracellular signaling by recruiting and phosphorylating kinases such as Syk kinase. TREM2/DAP12 signaling regulates activities such as phagocytosis, cell growth and survival, secretion of pro-inflammatory cytokines, and migration of cells such as microglia and macrophages. TREM2 undergoes regulated intramembrane proteolysis, in which the membrane-associated full-length TREM2 is cleaved by the metalloprotease ADAM10 into the sTREM2 part shed from the cell and the membrane further degraded by γ-secretase retains the C-terminal fragment. It has been reported that the level of sTREM2 changes in patients suffering from Alzheimer's disease or frontotemporal dementia and having a TREM2 mutation. In addition, mutations in TREM2 are associated with functional changes such as impaired phagocytosis and decreased microglia function.

As detailed in the Examples section below, antibodies that specifically bind to human TREM2 and modulate one or more downstream functions of the TREM2/DAP12 signaling complex have been produced. In certain embodiments, the antibodies further comprise Fc polypeptides containing mutations that allow the Fc polypeptide to bind to transferrin receptors (from, for example, human TfR). In some aspects, the antibodies disclosed herein are capable of binding to transferrin receptor protein via a modified Fc polypeptide (for example, expressed on the surface of brain endothelial cells (BEC)), and thus may be less TfR binding to Fc The mutant antibody crosses the blood-brain barrier (BBB) more effectively. In certain embodiments, the antibodies disclosed herein include mutations in Fc polypeptides that reduce or eliminate effector functions and, for example, mutations that increase the binding of the antibody Fc to the Fc neonatal receptor (FcRn) to extend the half-life in vivo.

In some embodiments, the anti-TREM2 antibody enhances TREM2 activity, such as enhancing phagocytosis or enhancing the differentiation, function, migration, or survival of bone marrow cells, microglia or macrophages. Therefore, in another aspect, a method is provided for enhancing TREM2 activity in, for example, individuals suffering from neurodegenerative diseases.

In some embodiments, anti-TREM2 antibodies reduce sTREM2 shedding. Therefore, in another aspect, a method is provided for reducing sTREM2 levels in, for example, individuals suffering from neurodegenerative diseases. II. Definition

Unless the content clearly indicates otherwise, as used herein, the singular forms "一 (a, an)" and "the (the)" include plural indicators. Thus, for example, reference to "antibody" includes the combination of two or more of these molecules and the like as appropriate.

As used herein, the terms "about" and "approximately" when used to modify an amount specified in a numerical value or range indicate the numerical value and the reasonable deviation from the value known to those skilled in the art (for example, ± 20%, ± 10% or ± 5%) is within the expected meaning of the listed value.

As used herein, the term "TREM2 protein" refers to the trigger receptor 2 protein expressed on bone marrow cells encoded by the gene TREM2. As used herein, "TREM2 protein" refers to the natural (ie wild-type) TREM2 protein of any vertebrate, such as (but not limited to) humans, non-human primates (such as cynomolgus monkeys), rodents (such as Mice, rats) and other mammals. In some embodiments, the TREM2 protein is a human TREM2 protein with the sequence identified in UniprotKB accession number Q9NZC2 (SEQ ID NO: 1).

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

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

As used herein, the term "anti-TREM2 antigen binding portion" refers to an antigen binding segment or entity that specifically binds to a TREM2 protein (such as human TREM2). The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retains specific binding to an antigen (such as TREM2 protein) via the variable region of the antibody. ) Of the ability. Examples of antigen-binding fragments include (but are not limited to) Fab fragments ( _{monovalent fragments composed of VL} , _VH , CL and CH1 domains), F(ab') ₂ fragments (including two disulfide bridges in the hinge region). bivalent fragment Fab fragments of connection), single chain Fv (scFv), Fv (dsFv ) disulfide-linked, the complementarity determining region (CDR), V _L (light chain variable region) and V _H (heavy chain Variable area).

The term "variable region" or "variable domain" refers to germline variable (V) genes, diversity (D) genes, or junction (J) genes (and not from constant (Cμ and Cδ) genes) Segment) is a domain in the heavy or light chain of an antibody, and which gives the antibody the specificity for binding to the antigen. Generally, the variable region of an antibody contains four conserved "framework" regions, which are arranged alternately with three hypervariable "complementarity determining regions".

The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain, which interrupt the four framework regions established by the light chain and heavy chain variable regions. CDR is mainly responsible for the binding of antibody and antigen epitope. The CDRs of each chain are usually called CDR1, CDR2, and CDR3, which are numbered sequentially from the N-terminus, and are usually also identified by the chain in which the specific CDR is located. Thus, V _H CDR3 is located or found in CDR-H3 of an antibody variable region of the heavy chain, and V _L CDR1-based or CDR-L1 found from which the CDR1 of the light chain variable region of an antibody.

The "framework regions" or "FR" of different light or heavy chains are relatively conserved within a species. The framework region of the antibody (that is, the combined framework region of the constitutive light chain and the heavy chain) is used to locate and align the CDRs in the three-dimensional space. Framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database of human and mouse sequences.

Various well-known definitions in this technology can be used to determine the amino acid sequence of the CDR and framework regions, such as Kabat, Chothia, International ImmunoGeneTics Database (IMGT), AbM, and observed antigen contacts ("contacts"). 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 Kabat, Chothia, and/or contact CDR definitions.

The term "antigenic determinant" refers to the region or region in the antigen specifically bound by the antibody CDR, and may include several amino acids or parts of several amino acids, such as 5 or 6 or more (e.g., 20 Or more) amino acids or parts of those amino acids. For example, if the target is a protein, the epitope may include consecutive amino acids (such as linear epitopes), or amino acids derived from different parts of the protein and approached by protein folding (such as discontinuous or structural epitopes). Shape epitope). In some embodiments, the epitope is phosphorylated at an amino acid (e.g., at a serine or threonine residue).

As used herein, the phrase "recognizing an epitope" used in relation to an anti-TREM2 antibody means that the CDR of the antibody interacts with the antigen (ie, TREM2 protein) at the epitope or part of the antigen containing the epitope. Role or its specific binding.

A "monoclonal antibody" refers to an antibody that is produced by a single cell line or a single cell line and is composed of or essentially composed of antibody molecules with the same primary amino acid sequence.

"Multiple antibodies" refers to antibodies obtained from a heterogeneous antibody population, where different antibodies in the population bind to different epitopes of the antigen.

"Chimeric antibody" refers to an antibody molecule in which the constant region or a part thereof is changed, substituted or exchanged so that the antigen binding site (that is, the variable region, CDR or a part thereof) is connected to a different or changed category, effect The function and/or the constant region of the species, or the variable region or part thereof, has been altered, substituted or exchanged by variable regions with different or altered antigen specificities (such as CDRs and framework regions from different species). In some embodiments, a chimeric antibody system monoclonal antibody comprises a variable region derived from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human). This technique describes methods for generating chimeric antibodies.

"Humanized antibodies" are chimeric immunoglobulins derived from non-human sources (such as murine), which contain the smallest amount of non-human immunoglobulin-derived sequences outside of the CDRs. Generally speaking, a humanized antibody will comprise at least one (e.g., two) antigen-binding variable domains, wherein the CDR regions substantially correspond to those of non-human immunoglobulins and the framework regions substantially correspond to human immunoglobulins. The framework regions of the protein sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) (usually a human immunoglobulin sequence). Methods of antibody humanization are known in the art.

"Human antibodies" or "fully human antibodies" are antibodies with human heavy and light chain sequences usually derived from human germline genes. In some embodiments, the anti-system is produced by human cells, by non-human animals (such as genetically engineered mice that are genetically engineered to express human antibody sequences) using a human antibody library, or by a phage display platform.

The term "specifically binds" refers to a molecule that binds to an epitope or target (e.g., an antibody or antigen-binding portion thereof) in comparison with another epitope or a non-target compound (e.g., an antigen that is structurally different) It binds to the epitope or target in the sample with greater affinity, greater affinity and/or longer duration. In some embodiments, the antibody (or antigen-binding portion thereof) that specifically binds to an epitope or target has a binding affinity to the epitope or target that is at least 5 times lower than the affinity to other epitopes or non-target compounds. (E.g., at least 5 times, 10 times, 100 times, 1,000 times, 10,000 times or more) antibodies (or antigen-binding portions thereof). As used herein, the term "specifically binds to a specific epitope or target", "specifically binds to a specific epitope or target" or "specific to a specific epitope or target" can, for example, be determined by a molecular pair _{The equilibrium dissociation constant K D} of its bound epitope or target is, for example, 10 ^-4 M or less (e.g., 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M or 10 ^-12 M) to show. Those familiar with the art will recognize that antibodies that specifically bind to a target from one species (e.g., TREM2 protein) can also specifically bind to orthologs of that target (e.g., TREM2 protein).

The term "binding affinity" is used herein to refer to the strength of the non-covalent interaction between two molecules (eg, between an antibody (or antigen-binding portion thereof) and an antigen). Thus, for example, unless otherwise indicated or obvious from the context, the term may refer to a 1:1 interaction between an antibody (or an antigen-binding portion thereof) and an antigen. The binding affinity may be measured by solution equilibrium dissociation constant (K _D) to quantify, which means constant dissociation rate constant (k _d, ^time-1) divided by the association rate constant (k _a, time ^-1 M ^-1). K _D can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods, such as the Biacore™ system; kinetic rejection analysis, such as KinExA ^® ; and biological layer interference (such as Use ForteBio ^® Octet platform). As used herein, "binding affinity" not only includes formal binding affinity, such as the affinity that reflects the 1:1 interaction between the antibody (or its antigen-binding portion) and the antigen, but also includes the K _D value calculated to reflect Apparent affinity of affinity binding.

As used herein, the term "cross-reactivity" refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody is directed. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from a species different from the antigen against which the antibody is directed. As a non-limiting example, anti-TREM2 antibodies directed against human TREM2 peptides as described herein can exhibit cross-reactivity with TREM2 peptides or proteins from different species (e.g., monkey or mouse).

As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is listed in SEQ ID NO:62. The transferrin receptor protein 1 sequence from other species is also known (e.g. chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768. 1; Rat, NP_073203.1; and Chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences (such as human sequences), which are encoded by the gene at the chromosome 1 locus of the transferrin receptor protein. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, 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 sequence of the apical domain of human transferrin receptor 1 is listed in SEQ ID NO:55.

The terms "CH3 domain" and "CH2 domain" as used herein refer to immunoglobulin constant region domain polypeptides. In the case of IgG antibodies, the CH3 domain polypeptide refers to the amino acid segment from about position 341 to about position 447 as numbered according to the EU numbering scheme, and the CH2 domain polypeptide refers to the amino acid segment as numbered according to the EU numbering scheme The amino acid segment from about position 231 to about position 340. The CH2 and CH3 domain polypeptides can also be numbered by the IMGT (ImMunoGeneTics) numbering scheme, where according to IMGT Scientific chart numbering (IMGT website), the CH2 domain numbering is 1-110 and the CH3 domain numbering is 1-107. The CH2 and CH3 domains are part of the Fc region of immunoglobulins. In the case of IgG antibodies, the Fc region refers to the amino acid segment from about position 231 to about position 447 as numbered according to the EU numbering scheme. As used herein, the term "Fc region" can also include at least a portion of the hinge region of an antibody. An exemplary partial hinge region sequence is listed in SEQ ID NO:57.

When used in the context of identifying a given amino acid residue in a polypeptide sequence, the term "corresponds to", "reference...determined" or "reference...number" refers to when given When aligning and comparing the amino acid sequence with the reference sequence for maximum alignment and comparison, the position of the residue in the reference sequence is specified. Therefore, for example, when optimally aligned with SEQ ID NO: 38, when the amino acid residue in the polypeptide is aligned with the amino acid in the region from amino acid 111 to 217 in SEQ ID NO: 38 When, the residue "corresponds to" the amino acid in SEQ ID NO:38. The polypeptide aligned with the reference sequence need not be the same length as the reference sequence.

As used herein, the term "Fc polypeptide" refers to the C-terminal region of a natural immunoglobulin heavy chain polypeptide, which is characterized by an Ig fold as a domain. An Fc polypeptide contains a constant region sequence including at least a CH2 domain and/or a CH3 domain, and may contain at least a part of a hinge region, but does not contain a variable region.

A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation (such as a 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 natural Fc polypeptide.

The term "isolated" as used with regard to a nucleic acid or protein (such as an antibody) means that the nucleic acid or protein is substantially free of other cellular components associated with it in its natural state. Analytical chemistry techniques such as electrophoresis (for example, polyacrylamide gel electrophoresis) or chromatography (for example, high performance liquid chromatography) are usually used to determine purity and homogeneity. In some embodiments, the isolated nucleic acid or protein (eg, antibody) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act similarly to natural amino acids. Natural amino acids are those amino acids encoded by the genetic code, and subsequently modified amino acids, 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), glutamine (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), aspartame Amide (Asn), Proline (Pro), Glucomine (Gln), Serine (Ser), Threonine (Thr), Valine (Val), Tryptophan (Trp), Tyrosine Amino acid (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-glutamine (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-aspartamide (D-Asn ), D-Proline (D-Pro), D-Granine (D-Gln), D-Serine (D-Ser), D-Threonine (D-Thr), D-Val Amino acid (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr) and combinations thereof. "Amino acid analogues" refer to compounds that have the same basic chemical structure as natural amino acids (that is, the alpha carbon is bonded to hydrogen, carboxyl, amino and R groups), such as homoserine, leucine, Sulfur methionine, methyl thiomethionine. These analogs have modified R groups (for example, ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. "Amino acid mimics" refer to compounds whose structure is different from the general chemical structure of amino acids, but whose mode of action is similar to that of natural amino acids. Amino acids can be referred to herein by their commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. These terms apply to amino acid polymers in which one or more of the amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as natural amino acid polymers and non-natural amino acid polymers. The amino acid polymer may include a complete L-amino acid, a complete D-amino acid, or a mixture of L and D amino acids.

The term "protein" as used herein refers to a dimer (ie, two) or a multimer (ie, three or more) of a polypeptide or a single-chain polypeptide. The single-chain polypeptides of proteins can be linked by covalent bonds (such as disulfide bonds) or non-covalent interactions.

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

The terms "conservative substitution" and "conservative mutation" refer to a change that allows an amino acid to be classified as a substitution of another amino acid with similar characteristics. Examples of conserved amino acid group categories defined in this way can include: "charged/polar group", including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (aspartic acid) Amine or N), Gln (glutamic acid or Q), Lys (lysine or K), Arg (arginine or R) and His (histidine or H); "aromatic group", including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group", including Gly (glycine or G ), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser ( Serine or S), Thr (threonine or T) and Cys (cysteine or C). Within each group, subgroups can also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups including the following subgroups: "positively charged subgroup", which includes Lys, Arg, and His; "negatively charged subgroup", which includes Glu and Asp ; And the "polar subgroup", which includes Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including: "nitrogen ring subgroup", which includes Pro, His, and Trp; and "phenyl subgroup", which includes Phe and Tyr. In another further example, the aliphatic group can be subdivided into subgroups, such as the "aliphatic non-polar subgroup", which includes Val, Leu, Gly, and Ala; and the "aliphatic slightly polar subgroup", which includes Met, Ser, Thr and Cys. Examples of conservative mutation classes include amino acid substitutions of amino acids in the above subgroups, such as (but not limited to): Lys replaces Arg or vice versa so that the positive charge can be maintained; Glu replaces Asp or vice versa, so that The negative charge is maintained; Ser replaces Thr or vice versa so that free -OH can be maintained; and Gln replaces Asn or vice versa, so that free -NH ₂ can be maintained. In some embodiments, the hydrophobic amino acid replaces the natural hydrophobic amino acid (e.g., in the active site) to maintain hydrophobicity.

In the case of two or more polypeptide sequences, the term "identity" or "identity" percentage refers to when comparing and aligning within a comparison window or a designated area to obtain the maximum correspondence, such as using a sequence comparison algorithm Or by manual alignment and visual inspection, two or more sequences or subsequences are the same or have a specified percentage in a specified area (for example, 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 greater) of identical amino acid residues.

For sequence comparison of polypeptides, usually an amino acid sequence is used as a reference sequence, and candidate sequences are compared with it. Various methods available to those familiar with the technology can be used to implement alignment, such as visual alignment or using publicly available software using known algorithms to achieve maximum alignment. These programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). Those familiar with the technology can determine the alignment parameters used to achieve the maximum alignment. For the purpose of this application, for sequence comparison of polypeptide sequences, the BLASTP algorithm is used, which is a standard protein BLAST that uses preset parameters to align two protein sequences.

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

The terms "treating, treatment" and the like are generally used herein to mean obtaining a desired pharmacological and/or physiological effect. "Treating or treatment" can refer to any indicator of success in treating or improving a neurodegenerative disease (such as Alzheimer's disease or another neurodegenerative disease described herein), including any objective or subjective Parameters such as reduction, remission, improved patient survival, increased survival time or survival rate, decreased symptoms or made the patient more tolerant of the disease, slowed the rate of degeneration or decline, or improved the patient's physical or mental health. The treatment or improvement of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared with individuals or groups of individuals who do not receive the treatment, or with the same patient at different times before or during treatment.

The term "pharmaceutically acceptable excipients" refers to inactive pharmaceutical ingredients that are biologically or pharmacologically suitable for use in humans or animals, such as (but not limited to) buffers, carriers, or preservatives.

As used herein, the "therapeutic amount" or "therapeutically effective amount" of an agent (eg, an antibody as described herein) is the amount of the agent that treats, alleviates, alleviates, or reduces the severity of the symptoms of an individual's disease. A "therapeutic amount" of an agent (e.g., an antibody as described herein) can improve patient survival, increase survival time or survival rate, reduce symptoms, make injury, disease or disease (e.g., neurodegenerative disease) more tolerant, and slow down degradation Or the rate of decline or the improvement of the patient’s physical or mental health.

The term "administration" refers to a method of delivering an agent, compound, or composition to a desired site of biological action. Such methods include, but are not limited to, local delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the antibody as described herein is administered intravenously.

The term "control" or "control value" refers to a reference value or a baseline value. Those who are familiar with this technique can determine the appropriate control. In some cases, the control value can be determined relative to the baseline in the same individual or experiment. For example, the sTREM2 measurement value obtained before treatment with the anti-TREM2 antibody can be the control value of the sTREM2 level measurement value after treatment in the same individual value. In other cases, it may be relative to the average value in a control individual (e.g., healthy control or disease control) or a population of control individuals (e.g., healthy control or disease control, such as 10, 20, 50, 100, 200, 500, 1000 or A population of more control individuals) to determine the control value. For example, the measured value of the sTREM2 level of the individual at baseline or after treatment can be compared with the healthy control value. III. Anti-TREM2 antibody

In one aspect, an antibody that specifically binds to the TREM2 protein is provided. In some embodiments, the antibody specifically binds to human TREM2 protein. In some embodiments, anti-TREM2 antibodies are more selective for TREM2 than other TREM-like receptors (such as TREM1).

In some embodiments, the anti-TREM2 antibody system comprises antibodies with one or more complementarity determining region (CDR), heavy chain variable region, and/or light chain variable region sequences as disclosed herein. In some embodiments, the anti-TREM2 antibody comprises one or more CDR, heavy chain variable region and/or light chain variable region sequences as disclosed herein, and further comprises one or more functional features as disclosed herein, For example, antibodies that enhance TREM2 activity (for example, enhance phagocytosis or enhance the migration, differentiation, function, or survival of cells such as bone marrow cells, microglia or macrophages) or antibodies that reduce sTREM2 levels. In some embodiments, anti-TREM2 antibodies comprise Fc polypeptides that comprise one or more modifications as described herein.

In some embodiments, the anti-TREM2 antibody system chimeric antibody. In some embodiments, the anti-TREM2 antibody system is a humanized and/or affinity matured antibody. 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 an anti-TREM2 antibody described herein (eg, a pure CL0020306, a pure CL0020188, or a pure CL0020307). The CDR, heavy chain variable region and light chain variable region amino acid sequences of these pure lines are listed in Table 8.

In some embodiments, the anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of: (a) Have at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 4 and 12 or have the most with respect to the amino acid sequence of any one of SEQ ID NO: 4 and 12 Heavy chain CDR1 (CDR-H1) sequence with two amino acid substitutions; (b) It has at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 5, 13 and 25 or is relative to the amino acid sequence of any one of SEQ ID NO: 5, 13 and 25 The acid sequence has a heavy chain CDR2 (CDR-H2) sequence with a maximum of two amino acid substitutions; (c) Have at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 6, 14 and 17 or relative to the amino acid sequence of any one of SEQ ID NO: 6, 14 and 17 The acid sequence has a heavy chain CDR3 (CDR-H3) sequence with a maximum of two amino acid substitutions; (d) Have at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 7 and 23 or have the most relative to the amino acid sequence of any one of SEQ ID NO: 7 and 23 Light chain CDR1 (CDR-L1) sequence with two amino acid substitutions; (e) Light chain CDR2 (CDR-L2) that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 8 or has at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 8 ) Sequence; and (f) Have at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 9 and 18 or have the most relative to the amino acid sequence of any one of SEQ ID NO: 9 and 18 Light chain CDR3 (CDR-L3) sequence with two amino acid substitutions.

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 (a) CDR-H1, (b) CDR-H2, and (c) CDR-H3. In some embodiments, the anti-TREM2 antibody comprises (d) CDR-L1, (e) CDR-L2, and (f) CDR-L3. In some embodiments, CDRs with up to two amino acid substitutions have one amino acid substitution relative to the reference sequence. In some embodiments, CDRs with up to two amino acid substitutions have two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

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

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 (a) CDR-H1, (b) CDR-H2, and (c) CDR-H3. In some embodiments, the anti-TREM2 antibody comprises (d) CDR-L1, (e) CDR-L2, and (f) CDR-L3.

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

In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising at least 85% of SEQ ID NO: 2, 10, 15, 19, 21, 24, and 26. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the anti-TREM2 comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26.

In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising at least 85%, 90% of SEQ ID NO: 3, 11, 16, 20, 22, and 27. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27.

In some embodiments, the anti-TREM2 antibody comprises: a heavy chain variable region, which comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence; the light chain variable region, which comprises the same as SEQ ID NO: 3, Any one of 11, 16, 20, 22, and 27 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity The amino acid sequence of sex. In some embodiments, the anti-TREM2 comprises: a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26; and a light chain can A variable region comprising the amino acid sequence of any one of SEQ ID NO: 3, 11, 16, 20, 22, and 27.

In some embodiments, the anti TREM2 antibody comprising: (a) and SEQ ID NO: 2 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 3 having at least 85% sequence identity to the V _L sequence; or (b) and SEQ ID NO: 10 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 11 having at least 85% sequence identity to the sequence of the V _L; or (c) and SEQ ID NO: 15 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 16 having at least 85% sequence identity to the sequence of the V _L; or (d) and SEQ ID NO: 19 having at least 85% sequence identity to the V _{H Sequence and a VL} sequence having at least 85% sequence identity with SEQ ID NO: 20 _{; or (e) a V H} sequence having at least 85% sequence identity with SEQ ID NO: 21 and a V H sequence having at least 85% sequence identity with SEQ ID NO: 20 85% sequence identity to the sequence of the V _L; or (f) and SEQ ID NO: 19 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 22 having at least 85% sequence identity to the V _L sequence; or (g) and SEQ ID NO: 21 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 22 having at least 85% sequence identity to the sequence of the V _L; or (h) and SEQ ID NO: 24 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 20 having at least 85% sequence identity to the sequence of the V _L; or (i) and SEQ ID NO: 26 having at least 85% sequence identity to the V _{H Sequence and a VL} sequence having at least 85% sequence identity with SEQ ID NO: 20 _{; or (j) a V H} sequence having at least 85% sequence identity with SEQ ID NO: 24 and a V H sequence having at least 85% sequence identity with SEQ ID NO: 22 85% sequence identity to the sequence of the V _L; or (k) and SEQ ID NO: 26 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 22 having at least 85% sequence identity to the V _L sequence; or (l) and SEQ ID NO: 24 V _H sequence having at least 85% sequence identity with and of SEQ ID NO: 27 having at least 85% sequence identity to the V _L sequence.

In some embodiments, the anti-TREM2 antibody comprises one or more sequences encompassed by the consensus sequences disclosed herein. As a non-limiting example, consensus sequences can be identified by aligning heavy or light chain sequences (e.g., CDRs) of antibodies from the same (or similar) germline. In some embodiments, consensus sequences can be generated from antibodies that contain the same (or similar) length and/or have at least one highly similar CDR (eg, highly similar CDR3) sequence. In some embodiments, these sequences in the antibodies can be aligned and compared to identify conserved amino acids or motifs (that is, changes in the sequence may change the function of the protein) and/or sequence Region of variation (that is, a situation where sequence variation is unlikely to significantly affect protein function). Alternatively, by aligning the heavy or light chain sequences (e.g. CDR) of antibodies that bind to the same or similar (e.g., overlapping) epitopes, the consensus sequence can be identified to determine conserved amino acids or motifs (i.e., Changes in the sequence may change the function of the protein) and regions where the alignment of the sequence is changed (that is, the change in the sequence is unlikely to significantly affect the function of the protein). In some embodiments, one or more consensus sequences can be identified in antibodies that recognize the same or similar epitopes as the anti-TREM2 antibodies as disclosed herein. Exemplary consensus sequences include SEQ ID NO: 28-32. In the consensus sequence of SEQ ID NO: 28-32, capital letters represent amino acid residues that are absolutely conserved in aligned sequences (such as aligned CDR sequences), and "X" or Greek letters (such as "α", "β ", "γ", "δ", "ε" or "φ") represent amino acid residues that are not absolutely conserved in the aligned sequence. It should be understood that when selecting the amino acid inserted at the position marked by "X" or the Greek letter, in some embodiments, the amino acid is selected from those amino acids found at the corresponding positions of the aligned sequence acid. Pure variants of CL0020188, CL0020306, CL0020307 and CL0020188

In some embodiments, the anti-TREM2 antibody comprises: (a) CDR-H1 sequence, which comprises the sequence of GFTFT- α ₆ -FYMS (SEQ ID NO: 28), wherein α ₆ is D or N; (b) CDR- H2 sequence, which includes the sequence of VIRN- β ₅ - β ₆ -N- β ₈ -YT- β ₁₁ - β ₁₂ -YNPSVKG (SEQ ID NO: 29), wherein β ₅ is K or R; β ₆ is A or P; β ₈ is G or A; β ₁₁ is A or T; and β ₁₂ is G or D; (c) CDR-H3 sequence, which includes γ ₁ -RL- γ ₄ -YGFDY (SEQ ID NO: 30) The sequence of γ ₁ is A or T; and γ ₄ is T or S; (d) CDR-L1 sequence, which includes the sequence of QSSKSLLHS- δ ₁₀ -GKTYLN (SEQ ID NO: 31), wherein δ ₁₀ is N Or T; (e) CDR-L2 sequence, which includes the sequence of WMSTRAS (SEQ ID NO: 8); and (f) CDR-L3 sequence, which includes the sequence of QQFLE- ϕ _6- PFT (SEQ ID NO: 32) , Where ϕ ₆ is Y or F.

In some embodiments, the anti-TREM2 antibody comprises a CDR-H1 sequence selected from SEQ ID NO: 4 and 12. In some embodiments, the anti-TREM2 antibody comprises a CDR-H2 sequence selected from SEQ ID NO: 5, 13 and 25. In some embodiments, the anti-TREM2 antibody comprises a CDR-H3 sequence selected from SEQ ID NO: 6, 14 and 17. In some embodiments, the anti-TREM2 antibody comprises a CDR-L1 sequence selected from SEQ ID NOs: 7 and 23. In some embodiments, the anti-TREM2 antibody comprises a CDR-L3 sequence selected from SEQ ID NO: 9 and 18.

In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising at least 85% of SEQ ID NO: 2, 10, 15, 19, 21, 24, and 26. Sequence identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) amino acid sequence. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26 .

In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising at least 85% sequence identity with any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27 (E.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) amino acid sequence. In some embodiments, the anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27. Pure line CL0020188

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

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

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

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

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

In some embodiments, the anti-TREM2 antibody and the antibody as described herein (e.g., comprise heavy chain CDR1-3 and the amino acid sequence comprising SEQ ID NO: 4, 5, 17, 7, 8 and 18, respectively) Light chain CDR1-3 antibodies, or antibodies comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 16) competition Combine.

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

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising at least 85% sequence identity with SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97%). % Sequence identity) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a light chain variable region comprising at least 85% sequence identity with SEQ ID NO: 22 (e.g., at least 90%, 95%, or 97%). % Sequence identity) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen binding portion comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:22.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising at least 85% sequence identity with SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97%). % Sequence identity) amino acid sequence; and a light chain variable region comprising at least 85% sequence identity with SEQ ID NO: 22 (for example, at least 90%, 95%, or 97% sequence Consistency) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24; and a light chain variable region, the light chain variable The region contains the amino acid sequence of SEQ ID NO:22.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequence of SEQ ID NO: 4, 25 and 17, respectively , And it has at least 85% sequence identity with SEQ ID NO: 24 (for example, at least 90%, 95%, or 97% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequence of SEQ ID NO: 23, 8 and 18, respectively , And it has at least 85% sequence identity with SEQ ID NO: 22 (for example, at least 90%, 95%, or 97% sequence identity).

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

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises the CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 4, the CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 25, The CDR-H3 sequence of the amino acid sequence of SEQ ID NO: 17, the CDR-L1 sequence of the amino acid sequence of SEQ ID NO: 7, the CDR-L2 sequence of the amino acid sequence of SEQ ID NO: 8 and The CDR-L3 sequence including the amino acid sequence of SEQ ID NO:9.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising at least 85% sequence identity with SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97%). % Sequence identity) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a light chain variable region comprising at least 85% sequence identity with SEQ ID NO: 27 (e.g., at least 90%, 95%, or 97%). % Sequence identity) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen binding portion comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:27.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising at least 85% sequence identity with SEQ ID NO: 24 (e.g., at least 90%, 95%, or 97%). % Sequence identity) amino acid sequence; and a light chain variable region comprising at least 85% sequence identity (for example, at least 90%, 95%, or 97% sequence identity with SEQ ID NO: 27) Consistency) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24; and a light chain variable region, the light chain variable The region contains the amino acid sequence of SEQ ID NO:27.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a heavy chain variable region comprising heavy chain CDR1-3 comprising the amino acid sequence of SEQ ID NO: 4, 25 and 17, respectively , And it has at least 85% sequence identity with SEQ ID NO: 24 (for example, at least 90%, 95%, or 97% sequence identity). In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises a light chain variable region comprising light chain CDR1-3 comprising the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively , And it has at least 85% sequence identity with SEQ ID NO: 27 (for example, at least 90%, 95%, or 97% sequence identity).

In some embodiments, the anti-TREM2 antibody or antigen-binding portion is the same as the antibody as described herein (e.g., comprising the amino acid sequence comprising SEQ ID NO: 4, 25, 17, 7, 8 and 9 respectively). The antibody of the chain CDR1-3 and the light chain CDR1-3, or the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 Region antibody) competing for binding antibody. Pure line CL0020306

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

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

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

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

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

In some embodiments, the anti-TREM2 antibody and the antibody as described herein (e.g., comprise heavy chain CDR1-3 and the amino acid sequence comprising SEQ ID NO: 4, 5, 6, 7, 8 and 9, respectively) Light chain CDR1-3 antibodies, or antibodies comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 3) competition Combine. Pure line CL0020307

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

In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising 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) of the amino acid sequence. In some embodiments, the anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10.

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

In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising 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) of the amino acid sequence; and the light chain variable region, the light chain variable region comprising at least 85% sequence identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) amino acid sequence. In some embodiments, the anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising SEQ ID The amino acid sequence of NO:11.

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

In some embodiments, the anti-TREM2 antibody system and the antibody as described herein (e.g., include heavy chain CDR1-3 comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, 7, 8 and 9, respectively) And light chain CDR1-3 antibodies, or antibodies comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 11) Antibodies that compete for binding. Binding characteristics of anti-TREM2 antibodies

In some embodiments, an antibody that specifically binds to TREM2 protein as described herein binds to TREM2 expressed on cells (e.g., primary cells or cell lines that endogenously express TREM2, such as human macrophages, or ( For example) Primary cells or cell lines that have been engineered to express TREM2 as described in the Examples section below). In some embodiments, an antibody that specifically binds to TREM2 protein as described herein binds to purified or recombinant TREM2 protein or a portion thereof, or to a chimeric protein comprising TREM2 or a portion thereof (e.g., Fc containing TREM2) Fusion protein or Fc fusion protein containing the extracellular domain of TREM2).

In some embodiments, antibodies that specifically bind to human TREM2 proteins exhibit cross-reactivity with one or more other TREM2 proteins of another species. In some embodiments, antibodies that specifically bind to human TREM2 protein exhibit cross-reactivity with cynomolgus monkey ("cyno") TREM2 protein. In some embodiments, antibodies that specifically bind to 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.

Methods for analyzing binding affinity, binding kinetics and cross-reactivity are known in the art. Such methods include (but are not limited to) solid phase binding analysis (e.g. ELISA analysis), immunoprecipitation, surface plasmon resonance (e.g. Biacore ^™ (GE Healthcare, Piscataway, NJ)), kinetic rejection analysis (e.g. KinExA ^® ) , Flow cytometry, fluorescence activated cell sorting (FACS), biological layer interference (such as Octet ^™ (FortéBio, Inc., Menlo Park, CA)) and western blot analysis. In some embodiments, ELISA is used to determine binding affinity and/or cross-reactivity. The method used to perform the ELISA analysis is known in the art and is 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, kinetic rejection analysis is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, biological layer interference analysis is used to determine binding affinity, binding kinetics, and/or cross-reactivity. Epitope recognized by anti-TREM2 antibody

In some embodiments, the anti-TREM2 antibody recognizes the same or substantially the same epitope in human TREM2 as the epitope recognized by the antibody clone as described herein. As used herein, the term "substantially the same" used with regard to the epitope recognized by the antibody clone as described herein means that the anti-TREM2 antibody recognition is consistent with the epitope recognized by the antibody clone as described herein. It is internally or almost identical to it (for example, it has at least 90% sequence identity with it, or has one, two or three amino acid substitutions relative to it, such as conservative substitutions) or is recognized by the antibody clone as described herein The epitopes have a large amount of overlap (for example, at least 50%, 60%, 70%, 80%, 90%, or 95% overlap) epitopes.

In some embodiments, the anti-TREM2 antibody recognizes an epitope in human TREM2 that is the same or substantially the same as the epitope recognized by an antibody selected from the group consisting of pure CL0020306, pure CL0020188, pure CL0020307 and variants thereof.

In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope in the stalk region of TREM2. In some embodiments, the anti-TREM2 antibody recognizes the epitope in human TREM2 comprising residues 129-172 or residues 131-169 of SEQ ID NO:1, within or consisting of these residues. In some embodiments, the anti-TREM2 antibody recognizes residues 129-148 of SEQ ID NO:1 (e.g., 143-148 of SEQ ID NO:1) in human TREM2, within or by residues The epitope of composition. In some embodiments, the anti-TREM2 antibody system activates TREM2/DAP12 signaling (for example, by inducing phosphorylation of kinases such as Syk) and binds to human TREM2 at an epitope in the stem region of TREM2. Agent. In some embodiments, the anti-TREM2 antibody binds to human TREM2 at an epitope in the stem region of TREM2 and inhibits the cleavage of TREM2 by a protease (eg, ADAM17). Functional characteristics of anti-TREM2 antibodies

In some embodiments, an anti-TREM2 antibody (eg, an antibody having one or more CDR, heavy chain variable region, and/or light chain variable region sequences as disclosed) has one or more TREM2 activities as disclosed herein Play a role. For example, in some embodiments, the anti-TREM2 antibody modulates the level of sTREM2 protein (e.g., the level of sTREM2 shed from the cell surface into an extracellular sample), and modulates the kinase that interacts with the TREM2/DAP12 signaling complex (e.g., Syk kinase) recruits or phosphorylates and/or regulates one or more downstream activities of signal transduction complexes, such as phagocytosis, cell growth, cell survival, cell differentiation, cytokine secretion, or cell migration.

In some embodiments, an anti-TREM2 antibody enhances one or more TREM2 activities (such as those described herein) induced by a ligand. In some embodiments, the coordination system is a lipid ligand. Examples of TREM2 lipid ligands include (but are not limited to) 1-palmitoyl-2-(5'-pendyl-pentanyl)-sn-glycerol-3-phosphocholine (POVPC), 2-peanut Tetraenylglycerol (2-AG), 7-ketocholesterol (7-KC), 24(S)hydroxycholesterol (24OHC), 25(S)hydroxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), Carnitine (AC), Alkylglycerophosphocholine (PAF), α-Galactosylceramide (KRN7000), Bis (monoglycerol) phosphate (BMP), Cardiolipin (CL) , Ceramide, Ceramide-1-phosphate (C1P), Cholesteryl Ester (CE), Cholesterol Phosphate (CP), Diglycerol 34:1 (DG 34:1), Diglycerol 38 :4 (DG 38:4), diglyceryl pyrophosphate (DGPP), dihydroceramide (DhCer), dihydrosphingomyelin (DhSM), ether phosphatidylcholine (PCe), free cholesterol ( FC), GalCer, GalSo Salt Solution (Hank's Balanced Salt Solution, HBSS), Kdo2-lipid A (KLA), lactose ceramide (LacCer), lysoalkyl glycerophosphocholine (LPAF), lysophosphatidic acid (LPA), lysophosphate Choline (LPC), lysophospholipid ethanolamine (LPE), lysophospholipid glycerol (LPG), lysophospholipid inositol (LPI), lysosphingomyelin (LSM), lysophospholipid serine (LPS) , N-amino-phospholipid ethanolamine (NAPE), N-amino-serine (NSer), oxidized phospholipid choline (oxPC), palmitic acid-9-hydroxy-stearic acid (PAHSA), phospholipid Ethanolamine (PE), phospholipid alcohol (PEtOH), phosphatidic acid (PA), phospholipid choline (PC), phospholipid glycerol (PG), phospholipid inositol (PI), phospholipid seramine Acid (PS), Dihydrosphingosine, Dihydrosphingosine-1-phosphate (Sa1P), Sphingomyelin (SM), Sphingosine, Sphingosine-1-phosphate (So1P) and sulfur fat. Regulation of sTREM2 shedding

In some embodiments, the anti-TREM2 antibody changes the level of sTREM2 protein in the sample, for example, the level of sTREM2 shed from the cell surface to the extracellular sample. In some embodiments, the anti-TREM2 antibody reduces the level of sTREM2.

In some embodiments, if the amount of sTREM2 in the processed sample is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% compared to the control value. %, at least 90% or more, the anti-TREM2 antibody reduces the level of sTREM2. In some embodiments, if the amount of sTREM2 in the processed sample is reduced by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more compared to the control value , The anti-TREM2 antibody reduces the level of sTREM2. In some embodiments, the control value is an untreated sample (e.g., a sample from a TREM2 expressing cell supernatant that has not been treated with an anti-TREM2 antibody, or a sample from an individual that has not been treated with an anti-TREM2 antibody) or an appropriate non-TREM2 The amount of sTREM2 in the sample that is bound to the antibody.

In some embodiments, a fluid-containing sample (such as blood, plasma, serum, urine, or cerebrospinal fluid) is used to measure sTREM2 shedding. In some embodiments, the sample comprises cerebrospinal fluid. In some embodiments, the sample comprises supernatant from a cell culture (e.g., from primary cells or cell lines that endogenously express TREM2 (such as human macrophages) or (e.g.) as described in the Examples section below Primary cells or cell line supernatants that have been engineered to express TREM2).

In some embodiments, immunoassay is used to measure the level of sTREM2 in the sample. Immunoassays are known in the art and include (but are not limited to) enzyme immunoassays (EIA), such as enzyme multiplier 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, sTREM2 levels are measured using ELISA analysis. In some embodiments, sTREM2 levels are measured using the ELISA analysis as described in the Examples section below. Regulation of kinase recruitment or phosphorylation

In some embodiments, the anti-TREM2 antibody induces phosphorylation of kinases (such as (but not limited to) Syk, ZAP70, PI3K, Erk, AKT, or GSK3b) that interact with the TREM2/DAP12 signaling complex. In some embodiments, anti-TREM2 antibodies induce phosphorylation of kinases that interact with the TREM2/DAP12 signaling complex without blocking the binding of natural TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance the phosphorylation of kinases that interact with the TREM2/DAP12 signaling complex induced by TREM2 ligands (eg, lipid ligands). In some embodiments, the anti-TREM2 antibody induces or enhances the phosphorylation of Syk. In some embodiments, if the level of Syk phosphorylation in a sample treated with an anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, At least 70%, at least 80%, at least 90% or more, the anti-TREM2 antibody induces or enhances the phosphorylation of Syk. In some embodiments, if the level of Syk phosphorylation in the sample treated with the anti-TREM2 antibody is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times the control value. Times or more, the anti-TREM2 antibody induces the phosphorylation of Syk. In some embodiments, the control value is an untreated sample (for example, a sample containing TREM2 expressing cells that has not been treated with an anti-TREM2 antibody, or a sample from an individual that has not been treated with an anti-TREM2 antibody), or has been TREM2 ligand It is not the level of Syk phosphorylation in samples treated with non-anti-TREM2 antibodies, or samples treated with appropriate non-TREM2 binding antibodies.

To detect and/or quantify phosphorylation (eg, Syk phosphorylation) in a sample, in some embodiments, immunoassays are used. In some embodiments, the immunoassay system is enzyme immunoassay (EIA), enzyme doubled immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microenzyme immunoassay (MEIA), immunohistochemistry (IHC), immune cell Chemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL) or electrochemiluminescence immunoassay (ECL). In some embodiments, immunoassays using amplified luminescence proximity homogeneous analysis (AlphaLISA®, PerkinElmer Inc.) are used to detect and/or quantify phosphorylation.

In some embodiments, phosphorylation is measured using a sample containing one or more cells, such as one or more TREM2 expressing cells (e.g., primary cells or cell lines endogenously expressing TREM2, such as human giant cells). Phage or iPSC-derived microglial cells, or, for example, primary cells or cell lines that have been engineered to express TREM2 as described in the Examples section below). In some embodiments, the sample contains fluid, such as blood, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the sample comprises tissue (e.g., lung, brain, kidney, spleen, nerve tissue, or skeletal muscle) or cells from this tissue. In some embodiments, the sample contains endogenous fluids, tissues, or cells (e.g., from a human or non-human individual). Regulation of phagocytosis

In some embodiments, the anti-TREM2 antibody enhances the phagocytosis of dead cell debris, tissue debris, starch-like beta particles, or foreign materials. In some embodiments, anti-TREM2 antibodies enhance phagocytosis without blocking the binding of natural TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance phagocytosis induced by TREM2 ligands (eg, lipid ligands). In some embodiments, if the level of phagocytosis in a sample treated with an anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, At least 70%, at least 80%, at least 90% or more, the anti-TREM2 antibody enhances phagocytosis. In some embodiments, if the phagocytosis level in the sample treated with the anti-TREM2 antibody is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times the control value, Times or more, the anti-TREM2 antibody enhances phagocytosis. In some embodiments, the control value is the level of phagocytosis in an untreated sample, a sample that has been treated with a TREM2 ligand instead of an anti-TREM2 antibody, or a sample treated with an appropriate non-TREM2 binding antibody.

In some embodiments, phagocytosis assays are used to measure phagocytosis using labeled masses. Phagocytosis analysis is known in the art. In some embodiments, phagocytosis analysis is performed on samples containing cells that endogenously express TREM2, such as human macrophages or microglia. In some embodiments, phagocytosis analysis is performed on samples containing cells that have been engineered to express TREM2. In some embodiments, phagocytosis is measured using the human macrophage phagocytosis analysis as described in the Examples section below. Regulation of cell differentiation, function, migration and survival

In some embodiments, the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation (e.g., bone marrow cells, macrophages, and microglia, including iPSC-derived microglia and disease-related microglia cell). Disease-related microglia and methods for detecting disease-related 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 (eg, bone marrow cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance cell survival of one or more cell types (eg, bone marrow cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance the cellular function of one or more cell types (eg, bone marrow cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance cell differentiation of one or more cell types (eg, bone marrow cells, macrophages, or microglia). In some embodiments, anti-TREM2 antibodies enhance the migration, survival, function, and/or differentiation of bone marrow 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 cell activation. In some embodiments, anti-TREM2 antibodies enhance the migration, survival, function, and/or differentiation of disease-related microglia. In some embodiments, anti-TREM2 antibodies enhance cell migration, cell survival, cell function, or cell differentiation without blocking the binding of natural TREM2 ligands. In some embodiments, anti-TREM2 antibodies enhance cell migration, cell survival, cell function, or cell differentiation induced by TREM2 ligands (eg, lipid ligands).

In some embodiments, if the level of activity (such as migration, survival, function, or differentiation) in the sample treated with the anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, At least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, the anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation. In some embodiments, if the level of activity (such as migration, survival, function, or differentiation) in the sample treated with the anti-TREM2 antibody is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold the control value , 8 times, 9 times, 10 times or more, the anti-TREM2 antibody enhances cell migration, cell survival, cell function or cell differentiation. In some embodiments, the control value is an untreated sample (for example, a sample that has not been treated with an anti-TREM2 antibody), a sample that has been treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with an appropriate non-TREM2 binding antibody The level of activity (e.g., migration, survival, function, or differentiation).

In some embodiments, chemotaxis analysis is used to measure cell migration. Chemotaxis analysis is known in the art. In some embodiments, cell migration analysis (e.g., chemotaxis analysis) is performed on a sample containing cells that endogenously express TREM2 (such as human macrophages). In some embodiments, cell migration analysis (e.g., chemotaxis analysis) is performed on a sample containing cells that have been engineered to express TREM2. In some embodiments, the human macrophage chemotaxis assay as described in the Examples section below is used to measure cell migration.

In some embodiments, cell survival analysis is used to measure cell survival. Cell viability analysis is known in the art. In some embodiments, cell survival analysis (e.g., cell survival rate analysis) is performed on a sample containing cells that endogenously express TREM2 (such as human macrophages). In some embodiments, cell survival analysis (e.g., cell survival analysis) is performed on a sample containing cells that have been engineered to express TREM2. In some embodiments, cell survival is measured using the human macrophage survival rate analysis as described in the Examples section below.

In some embodiments, the function of the cell is measured using a functional analysis suitable for the cell being measured. For example, in some embodiments, macrophage function is assessed using, for example, a phagocytosis analysis as described in the Examples section below.

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

In some embodiments, the activation of microglia is measured in vivo. In some embodiments, TSPO-PET imaging is used to measure microglial cell activation. The TSPO-PET imaging method is known in the art.

In some embodiments, anti-TREM2 antibodies enhance microglial cell function without increasing nerve inflammation. The level of nerve inflammation can be determined by measuring the level of cytokines (such as inflammatory cytokines), such as (but not limited to) TNF-α, IL-1β, IL-6, and IL- 1ra, TGFβ, IL-15 or IFN-γ. In some embodiments, immunoassays are used to measure the level of cytokines, such as enzyme immunoassay (EIA), enzyme multiplier 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). IV. FC polypeptide mutation of protein with anti-TREM2 antigen binding portion

In some aspects, the anti-TREM2 antibody comprises two Fc polypeptides, one or both of which may each comprise an independently selected modification (e.g. mutation) or may be a wild-type Fc polypeptide, such as a human IgG1 Fc polypeptide. Non-limiting examples of mutations that can be introduced into one or two Fc polypeptides include, for example, allowing Fc polypeptides (or antibodies comprising them) to bind to BBB receptors (such as transferrin receptor (TfR) proteins (such as humans)). Or cynomolgus monkey TfR, such as can be expressed on brain endothelial cells)) mutations, improve serum stability, modulate effector functions, affect glycosylation, reduce immunogenicity in humans and/or provide Fc polypeptides The mutation of polymerization. Transferrin receptor binding mutation

In some embodiments, anti-TREM2 antibodies include Fc polypeptides that include modifications (e.g., amino acid substitutions) that allow the Fc polypeptide to bind to the TfR protein. In short, binding to a TfR protein (such as its apical domain) expressed on, for example, brain endothelial cells, in some embodiments may allow the modified Fc polypeptide of the present disclosure or an antibody containing it to be mediated by receptors. The endocytosis is guided through the blood-brain barrier. In certain embodiments, receptor-mediated endocytosis can enhance or improve the ability of Fc polypeptide-containing proteins to exist in the brain (ie, located on the lumen side of the blood-brain barrier), which can allow for improvement Binding to TREM2 in the CNS and other functions, such as target clearance, neutralization or immune depletion or the like.

Exemplary TfR binding amino acid modifications for Fc (e.g. CH2 and/or CH3 parts, fragments or domains) and Fc polypeptides comprising these amino acid modifications and a portion thereof are described in PCT Patent Publication No. WO 2018/152326A1 middle. These amino acid modifications, TfR-binding Fc polypeptide sequences, and TfR-binding Fc polypeptides, and techniques for producing and testing them are incorporated herein by reference. One or both of the Fc polypeptides of the Fc dimer of the present disclosure can be engineered to include modifications that allow binding to TfR. In certain embodiments, one Fc polypeptide of the Fc dimer contains modifications that allow binding to TfR, while the other Fc polypeptide does not contain such modifications.

In some embodiments, the modified Fc polypeptide comprises a YxTEWSS (SEQ ID NO: 58) motif. In some embodiments, the modified Fc polypeptide comprises a TxxExxxxF (SEQ ID NO:59) motif. In some embodiments, the modified Fc polypeptide comprises YxTEWSS (SEQ ID NO: 58) and TxxExxxxF (SEQ ID NO: 59) motifs.

In some embodiments, according to EU numbering, the modified Fc polypeptide comprises wild-type amino acid residues at positions 380, 389, 390, and 415, wherein the wild-type amino acid residue is in SEQ ID NO: 38 Found at the corresponding location.

In some embodiments, according to EU numbering, anti-TREM2 antibodies include Fc polypeptides with the following amino acids: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; and Thr at position 387 Glu at position 388; 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, according to EU numbering, the anti-TREM2 antibody includes an Fc polypeptide with the following amino acids: Trp at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; 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.

In some embodiments, according to EU numbering, the anti-TREM2 antibody includes an Fc polypeptide with the following amino acids: Glu at position 380; Phe at position 384; Thr at position 386; Glu at position 387; Ser at position 389; Ser at position 390; Ser at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

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

In some embodiments, according to the EU numbering, the anti-TREM2 antibody includes an Fc polypeptide with the following amino acids: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Ser at position 389; Ser at position 390; Ser at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

In some embodiments, the modified Fc polypeptide comprises a sequence that has at least 90% identity with the amino acid sequence listed in any one of SEQ ID NO: 40, 43, 46, and 49. In some embodiments, the modified Fc polypeptide comprises or consists of the amino acid sequence listed in any one of SEQ ID NO: 41, 44, 47, and 50.

Other examples of modified Fc polypeptides are set forth in Table 8. Mutations that promote heterodimerization of Fc polypeptides

In some embodiments, the Fc polypeptides present in the anti-TREM2 antibodies as disclosed herein include knob and socket mutations to promote heterodimer formation and hinder homodimer formation. Generally, these modifications introduce protrusions ("pustle") at the interface of the first polypeptide and corresponding cavities ("mortar") at the interface of the second polypeptide, so that the protrusions can be positioned in the cavity to facilitate Heterodimer formation and thus hinder homodimer formation. The protrusions are constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). By substituting a smaller amino acid side chain (such as alanine or threonine) for a larger amino acid side chain, a compensating cavity with the same or similar size as the protrusion is created in the interface of the second polypeptide. In some embodiments, the location of these other mutations in the Fc polypeptide does not have a negative (e.g., inhibitory) effect on the binding of the Fc polypeptide to BBB receptors (e.g., TfR).

In an illustrative embodiment of the mortar-and-mortar method for dimerization, position 366 (numbered according to the EU numbering scheme) of an Fc polypeptide present in the protein described herein contains tryptophan instead of natural threonine. The other Fc polypeptide in the dimer has valine at position 407 (numbered according to the EU numbering scheme) instead of natural tyrosine. Another Fc polypeptide may further comprise substitutions in which the natural threonine at position 366 (numbered according to the EU numbering scheme) is substituted with serine and the natural leucine at position 368 (numbered according to the EU numbering scheme) is substituted with alanine . Therefore, one Fc polypeptide of the anti-TREM2 protein of the present disclosure has a T366W knob mutation and the other Fc polypeptide has a Y407V mutation, which is usually accompanied by T366S and L368A hole mutations.

In some embodiments, one or two Fc polypeptides can also be engineered to contain other modifications for heterodimerization, such as the presence of naturally charged or hydrophobic amino acid fragments in the CH3-CH3 interface. ) Electrostatic engineering of modified contact residues.

In some embodiments, modifications that extend serum half-life can be introduced. For example, in some embodiments, one or two Fc polypeptides present in the anti-TREM2 protein of the present disclosure may comprise tyrosine at position 252, threon at position 254 as numbered according to the EU numbering scheme. Amino acid and glutamic acid at position 256. Therefore, one or two Fc polypeptides can have M252Y, S254T, and T256E substitutions. Alternatively, according to EU numbering, one or two Fc polypeptides may have M428L and/or N434S substitutions. Alternatively, one or two Fc polypeptides can have N434S or N434A substitutions. Fc effector function

In some embodiments, one or both of the Fc polypeptides in the anti-TREM2 protein disclosed herein may include a modification to reduce effector function, that is, when binding to an Fc receptor that is expressed on an effector cell that mediates an effector function Reduce the ability to induce certain biological functions. Examples of antibody effector functions include (but are not limited to) C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulate cell surface receptors (such as B cell receptors) and B cell activation. The effector function can vary with the class of antibody. For example, natural human IgG1 and IgG3 antibodies can trigger ADCC and CDC activities when bound to appropriate Fc receptors present on immune system cells; and natural human IgG1, IgG2, IgG3, and IgG4 can bind to appropriate Fc receptors present on immune cells. The ADCP function can be triggered when the appropriate Fc receptor is on.

In some embodiments, one or both Fc polypeptides may include modifications that modulate effector functions.

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

Other Fc polypeptide mutations that modulate effector functions include (but are not limited to) the following: position 329 can have proline via glycine, arginine, serine or large enough to destroy the proline 329 and FcγRIII in Fc Tryptophan residues Trp 87 and Trp 110 formed between the Fc/Fcγ receptor interface amino acid residue substitution mutation. According to the EU numbering plan, other illustrative substitutions include S228P, E233P, L235E, N297A, N297D, N297G, and P331S. There may also be multiple substitutions. For example, according to the EU numbering scheme, L234A and L235A in the Fc region of human IgG1; L234A, L235A and P329G in the Fc region of human IgG1; L234A, L235A and P329S in the Fc region of human IgG1; and S228P in the Fc region of human IgG4 And L235E; human IgG1 Fc region L234A and G237A; human IgG1 Fc region L234A, L235A and G237A; human IgG2 Fc region V234A and G237A; human IgG4 Fc region L235A, G237A and E318A; and human IgG4 Fc region S228P And L236E. In some embodiments, one or two Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, such as substitutions at positions 298, 333, and/or 334 according to the EU numbering scheme. Mutations in FcRn binding site and prolonging serum half-life

In certain aspects, the Fc polypeptide (e.g., a modified Fc polypeptide) present in the anti-TREM2 protein of the present disclosure may include an FcRn binding site. In some embodiments, the FcRn binding site is within an Fc polypeptide or fragment thereof.

In some embodiments, the FcRn binding site comprises a natural FcRn binding site. In some embodiments, the FcRn binding site does not contain amino acid changes relative to the amino acid sequence of the native FcRn binding site. In some embodiments, the natural FcRn binding site is an IgG binding site, such as a human IgG binding site. In some embodiments, the FcRn binding site comprises a modification that alters FcRn binding.

In some embodiments, the FcRn binding site has one or more mutated (e.g., substituted) amino acid residues, wherein the (etc.) mutation prolongs the serum half-life or does not substantially shorten the serum half-life (i.e., When analyzed under the same conditions, the serum half-life is shortened by no more than 25% compared to the counterpart Fc polypeptide with wild-type residues at the mutation position). In some embodiments, according to the EU numbering scheme, the FcRn binding site has one or more amino acid residues substituted at positions 251-256, 428, and 433-436.

In some embodiments, relative to the natural human IgG sequence, one or more residues at or near the FcRn binding site are mutated to extend the serum half-life of the polypeptide. In some embodiments, mutations are introduced into 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 mutations M252Y, S254T, and T256E. In a specific embodiment, one or two Fc polypeptides present in the anti-TREM2 protein of the present disclosure may comprise tyrosine at position 252, threonine at position 254, and position as numbered according to the EU numbering scheme Glutamate at 256 places. Therefore, one or two Fc polypeptides can have M252Y, S254T, and T256E substitutions.

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, according to the EU numbering scheme, the Fc polypeptide comprises mutations at one, two, or all three of positions T307, E380, and N434. In some embodiments, the mutations are T307Q and N434A. In some embodiments, the Fc polypeptide comprises mutations T307A, E380A, and N434A. In some embodiments, according to the EU numbering scheme, the Fc polypeptide comprises mutations at positions T250 and M428. In some embodiments, the Fc polypeptide comprises the mutations T250Q and/or M428L. In some embodiments, according to the EU numbering scheme, the Fc polypeptide contains mutations at positions M428 and N434. In some embodiments, the Fc polypeptide comprises the mutations M428L and N434S. In some embodiments, the antibodies of the present disclosure may comprise two Fc polypeptides, wherein the two Fc polypeptides each comprise M428L and/or N434S substitutions. In some embodiments, the Fc polypeptide comprises the N434S or N434A mutation. In some embodiments, the antibodies of the present disclosure may comprise two Fc polypeptides, wherein the two Fc polypeptides each comprise a N434S or N434A substitution. V. Preparation of antibodies

In some embodiments, antibodies are prepared by immunizing one or more animals (e.g., mice, rabbits, or rats) with an antigen or a mixture of antigens 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 booster injections of the one or more antigens can be administered to improve antibody production. After immunization, antigen-specific B cells are harvested from, for example, the spleen and/or lymphoid tissue. To produce monoclonal antibodies, B cells are fused with myeloma cells, and then they are screened for antigen-specificity. Methods of preparing antibodies are also described in the Examples section below.

The genes encoding the heavy and light chains of the antibody of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Alternatively, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to the selected antigen. The antibody can also be bispecific, that is, it can recognize two different antigens. Antibodies can also be heterologous conjugates (e.g., two covalently linked antibodies) or immunotoxins.

Antibodies can be produced using any number of expression systems (including prokaryotic and eukaryotic expression systems). In some embodiments, the expression system is a mammalian cell expression system, such as a hybridoma or CHO cell expression system. Many of these systems are widely available from commercial suppliers. In both embodiments the antibody comprises the V _H and V _L regions, a single vector may be used (e.g.) polycistronic expression unit or expression V _H and V _L region under the control of different promoters of the two cis. In other embodiments, expression vectors may be used singly V _H and V _L regions. The V _H or V _L regions set forth herein may optionally contain the methionine at the N-terminus.

In some embodiments, anti-system chimeric antibodies. Methods of preparing chimeric antibodies are known in the art. For example, it can be prepared in which the antigen binding region (heavy chain variable region and light chain variable region) from one species (such as mouse) is fused with the effector region (constant domain) of another species (such as human) Chimeric antibody. As another example, a "class-switched" chimeric antibody in which the effector region of one antibody is replaced by the effector region of a different immunoglobulin class or subclass can be prepared.

In some embodiments, the anti-systemic humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity. Humanized antibodies usually include one or more non-human variable regions (such as CDRs) or parts thereof (such as derived from mouse variable region sequences), and may include some non-human framework regions or parts thereof, and further include one or more Multiple constant regions derived from human antibody sequences. Methods of humanizing non-human antibodies are known in the art. Transgenic mice or other organisms (such as other mammals) can be used to express humanized or human antibodies. Other methods for humanizing antibodies include, for example, variable domain resurfacing, CDR grafting, grafting specificity determining residues (SDR), targeted selection, and framework shuffling.

As an alternative to humanization, fully human antibodies can be produced. As a non-limiting example, genetically transgenic animals (such as mice) can be generated that can produce a complete human antibody library without producing endogenous immunoglobulins after immunization. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of human germline immunoglobulin gene arrays to these germline mutant mice will allow the production of human antibodies after being attacked by antigens. As another example, human antibodies can be produced by hybridoma-based methods, such as by using primary human B cells for the production of cell lines that produce human monoclonal antibodies.

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

In some embodiments, antibody fragments (such as Fab, Fab', F(ab') ₂ , scFv, _VH, or _VHH ) are produced. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies. However, recombinant host cells can now be used directly to produce these fragments. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab') ₂ fragments. According to another method, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be obvious to those familiar with this technique.

In some embodiments, the antibody or antibody fragment is bound to another molecule (e.g., polyethylene glycol (pegylation) or serum albumin) to provide an extended half-life in vivo. VI. Nucleic acids, vectors and host cells

In some embodiments, recombinant methods are used to prepare anti-TREM2 antibodies as disclosed herein. Therefore, in some aspects, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding any anti-TREM2 antibody as described herein (e.g., the CDRs, heavy chain variable regions, and light chain variable regions described herein). Any one or more of the regions); a vector containing these nucleic acids; and a host cell into which the nucleic acids are introduced, which are used to replicate antibody-encoding nucleic acids and/or express the antibodies.

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

Suitable vectors containing polynucleotides encoding the antibodies or fragments thereof of the present disclosure include selection vectors and expression vectors. Although the selected cloning vector can vary according to the host cell to be used, the available cloning vector is usually capable of self-replication, can have a single target for a specific restriction endonuclease and/or can carry a clone that can be used to select the vector containing the vector. The gene of the marker. Examples include plastids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (such as pSA3 and pAT28). These and many other selection vectors are available from commercial suppliers, such as BioRad, Strategene, and Invitrogen.

The expression vector is usually a replicable polynucleotide construct containing the nucleic acid of the present disclosure. The expression vector can be replicated in the host cell as an episome or as a component of chromosomal DNA. Suitable expression vectors include (but are not limited to) plastids, viral vectors (including adenovirus, adeno-associated virus, retrovirus) and any other vectors.

Suitable host cells for the selection or expression of polynucleotides or vectors as described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell line is prokaryotic. In some embodiments, the host cell line is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells. In some embodiments, the host cell line is a human cell, such as a human embryonic kidney (HEK) cell.

In another aspect, a method of preparing an anti-TREM2 antibody as described herein is provided. In some embodiments, the method includes culturing a host cell as described herein (e.g., a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expressing the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium). VII. Treatment methods using anti-TREM2 antibodies

In another aspect, a method of treatment using an anti-TREM2 antibody as disclosed herein (eg, an anti-TREM2 antibody as described in section III above) is provided. In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, methods are provided for modulating one or more TREM2 activities (e.g., in individuals with neurodegenerative diseases).

In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, primary age-related tau disease, progressive supranuclear palsy (PSP), frontotemporal dementia Disease, frontotemporal dementia with Parkinson’s disease related to chromosome 17, argyrophilic granular dementia, amyotrophic lateral sclerosis, Guam-type amyotrophic lateral sclerosis/Parkinson’s disease-loss Mental Syndrome (ALS-PDC), Cortical Basal Nucleus Degeneration, Chronic Traumatic Encephalopathy, Kuja's Disease, Boxer Dementia, Diffuse Neurofibrillary Tangles with Calcification, Down Syndrome, Familial British Type Dementia, familial Danish dementia, Jetzman-Strusler-Shinker disease, globular glial tau disease, Guadeloupe Parkinson's disease with dementia, Guadeloupe PSP, Hallerwarden-Spatz disease, hereditary diffuse white matter disease with spheroids (HDLS), Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu- Hakola disease, neurofibrillary tangles dominant dementia, Niemann-Pick disease type C, globus pallidus-pontine-substantia nigra degeneration, Parkinson's disease, Pick's disease, Parkinson's disease after encephalitis, Prion protein brain Amyloid angiopathy, progressive subcortical glioma, subacute sclerosing panencephalitis, and tangled-only dementia. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neurodegenerative disease is Nasu-Hakola disease. In some embodiments, the neurodegenerative disease is frontotemporal dementia. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the method includes administering to the individual an isolated antibody or antigen-binding fragment thereof that specifically binds to human TREM2 protein (e.g., an anti-TREM2 antibody as described herein) or an anti-TREM2 antibody as described herein. Antibody pharmaceutical composition.

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

In some embodiments, methods are provided for modulating one or more TREM2 activities in an individual (e.g., an individual with a neurodegenerative disease). In some embodiments, the method includes regulating the level of sTREM2; regulating the recruitment or phosphorylation of kinases (for example, Syk kinase) that interact with the TREM2/DAP12 signaling complex; regulating phagocytosis (for example, on cell debris, starch-like β Phagocytosis of particles, etc.); regulating cell migration (for example, migration of bone marrow cells, macrophages, microglial cells and disease-related microglial cells); and/or regulating cell differentiation (for example, for bone marrow cells, macrophages) , Microglia and disease-related microglia). In some embodiments, methods are provided for enhancing one or more TREM2 activities in individuals with neurodegenerative diseases. In some embodiments, methods are provided for reducing sTREM2 levels in individuals with neurodegenerative diseases. In some embodiments, the method of modulating one or more TREM2 activities in an individual comprises administering to the individual an isolated antibody or antigen-binding portion thereof that specifically binds to human TREM2 protein (e.g., an anti-TREM2 antibody as described herein). ) Or a pharmaceutical composition comprising an anti-TREM2 antibody as described herein.

In some embodiments, the individual to be treated is a human being, such as an adult or a child.

In some embodiments, methods are provided to reduce plaque accumulation in individuals with neurodegenerative diseases. In some embodiments, the method includes administering to the individual an antibody or pharmaceutical composition as described herein. In some embodiments, the individual has Alzheimer's disease. In some embodiments, the animal model of neurodegenerative disease of the system (for example, 5XFAD or APP/PS1 mouse model). In some embodiments, plaque accumulation is measured by amyloid plaque imaging and/or Tau imaging, for example, using positron emission tomography (PET) scanning. In some embodiments, administration of anti-TREM2 antibody reduces plaque accumulation by at least 20%, at least 30%, or at least 40% compared to a baseline value (eg, the level of plaque accumulation in the individual prior to administration of anti-TREM2 antibody) , At least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

In some embodiments, the anti-TREM2 antibody is administered to the individual in a therapeutically effective amount or dose. However, the dosage may vary based on several factors, including the chosen route of administration, formulation of the composition, patient response, severity of the disease, individual weight, and the judgment of the prescribing physician. The dose can be increased or decreased over time depending on the needs of individual patients. In some cases, a lower dose is initially given to the patient, and then it is increased to an effective dose that the patient can tolerate. The determination of the effective amount must be within the abilities of those who are familiar with the technology.

The route of administration of the anti-TREM2 antibody as described herein can be oral, intraperitoneal, percutaneous, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intralesional, transrectal, intrabronchial, transnasal , Transmucosal, intestinal, transocular, or transaural delivery, or any other method known in the art. In some embodiments, the antibody is administered orally, intravenously, or intraperitoneally.

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

In another aspect, there are provided pharmaceutical compositions and kits comprising antibodies that specifically bind to human TREM2 protein. In some embodiments, the pharmaceutical compositions and kits are used to treat neurodegenerative diseases. In some embodiments, the pharmaceutical compositions and kits are used to modulate (e.g., enhance or inhibit) one or more TREM2 activities, such as Syk phosphorylation. In some embodiments, the pharmaceutical compositions and kits are used to adjust (e.g. lower) sTREM2 levels. Pharmaceutical composition

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

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

In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration. The pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds for use in, for example, stabilizing the composition or increasing or decreasing the absorption of the active agent. Physiologically acceptable compounds may include, for example, carbohydrates, such as glucose, sucrose, or polydextrose; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; combinations that reduce the clearance or hydrolysis of active agents物; Or excipients or other stabilizers and/or buffers. Other pharmaceutically acceptable carriers and their formulations are well known in the art.

The pharmaceutical composition described herein can be manufactured in a manner known to those skilled in the art, for example, by conventional mixing, dissolution, granulation, sugar coating, emulsification, encapsulation, embedding or freeze-drying processes. The following methods and excipients are only exemplary and in no way restrictive.

For oral administration, it can be formulated by combining the anti-TREM2 antibody with a pharmaceutically acceptable carrier well known in the art. These carriers enable the compound to be formulated into tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, and the like, for the treatment of patients to be treated. Oral intake. Pharmaceutical preparations for oral use can be obtained by mixing the compound with solid excipients, grinding the resulting mixture as appropriate, and processing the mixture of particles after adding suitable adjuvants (if desired) to obtain lozenges or Dragee core. Suitable excipients include, for example, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl Cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose; and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (such as sodium alginate).

Anti-TREM2 antibodies can be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. For injection, one or more compounds can be dissolved, suspended or emulsified in aqueous or non-aqueous solvents (such as vegetable oils or other similar oils, synthetic fatty acid glycerides, higher fatty acids or esters of propylene glycol). It is formulated into a preparation; and if desired, it is formulated with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the compound may be formulated in an aqueous solution, for example, in a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or physiological saline buffer . Formulations for injection can be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with the addition of preservatives. The composition may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation agents such as suspending agents, stabilizers and/or dispersants.

Generally, the pharmaceutical composition used for in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, such as heat sterilization, steam sterilization, sterile filtration or irradiation.

The dosage and expected drug concentration of the pharmaceutical composition of the present disclosure can vary depending on the specific application envisaged. The determination of the appropriate dose or route of administration is well known to those familiar with the art. The appropriate dosage is also described in Section VII above. Set

In some embodiments, kits comprising anti-TREM2 antibodies are provided. In some embodiments, the anti-TREM2 antibody system is the antibody or antigen-binding fragment thereof as described in Section III above.

In some embodiments, the kit further includes one or more other therapeutic agents. For example, in some embodiments, the kit includes an anti-TREM2 antibody as described herein, and further includes one or more other therapeutic agents for the treatment of neurodegenerative diseases, such as Alzheimer's disease. In some embodiments, the therapeutic agent is an agent used to treat cognitive or behavioral symptoms of neurodegenerative diseases (for example, antidepressants, dopamine agonists, or antipsychotics). In some embodiments, the therapeutic agent is a neuroprotective agent (e.g., carbidopa/levodopa, anticholinergic, dopaminergic, monoamine oxidase B (MAO-B) inhibitor, infant Tea phenol-O-methyltransferase (COMT) inhibitors, glutamate, tissue protein deacetylase (HDAC) inhibitors, cannabinoids, caspase inhibitors, melatonin, anti-inflammatory Drugs, hormones (such as estrogen or progesterone) or vitamins).

In some embodiments, the kit includes an anti-TREM2 antibody as described herein, and further includes one or more reagents for measuring sTREM2 levels. In some embodiments, the kit includes an anti-TREM2 antibody as described herein, and further includes one or more reagents for measuring TREM2 activity (eg, for measuring Syk phosphorylation).

In some embodiments, the kit further includes instructional materials that contain instructions (ie solutions) for practicing the methods described herein (for example, use the kit for the methods described in section VI above). Instructions for treatment methods). Although instructional materials usually include written or printed materials, they are not limited to this. This disclosure considers any media that can store these instructions and deliver them to the end user. Such media include (but are not limited to) electronic storage media (such as magnetic disks, tapes, disk cartridges, chips), optical media (such as CD-ROM), and the like. Such media may include the addresses of Internet sites that provide such instructional materials. IX. Examples

This disclosure will be elaborated in more detail with the help of specific examples. The following examples are provided for illustrative purposes only, and are not intended to limit the present disclosure in any way. Example 1. Anti-TREM2 antibody production and initial characterization Recombinant expression and purification of mouse Fc fused to human TREM2 ECD

The extracellular domain (residues 19-172) of human TREM2 (UniProtKB ID-Q9NZC2) and the secretion signal amino acid 1-20 (UniProtKB ID-P01661) from mouse IgG κ chain V-III in the N-terminal region and The mouse Fc tag in the C-terminal region and the GGGGS between TREM2 ECD and Fc (SEQ ID NO: 34) were subcloned into the pRK vector together.

Using the Expi293F™ expression system kit, the purified plastids were transfected into Expi293F™ cells (Thermo Fisher) according to the manufacturer’s instructions. In order to inhibit the maturation of N-linked glycans and reduce glycosylation heterogeneity, the high mannosidase I inhibitor kifunensine (Sigma) was added to the culture at a concentration of 1 μg/mL immediately after transfection In. The transfected cells were incubated in an orbital shaker (Infors HT Multitron) at 125 rpm and 37°C in a humidified atmosphere of _{6% CO 2.} 16 hours after transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and 2 were added to the cells, and the culture supernatant was harvested 96 hours after transfection. The clarified supernatant was supplemented with EDTA-free protease inhibitor (Roche) and stored at -80°C.

In rhTREM2-Fc separation, the clarified medium supernatant was loaded on HiTrap MabSelect SuRe protein A affinity column (GE Healthcare Life Sciences), and washed with 200 mM arginine and 137 mM succinate buffer pH 5.0 . The fusion protein was eluted in 100 mM QB citrate buffer pH 3.0 and 50 mM NaCl. Immediately after elution, 1 M Tris-HCl buffer pH 8.0 was added to the protein solution to neutralize the pH. The protein aggregates were separated by particle size analysis chromatography (SEC) on a Superdex 200 increase 10/300 GL column (GE Healthcare Life Sciences). The SEC mobile phase buffer is maintained at 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 50 mM arginine, which is also a protein storage buffer. All chromatographic steps are implemented on the AKTA pure or AKTA Avant system (GE Healthcare Life Sciences). Recombination performance and purification of TREM2 ECD with His tag

The extracellular domain (residues 19-172) of TREM2 (UniProtKB-Q9NZC2) and the secretion signal amino acid 1-20 (UniProtKB ID-P01661) from the mouse Ig κ chain V-III in the N-terminal region and the C The 6X-His tag (SEQ ID NO: 35) of the terminal region was sub-populated in the pRK vector together. The insert was verified by sequencing and maxi prep plastid purification was performed.

Using the Expi293F™ expression system kit, the purified plastids were transfected into Expi293F™ cells (Thermo Fisher) according to the manufacturer’s instructions. The transfected cells were incubated in an orbital shaker (Infors HT Multitron) at 125 rpm and 37°C in a humidified atmosphere of _{6% CO 2.} 16 hours after transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and 2 were added to the cells, and the culture supernatant was harvested 96 hours after transfection.

The harvested medium was supplemented with 1 M imidazole 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 micron. The HisPur™ Ni-NTA resin (Thermo Fisher) was washed with MQ water and equilibrated with loading buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 10 mM imidazole). The gravity flow method was used to perform affinity purification. The harvested medium was loaded on the resin, and the non-specifically bound protein was washed with a loading buffer supplemented with 50 mM and 100 mM imidazole. The bound His-tagged TREM2 extracellular domain was eluted with 20 mM Tris pH 8.0, 150 mM NaCl, and 200 mM imidazole. The eluted protein was concentrated using an Amicon 10 kDa concentrator, and the concentrated protein was further purified by gel filtration chromatography using the AKTA Avant system (GE Healthcare Life Sciences). The protein was loaded on a HiLoad Superdex 200 16/600 (GE Healthcare Life Sciences) column equilibrated with 1×PBS, and 1×PBS was used as the running buffer for elution and fractionation. Under denatured and natural conditions, the eluted fraction was analyzed by polyacrylamide (PAGE) gel electrophoresis. The eluted part was further characterized by analytical particle size sieve chromatography and intact protein quality determination. The results from PAGE and analytical characterization were used to pool the highly glycosylated protein fractions, and these protein fractions were aliquoted and stored at -80°C. Antibody production

Using standard protocols, rodents (mice and rats) were immunized with rhTREM2-Fc immunogen or BWZ cells expressing full-length Trem2 receptors. Using serum collected at different time points, the titer was measured during the entire immunization process. Using flow cytometry, rhTREM2-Fc immunogen and live BWZ cells expressing full-length TREM2 were used to detect antigen-specific immune responses. The selection criteria for candidate antibodies include the specificity of rodent antibody production and binding to TREM2 as detected by flow cytometry. Antibody secreting cells are isolated from animal immune tissues (including spleen, lymph nodes and bone marrow).

The single cell suspension is analyzed to determine the binding properties of the secreted antibody. The antibody secreting cells are loaded into a microfluidic device and separated in a nanoliter volume reaction chamber to enable the detection of secreted antibodies using microscopy analysis based on fluorescence and brightfield images (for example, see US Patent No. 9,188,593 ). Perform binding analysis involving the binding of detection antibodies to antigen-coated microbeads, the binding of fluorescently labeled soluble antigens to the antibodies immobilized on the beads, and the binding of detection antibodies to antigens expressed on the cell surface. The antigen expressed on the cell surface includes both the recombinant form and the natural form of the antigen presented on the cell surface.

Image analysis is used to identify chambers exhibiting a positive fluorescent signal, which indicates the presence of single cells that produce antibodies with desired properties, and the contents of the chamber are recovered and lysed in a 384-well plate (for example, see US Patent No. 10,087,408). The single cell lysate is then subjected to RT-PCR to amplify the heavy and light chain variable region sequences. The resulting amplicon was then sequenced to determine the cDNA sequence of the paired heavy chain and light chain variable regions from the selected single cell. Manually check and analyze the obtained sequence to determine sequence diversity and somatic hypermutation. Select performance sequences based on screening data and sequence diversity. Test the expressed antibodies to confirm the antigen binding specificity. Example 2. Sequence optimization and humanization of anti-TREM2 antibodies

Sequence optimization and humanization of exemplary anti-TREM2 antibodies were performed, and then the binding kinetics and binding specificity were characterized.

By searching for residues that are easy to be chemically modified in the CDR sequence (such as aspartamide deamidation motif (NG), aspartic acid isomerization motif (DS), and potentially oxidizing residues (tryptophan (tryptophan ( W) and methionine (M)) are combined with conservative and germline residues for amino acid substitution to eliminate these sequence disadvantages for sequence optimization. Then use Biacore and dose titration cell binding to analyze anti-TREM2 antibodies Binding kinetics of humanized and sequence-optimized variants to HEK293-H6 cells (for representative protocols, see Example 5). Example 3. Production of anti-TREM2 antibodies with modified Fc polypeptides ("ATV:TREM2")

_{The Fd (V H} +CH1) region (SEQ ID NO: 22 and 24) of the humanized affinity matured anti-TREM2 antibody was selected and cloned into expression vectors, the expression vectors containing codes engineered to bind to human transferrin The sequence of the Fc polypeptide (CH3C.35.23.1.1, CH3C.35.23.3, CH3C.35.23.3 cisLALA or CH3C.35.24) of the receptor (TfR) or the Fc polypeptide ( CH3C.35.21) sequence. The Fc polypeptide coding sequence also contains a "knob" (T366W) mutation to prevent homodimerization and promote heterodimerization with an Fc polypeptide containing a "hole" (T366S/L368A/Y407V) mutation. The Fd region was also cloned into corresponding "hole" vectors, which contained sequences encoding Fc polypeptides containing hole mutations but lacking TfR binding mutations. The coding sequence (both Fd-knob-Fc and Fd-hole-Fc constructs) also contains mutations in the hinge region "LALA"(L234A; L235A) to reduce effector function (Wines et al., J. Immunol. 164 :5313-5318 (2000) and Fc CH3 region "LS"(M428L; N434S) mutation to increase binding to FcRn (for example, see Zalevsky et al., Nat. Biotech. 28 (2): 157-159 (2010)). Expressed by the vector The final encoded heavy chain sequence is listed in Table 1. Table 1. ATV: TREM2 sequence ATV: TREM2 First heavy chain Second heavy chain number 1 SEQ ID NO: 42 SEQ ID NO: 53 number 2 SEQ ID NO: 45 SEQ ID NO: 53 number 3 SEQ ID NO: 48 SEQ ID NO: 53 No 4 SEQ ID NO: 48 SEQ ID NO: 52 Number 5 SEQ ID NO: 51 SEQ ID NO: 53

The corresponding knob and socket vector mentioned above together with the corresponding light chain vector (SEQ ID NO: 54) were co-transfected into ExpiCHO or Expi293 cells at a 1:1:2 ratio of knob: socket: light chain. The expressed protein was purified by protein A chromatography followed by preparative particle size analysis (SEC) to isolate the purified anti-TREM2 protein.

The binding of the anti-TREM2 protein to the human transferrin receptor was determined as follows: the anti-human Fab was immobilized on the CM5 chip, and the anti-TREM2 protein was captured. The full-length human TfR or human TfR apical domain in serial dilutions (for example, a concentration of 1-1,000 nM) is flowed through the wafer (180 seconds association time), and then it is dissociated. Use 1:1 combination model to implement fitting. Example 4. Characterization of anti-TREM2 antibodies

The following sections describe the various analyses performed to evaluate the binding and functional characteristics of the anti-TREM2 antibodies produced. Affinity measurement by Biacore kinetic measurement

Surface plasmon resonance (Biacore™ 8K instrument) was used to measure the affinity of anti-TREM2 antibodies to human and cynomolgus TREM2 ECD. The human Fab capture kit (GE Healthcare Life Sciences, catalog number 28958325) was used to capture anti-TREM2 antibodies on the Biacore Series S CM5 sensor chip (GE Healthcare Life Sciences, catalog number 29149604). Inject 3 times serial dilutions of recombinant human or cynomolgus TREM2 at a flow rate of 30 µL/min. Monitor antibody binding for 300 seconds, and then monitor antibody dissociation in HBS-EP+ running buffer (GE Healthcare Life Sciences, catalog number BR100669) for 600+ seconds. Correct the binding reaction by subtracting the RU value from the blank flow cell. A 1:1 Languir model that simultaneously fits k- _association and k- _{dissociation is used for kinetic analysis.} Calculate the K _D binding value from k _association and k _{dissociation.} Evaluation of TREM2 binding in HEK cells expressing TREM2

The binding characteristics of anti-TREM2 antibodies were evaluated in HEK 293 cells expressing human TREM2 as follows.

The HEK 293 cell line stably expressing human TREM2/DAP12 was generated by transfecting cells with vectors expressing wild-type human TREM2 and DAP12 and DAP12 alone. Select stable expression clones, and evaluate the cell surface TREM2 expression by flow cytometry. APC-conjugated rat anti-human/mouse-TREM2 monoclonal antibody (R&D, catalog number MAB17291) was used to detect surface TREM2 expression. The pure line showing the highest expression level of wild-type TREM2 was selected and named "HEK293-H6". The pure line of DAP12 was stably expressed by western ink dot analysis, and the selected pure line was named "No. 1 HEK293-DAP12".

HEK 293 (HEK293-H6) expressing human TREM2 (HEK293-H6) and HEK 293 (B5) expressing GFP were harvested with 0.05% trypsin, and incubated at 37°C for 2 hours. After incubation, the cells were centrifuged and washed twice in FACS buffer (PBS + 0.5% BSA). Mixed cell line cells per 10 ⁶ / mL densitometry resuspended in a solution containing human Trustain FcX (Biolegend, Catalog No. 422302) of FACS buffer. The mixed cell line was seeded in a 96-well round bottom plate at 200,000 cells/well and incubated at room temperature for 20 minutes. After incubation, the cells were centrifuged and incubated with the titrated anti-TREM2 antibody for 45 minutes on ice. After incubation, the cells were centrifuged and washed three times with FACS buffer. The cells were then placed on ice with the secondary antibody (Alexa Fluor 647 AffiniPure F(ab') 2 fragment goat anti-human IgG (H+L), Jackson ImmunoResearch Laboratories, catalog number 109-606-088, 1:800 dilution) Incubate for 30 minutes. After incubation, the cells were washed three times with FACS buffer, resuspended in 100 μL FACS buffer, and analyzed by flow cytometry (BD FACSCanto II, San Jose, CA), and each sample obtained 30,000 events. Calculate the average fluorescence intensity of each cell by FlowJo software, and use it to generate a dose-response binding curve. TREM2-dependent activation of pSyk signaling

Using the commercially available AlphaLisa assay from Perkin-Elmer, the activation of TREM2-dependent pSyk signaling was measured in human macrophages or HEK293-H6 cells.

For all experiments involving the use of lipid vesicles containing 70% DOPC and 30% POPS, lipid vesicles were prepared within two weeks of the experiment as follows: 7 mg DOPC (1,2-dioleyl- sn -Glycerol-3-phosphocholine) and 3 mg POPS (1-palmitoyl-2-oleyl- sn -glycero-3-phospho-L-serine) combined in chloroform and under N ₂ flow Dry for 1-2 hours, or until completely dry. Resuspend the lipid mixture in 1 mL HBSS (final lipid concentration is about 10 mg/mL) and vortex for 2-3 minutes. Subsequently, an Avanti micro-extruder constructed with a 100-nm pore size membrane was used to extrude the lipid suspension to form small unilamellar vesicles at 10 mg/mL. 1. Administering antibodies in cells

On the day before analysis, human macrophages or HEK293-H6 cells were plated in a 96-well plate coated with poly-D-lysine at 100,000 cells/well or 40,000 cells/well, respectively. The antibody was diluted by a 10-point serial dilution, and each point was diluted 3 times in PBS. For the antagonist dose-response curve, the antibody/PBS mixture also includes lipid vesicles containing 70% DOPC and 30% POPS at a final concentration of 1 mg/mL. Use the Biotek 405/406 plate washer to wash the cells with HBSS 3 times, and then use the Hamilton Nimbus liquid handler to add 50 μL/well of antibody/PBS (with or without vesicles) solution. Then transfer the cell plate to a 37°C incubator for 5 minutes. Remove the liposome/antibody solution by gently flicking the plate, and use a liquid handler to add 40 μL of lysis buffer (Cell Signaling Technologies, CST) containing 1 μM PMSF. The lysate was then frozen at -80°C or immediately analyzed in AlphaLisa analysis.

The human macrophages for analysis were prepared as follows. Following the RosetteSep human monocyte enrichment mix protocol (Stemcell Technologies, reference number 15068), human monocytes were separated from fresh blood. The separated mononuclear spheres were washed in washing buffer (PBS+2% FBS) and resuspended in 10 mL of ACK lysis buffer (ThermoFisher Scientific, catalog number A10492) to solubilize red blood cells. Add twenty (20) mL wash buffer to stop cell lysis, and centrifuge the sample with culture medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acid, and 1% penicillin -Streptomycin) wash again. Then, human monocytes were differentiated into macrophages in a 250-mL flask in the presence of 50 ng/mL human recombinant M-CSF (Gibco, catalog number PHC9501) in the culture medium. Fresh human M-CSF was spiked on day 3, and then human macrophages were harvested on day 5 and used for analysis. 2. AlphaLisa analysis

The standard protocol of the Perkin Elmer pSyk AlphaLisa kit was used to analyze the pSyk in the cell lysate. Briefly, transfer 10 μL of lysate/well to white opaque 384-well Optiplate (Perkin Elmer). Next, add 5 μL of receptor mix (working solution containing receptor beads) to each well, then seal the plate with a foil seal and incubate at room temperature for 1 hour. Subsequently, 5 μL of the donor mixture (working solution containing donor beads) was added to each well under low light conditions. The plate was sealed again and incubated at room temperature for 1 hour. Finally, use the AlphaLisa on the Perkin Elmer EnVision plate reader to set up the plate reading. Survival analysis in human macrophages

Following the RosetteSep human mononuclear sphere enrichment mixture protocol (Stemcell Technologies, catalog number 15068), human mononuclear spheres were isolated. The separated mononuclear spheres were washed in washing buffer (PBS+2% FBS) and resuspended in 10 mL of ACK dissolving solution (ThermoFisher Scientific, catalog number A10492) to dissolve red blood cells. Add twenty (20) mL wash buffer to stop dissolution. The cell suspension was centrifuged and washed once with medium (RPMI 1640 + 10% FBS + penicillin/streptomycin). Cells at ¹⁰⁶ cells μL / mL of a density resuspended in culture medium and used as set forth below in the survival analysis.

The day before the analysis, the 96-well plate was pre-coated with a dose-titrated anti-TREM2 antibody or isotype control (45 μL/well, 12 points in total), and incubated overnight at 4°C. ^{After overnight incubation, the pre-coated plate was washed twice with PBS, and then loaded with human monocytes (10 5} cells) in the presence of a low concentration of human M-CSF (5 ng/mL, Gibco, catalog number PHC9501) /hole). After 5 days at 37°C, the medium was aspirated and 100 μL PBS + 100 μL Celltiter-glo medium (Promega, catalog number G7571) was added to each well. After incubating for 10 minutes, transfer the cell culture medium to a multi-well plate suitable for the use of a luminometer, and record the luminescence regarding cell viability. Lipid storage analysis

Before analysis, a commercially available kit (STEMdiff hematopoietic kit from StemCell Technologies) was used to differentiate induced human pluripotent stem cells (iPSC) into hematopoietic progenitor cells (HPC). The HPC was transferred to a plate containing primary human stellate cells and co-cultured for 14-21 days. After the floating cells in the co-culture were mainly identified as mature microglia (>80%), these microglia were used for analysis.

The cells (iPSC-derived human microglia cells, 30,000 cells/well) were plated in the whole serum medium on a 96-well plate coated with PDL. After 24 hours at 37°C, replace the medium with oleic acid-albumin (10 μM or 33 μM final concentration, Sigma O3008) or purified unlabeled myelin (50 μg/mL final concentration, use The method described in Safaiyan et al. (2016, Nature Neuroscience 19(8):995-998) is a whole serum medium purified from wild-type C57Bl/6 mouse brain (Jackson Laboratories). After lipid treatment at 37°C for 24 hours, the medium was changed to a medium containing anti-TREM2 antibody. For single point experiments, the anti-TREM2-antibody concentration used was 100 nM. For the dose-response curve, the medium containing 100 nM anti-TREM2 antibody was serially diluted 3-fold, for a total of 10 points. RSV was used as a control. As explained below, the cells were incubated for another 48 hours at 37°C, after which the cells were imaged using Bodipy stain, or the cells were extracted for liposomes.

To perform Bodipy imaging, remove the supernatant and place the cells in a 1:2500 solution (Thermo-Fisher D3922) containing 1 mg/mL Bodipy 493/503 in DMSO and 1 drop/mL Nucblue (ThermoFisher) at 37°C. , Catalog number R37605) live cell imaging buffer (Life Technologies, catalog number A14291DJ) for 30 minutes. After the incubation period, the staining solution was removed, and the cells were imaged in vivo or fixed in 4% paraformaldehyde. Use Opera Phoenix high-content confocal imager for Bodipy's Alexa 488 channel and DAPI illumination settings to image cells. Use the spot search algorithm on the Harmony software provided with the instrument to analyze lipid spots.

For liposome analysis, the cells were washed once with PBS while keeping the cells on ice. Add a volume of 70 µL of 9:1 methanol:water solution containing 1:100 internal standards to the cells in the 96-well plate. The plate was agitated on a shaker at 4°C and 1200 rpm for 20 minutes, and then centrifuged at 300×g for 5 minutes. Transfer 50 µL of the supernatant sample to the LCMS vial and keep it at -80°C until analysis on the instrument.

The lipid level was 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 on a BEH C18 1.7 µm, 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) at 55°C with a flow rate of 0.25 mL/min. For positive ionization mode, mobile phase A is composed of 60:40 acetonitrile/water (v/v) containing 10 mM ammonium formate + 0.1% formic acid; mobile phase B is composed of 90: 10 isopropanol/acetonitrile (v/v) composition. For negative ionization mode, mobile phase A is composed of 60:40 acetonitrile/water (v/v) containing 10 mM ammonium acetate; mobile phase B is composed of 90:10 isopropanol/acetonitrile (v/v) containing 10 mM ammonium acetate /v) Composition. The gradient programming is as follows: 0.0-8.0 min at 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B. Apply the following settings to perform electrospray ionization in positive or negative ion mode: curtain gas is 30; collision gas is set to medium; ion spray voltage is 5500 (positive mode) or 4500 (negative mode); temperature is 250°C (positive mode) or 600 ℃ (negative mode); ion source gas 1 is 50; ion source gas 2 is 60. Use Analyst 1.6.3 (Sciex) to implement data collection in multiple reaction monitoring mode (MRM), where the parameters are as follows: residence time (msec) and collision energy (CE); declustering potential (DP) is 80; entrance potential (EP) ) Is 10 (positive mode) or -10 (negative mode), and the collision cell exit potential (CXP) is 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 retention time and the MRM properties of a commercially available reference standard (Avanti Polar Lipids, Birmingham, AL, USA). TREM2-dependent activation of mTOR signaling

Wild-type iPSC-derived human microglia cells were cultured and treated with anti-TREM2 antibody (100 nM final concentration) and DMSO or a commercially available mTOR inhibitor (Selleckchem, catalog number AZD8055, 20 nM final concentration) for 96 hours. The treated cells were then lysed, and cell lysates were prepared for use in Western blots to study the phosphorylation of the main signaling targets in the mTOR pathway. The primary antibody system used in Western ink dots was obtained from Cell Signaling Technologies: (1) Phosphoric acid-mTOR (Ser2448), product number 5536T; (2) mTOR (7C10), Product number 2983T; (3) Phosphoric acid-AKT (Ser473) , Product number 9271T; (3) phospho-GSK-3β (Ser9), product number 5558T; (4) phospho-S6 ribosomal protein (S235/236), product number 4858T; (5) phosphate-4E-BP1 (Thr37 /46), product number 2855T; (6) β-actin, product number 58169S. Example 5. Results

The results of the binding characteristics analysis of the humanized and sequence-optimized variants of antibody CL0020188 are provided in Table 2 and Figures 1A to 1H. The NG motifs in the CL0020188 CDR-H2 sequence (SEQ ID NO: 5) and CDR-L1 sequence (SEQ ID NO: 7) were modified, transplanted to the human framework region and analyzed. _{Table 2 provides K D values as measured by Biacore and EC 50} values as measured by dose titration binding analysis in HEK293-H6 cells. Figures 1A to 1H include representative dose-response curves of humanized and sequence-optimized variants with TREM2 binding expressed by HEK293-H6 cells. Variants are represented by solid black circles (●), while isotype controls are represented by hollow white circles (°). Table 2. Binding characteristics of sequence optimized and humanized variants of CL0020188 Pure line h V _H h V _L K _D EC ₅₀ CL0020188-1 NG /graft NG /graft 2.3 nM 0.42 nM CL0020188-2 NG /3m NG /graft 3.4 nM 0.26 nM CL0020188-3 NG /graft TG /graft 6.8 nM 0.64 nM CL0020188-4 NG /3m TG /graft 4.8 nM 0.44 nM CL0020188-5 NA /graft NG /graft 5.1 nM 0.45 nM CL0020188-6 NA /3m NG /graft 4.0 nM 0.31 nM CL0020188-7 NA /graft TG /graft 10 nM 0.68 nM CL0020188-8 NA /3m TG /graft 7.3 nM 0.51 nM Parent 9.5 nM 0.44 nM 3m = A24G/L45P/V48L in _{V H}

As illustrated in Table 2, as measured by Biacore, the humanized and sequence-optimized clone of CL00201088 exhibited similar affinity to hTREM2 compared _{to the parent antibody (K D = 9.5 nM).} This is consistent with the cell binding results in HEK293-H6 cells described in Table 1, wherein the corresponding dose-response curves are provided in Figures 1A to 1H. Compared with the parental antibody (EC ₅₀ = 0.44 nM), the humanized and sequence-optimized pure line showed comparable affinity to TREM2 expressed in HEK293-H6 cells. In summary, these results indicate that the binding kinetics between the parental antibody and the humanized and sequence-optimized variants are comparable.

The results of the ATV:TREM2 variants described in Example 3 are summarized in Table 3 below. Figure 2 illustrates an exemplary cell binding curve based on the binding to HEK cells expressing human TREM2 and analysis by FACS. Table 3. ATV: Summary of TREM2 features ATV: TREM2 Biacore K _D (nM) EC50 (nM) Human TREM2 Cyno TREM2 Human TfR Human TREM2 HEK number 1 TBD TBD TBD TBD number 2 5.4 2.4 650 4.9 number 3 3.0 2.2 1400 4.6 No 4 2.0 2.2 TBD TBD Number 5 5.7 2.4 620 3.6 TBD = to be determined

The antibody's TREM2-dependent pSyk signaling in HEK-H6 cells, the ability to promote the survival of human macrophages, and the regulation of iPSC-derived human microglia (hereinafter referred to as "iPSC microglia" or "iMG ") The ability to accumulate lipids. Figure 3 illustrates the results of an ATV:TREM2 variant (No. 3 ATV:TREM2) and the corresponding anti-TREM2 antibody. ATV: TREM2 can activate pSyk signaling in HEK293-H6 cells expressing TREM2 to a significantly greater extent than the corresponding TREM2 antibody, indicating that the addition of ATV to the molecule can increase its efficacy (Figure 3). In addition, ATV:TREM2 induces macrophage survival with an EC ₅₀ of 4.1 + 0.3 nM. Finally, the ability to reduce the anti-antibody display TREM2 (FIGS. 4A and 4B) of lipid accumulation iMG myelin in the process, where inhibition of lipid storage IC ₅₀ of 0.20 nM (97.7 + 0.3% maximum inhibition).

Perform additional studies to study the ability of ATV:TREM2 to reduce lipid accumulation. Figures 5A to 5F and Figures 6A to 6C show that a representative ATV:TREM2 variant (No. 3 ATV:TREM2) reduces lipid accumulation and enhances fatty acid oxidation intermediates, which indicates that ATV:TREM2 is in enhancing mitochondrial function The potential role. As illustrated by Bodipy staining, cells (iMG) treated with oleic acid lipid challenge (33 μM) were able to reduce lipid accumulation after being incubated with ATV:TREM2 (Figure 5A and Figure 5B). The LCMS analysis of iMG treated with myelin for 24 hours after incubating with ATV:TREM2 for 48 hours indicated that ATV:TREM2 reduces triglyceride (TG) substances, and at the same time increases β-oxidation intermediate (carnitine) And TCA cycle intermediates (Figure 5C to Figure 5F). Figure 5C provides a heat map showing all TG, carnitine, and TCA cycle intermediate substances with a fold change> 1.5 (p <0.05). Or the change of the representative substance in the iMG cultivated with the vehicle and the myelin attack of the same type control. Figures 6A to 6C illustrate the changes in specific TG, carnitine and TCA cycle intermediate substances in iMG cultivated with ATV:TREM2 or isotype control after myelin attack. As shown in FIGS. 6A to 6C, ATV:TREM2 reduces all TG and ceramide substances, while increasing some short-chain carnitine substances, which indicates that ATV:TREM2 can enhance mitochondrial function.

Microglial cell proliferation is related to mTOR signal activation and engagement. Therefore, explore the role of ATV:TREM2 in downstream mTOR pathway signaling. By Western blot analysis, the phosphoric acid of the mTOR signal pathway target in wild-type iPSC microglial cells cultivated with representative ATV:TREM2 variants (ATV:TREM2 No. 3) in the presence and absence of mTOR inhibitors化 status. Figure 7A illustrates a representative western blot image of the mTOR signal path target. The quantification of western blot data is provided in Figure 7B to Figure 7E (phosphorylation levels are normalized with β-actin loading control). For each independent experiment (n=6), the phosphorylation level of each ATV:TREM2 treated sample was compared with the isotype antibody control treated sample. The results show that ATV:TREM2 activates mTOR pathway signaling. For example, compared with the isotype control, in the ATV:TREM2 treated samples, mTOR is at serine ²⁴⁸⁸ , AKT at serine ⁴⁷³ , ribosomal protein S6 (RPS6) ) As evidenced by the increased phosphorylation levels at serine ^235/236 and GSK3b at serine ⁹ (Figure 7B to Figure 7E; statistical summary: "ns"(p>0.05);"*"(p<0.01);"**" (p＜0.001)). RPS6 is the signal target downstream of the mTORC1 complex, and GSK3b is the signal target downstream of the mTORC2 complex. For all the data generated in FIG. 5A to FIG. 7E, the isotype control ("ISO") of No. 3 ATV:TREM2 contains the sequence provided in Table 5. Example 6. ATV: The role of TREM2 in lysosomal dysfunction

To study the role of ATV:TREM2 in lysosomal function. To evaluate the effect on lysosomal function, pre-granulin (PGRN) and bis(monoglycero)phosphate were measured in iPSC-derived microglial cells ("iMG") treated with ATV:TREM2 variants The level of ester (BMP).

To evaluate the effect of ATV:TREM2 on PGRN levels, iMG was spread in 96-well plates at a density of 30,000 cells/well and incubated with No. 3 ATV:TREM2 (100 nM) for 72 hours, and then the cell supernatant was collected. Liquid and cell lysate were analyzed using a colorimetric sandwich ELISA to analyze the level of granular protein precursor (PGRN).

To measure the PGRN level, a Thermo Scientific 384-well Maxisorp plate was coated with 4 µg/mL capture antibody (R&D anti-PGRN antibody from DuoSet ELISA kit, catalog number DY2420) diluted in phosphate buffered saline (PBS) and Incubate overnight at 4°C. Block the sample wells with 3% BSA in PBS for 90 minutes. The cell samples were diluted 1:10 in 3% BSA in PBS and added to each sample well on the plate, and then incubated at room temperature for 90 minutes. The detection antibody (R&D anti-PGRN antibody from DuoSet ELISA kit, catalog number DY2420) diluted to 125 ng/mL was then added to each sample well, and the plate was incubated at room temperature for 90 minutes. Finally, HRP-bound streptavidin (R&D SA-HRP from DuoSet ELISA kit, catalog number DY2420) was diluted 1:200 and added to each sample well. The plate was incubated at room temperature for 20 minutes. After washing the sample well with PBS, a color reagent (TMB substrate) was added and allowed to react for 5 minutes, and then the reaction was stopped _{with 4 NH 2} SO _4. The absorbance was measured with the BioTek Synergy Neo2 plate reader, and the PGRN level was determined by interpolation from the standard curve fitted with the four-parameter logistic curve. As illustrated in Figure 8, incubation with ATV:TREM2 increased PGRN levels in both the supernatant and cell lysate relative to the isotype control.

To evaluate the effect on BMP levels, iMG was challenged with myelin or vehicle for 24 hours, and then incubated with No. 3 ATV:TREM2 (100 nM) for 48 hours. Cell lipids were extracted by adding methanol containing a mixture of internal standards, and similar to those described in International PCT Publication No. WO 2020/112889, by liquid chromatography-mass spectrometry (LC-MS/MS) in Q-trap 6500 ( SCIEX) measured BMP abundance. BMP (14:0_14:0) was used as an internal standard to quantify BMP substances and identify them based on their residence time and MRM properties. After correcting for isotope overlap, use MultiQuant 3.02 (Sciex) to implement quantification. The BMP substance was normalized with the lipid content of all the substances measured. The protein concentration was measured using bicinchoninic acid (BCA) analysis (Pierce, Rockford, IL, USA). A representative BMP result is illustrated in Figure 9. As shown in Figure 9, iMG that was attacked by myelin and then treated with ATV:TREM2 lowered the level of BMP substances, indicating that myelin-induced lysosomal attack may be rescued.

As illustrated in Figures 8 and 9, the iMG was incubated with ATV:TREM2 to increase PGRN levels and correct myelin-induced BMP levels, which indicates the role of ATV:TREM2 in regulating lysosomal effects. In Figures 8 and 9, the isotype control (ISO) of No. 3 ATV: TREM2 contains the sequence provided in Table 5. Example 7. ATV: The role of TREM2 in mitochondrial respiration

In order to evaluate the effect on mitochondrial respiration, the Seahorse XFe96 analyzer (Agilent) and the materials and protocols from the Seahorse XF palmitate oxidation pressure kit (Agilent 103693) were used for the treatment of ATV:TREM2 variants or isotype controls. The oxygen consumption in iPSC-derived microglia ("iMG") was measured. Prior to the Seahorse experiment, cells were incubated on XF96 microplates (Agilent, catalog number 102416) with No. 3 ATV: TREM2 (100 nM) or isotype controls for 72 hours. The cells were subjected to a combination of supplementation with 0.5 mM glucose (Agilent 103577), 1 mM glutamic acid (Agilent 103579), 0.5 mM L-carnitine (part of the Seahorse XF palmitate oxidative pressure kit) and 1% Hyclone FBS. The substrate-restricted medium composed of Seahorse XF RPMI (Agilent 103576) for 16 hours, after which the Seahorse experiment was performed. Immediately before the experiment, the cells were administered palmitate-BSA conjugate (166 μM) or BSA control. Use sequential injections of (1) etomoxir (4 μM) (carnitine palmitoyl transferase 1 (CPT1) inhibitor) or vehicle, (2) oligomycin (1.5 μM) (ATP combined Enzyme complex V inhibitor), (3) carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, mitochondrial decoupling agent, 1 μM) and (4) rotenone/antimycin (each 0.5 mM, which is the inhibitor of complex I and complex III respectively) to evaluate the mitochondrial respiratory capacity of the cell. During the experiment, the oxygen consumption rate was measured by a Seahorse analyzer (Agilent). Table 4 provides a summary of the experimental conditions used to evaluate mitochondrial respiration. Table 4. Experimental conditions condition Antibody CPT1 inhibitor Recovery 1 Isotype control Moxa BSA (control) 2 ATV: TREM2 Vehicle BSA (control)) 3 Isotype control Moxa Palmitic acid (PAL) 4 ATV: TREM2 Vehicle Palmitic acid (PAL) 5 Isotype control Moxa BSA (control) 6 ATV: TREM2 Vehicle BSA (control)) 7 Isotype control Moxa Palmitic acid (PAL) 8 ATV: TREM2 Vehicle Palmitic acid (PAL)

A representative kinetic graph of oxygen consumption is illustrated in Figure 10A, and a bar graph illustrating the maximum respiratory capacity of cells is provided in Figure 10B. As shown in these figures, ATV:TREM2 increases maximum respiration, similar to the degree of fatty acid substrate palmitic acid (PAL). In the presence of CPT1 inhibitors, this effect is attenuated. These results indicate that ATV:TREM2 enhances maximal mitochondrial respiration, and this effect seems to be conferred by the enhanced fatty acid oxidation capacity. Example 8. ATV: TREM2 comparative properties

The properties of No. 1 ATV: TREM2 and No. 3 ATV: TREM2 were compared with those of the reference antibody that binds to TREM2 as described in WO 2019/028292. The heavy chain and light chain sequences of reference antibody No. 1 ("Ref. Ab. No. 1") are represented by SEQ ID NOs: 74 and 75, respectively. The heavy chain and light chain sequences of reference antibody No. 2 ("Ref. Ab. No. 2") are represented by SEQ ID NOs: 76 and 75, respectively. Table 5 provides the heavy chain and light chain sequences of the isotype control for each anti-TREM2 antibody. Table 5. Isotype control sequence Isotype control First heavy chain Second heavy chain Light chain ISO of Reference Antibody No. 1 and No. 2 SEQ ID NO: 77 SEQ ID NO: 77 SEQ ID NO: 78 ATV No. 1: ISO of TREM2 SEQ ID NO: 79 SEQ ID NO: 81 SEQ ID NO: 82 ATV No. 3: ISO of TREM2 SEQ ID NO: 80 SEQ ID NO: 81 SEQ ID NO: 82 ATV: TREM2 is more potent in vitro and induces less inflammation (less cytokine release) than the reference Ab

The human macrophage survival analysis described in Example 4 was used to evaluate anti-TREM2 antibodies. Figure 11A illustrates the cell viability dose-response curve of anti-TREM2 antibody, where "ISO" refers to the isotype control of No. 3 ATV: TREM2 (Table 5). The corresponding efficacy (EC50) and maximum response (Emax) values determined from the dose-response curve are provided in Figure 11B and Figure 11C; individual labels represent a single value from a human cell donor (n=3). The results provided in Figures 11A to 11C indicate that ATV:TREM2 No. 3 is more potent than reference antibodies No. 1 and No. 2 in promoting the survival of human macrophages in vitro.

In a separate experiment, human macrophages were treated with 100 nM surface-immobilized anti-TREM2 antibody for 5 days, and then the cell culture medium of each group of cells was collected and Luminex xMAP technology and a bead-based commercial multiplex analysis kit (Human Cytokine 42-Plex Discovery Assay®, Eve Technologies Corp.) analyzes the release of cytokines. Figure 12 illustrates a heat map of relative cytokinin release levels (Z-score is used for mapping). The results in Figure 12 indicate that ATV:TREM2 No. 3 induces less inflammation in human macrophages than the reference antibodies No. 1 and 2.

In summary, No. 3 ATV: TREM2, No. 1 reference antibody, and No. 2 reference antibody can promote the survival and proliferation of human macrophages (Figure 11A). However, No. 3 ATV: TREM2 showed stronger cell survival efficacy and reduced overall cytokine markers compared with No. 1 and No. 2 reference antibodies (Figure 11B, Figure 11C and Figure 12). ATV: TREM2 can reduce the level of triglycerides after an attack

Anti-TREM2 antibodies were evaluated by lipid storage analysis as described in Example 4 (using 10 μM oleic acid challenge). Figures 13A to 13E provide the results of No. 3 ATV: TREM2, No. 1 reference antibody, and No. 2 reference antibody.

Figures 13A to 13C illustrate volcano graphs with cut-off values of p<0.05 and multiple changes>1.5 for triglyceride substances in iPSC-derived microglial cells ("iMG") as quantified by LCMS. The data was normalized with the isotype control of each antibody. As shown in Figure 13A to Figure 13C, No. 3 ATV:TREM2 can adjust the triglyceride (TG) substance after the administration of oleic acid, while the reference antibody does not significantly change the TG substance level after oleic acid attack. Figure 13D and Figure 13E illustrate the bar graphs of representative TG material quality measurements, in which the data is normalized with the No. 3 ATV:TREM2 isotype control. ATV: TREM2 can regulate TREM2 levels in vitro

To evaluate how anti-TREM2 antibodies affect the TREM2 levels in iPSC-derived microglial cells ("iMG"), iMG was incubated with various concentrations of ATV:TREM2 or reference antibody No. 3 for 72 hours, and then iMG cells were measured. TREM2 levels in lysates and cell culture media. Measure TREM2 as follows. In brief, MSD small spot streptavidin plate (Meso Scale Discovery) was coated with biotinylated goat anti-hTREM2 multi-strain antibody (R&D Systems, BAF1828) for 1 hour at room temperature. Then the plate was blocked with MSD blocking A buffer (Meso Scale Discovery) at room temperature for 1 hour. Prepare/dilute samples and standards using assay buffer (25% MSD blocking A buffer in TBST), and load 30 μL of samples and standards into the plate after blocking. After incubating for 1 hour at room temperature, the plate was washed with TBST, and then combined with the primary antibody (No. 3 ATV: TREM2) for 1 hour at room temperature. Then, the diluted sulfo-tagged goat anti-human IgG (Southern Biotech, 2049-01) was added to the plate and incubated at room temperature for 1 hour. After washing with TBST, use 2× MSD reading buffer T to make the MSD plate color, and then use the MSD Sector plate reader for detection. By using Meso Scale Discovery software (Discovery Workbench) to fit the standard curve, the MSD value is converted into the absolute amount of TREM2.

Figures 14A and 14B show graphs showing changes in TREM2 levels with the antibody concentration in iMG cell lysates and cell culture media after incubation with anti-TREM2 antibodies. The TREM2 level of each antibody is normalized with a specific isotype control. In Figure 14A and Figure 14B, "ISO" represents the isotype control of No. 3 ATV:TREM2. As illustrated in Figure 14A, the level of total TREM2 increased with the increase in the amount of antibody in iMG cell lysates treated with No. 3 ATV: TREM2 and No. 1 reference antibody. In contrast, the level of soluble TREM2 decreases as the amount of antibody in the cell culture medium of the antibody-treated cells increases. ATV: TREM2 shows excellent pharmacokinetic profile in non-human primates

The pharmacokinetic (PK) properties of anti-TREM2 antibodies were studied in non-human primates. In short, a single dose of 30 mg/kg test substance was administered intravenously to young adult/adult male cynomolgus monkeys ranging in age from 36 to 53 months (Table 6, n=5 animals/group). CSF samples were collected 24, 168, and 336 hours after administration.

The results are shown in Table 6 and FIG. 15. Table 6 shows the PK values of anti-TREM2 antibodies in the cerebrospinal fluid (CSF) of non-human primates. The PK value of ATV: TREM2 variant showed that compared with TREM2 Ab (lack of binding ability to transferrin receptor; the heavy chain and light chain sequences are represented by SEQ ID NO: 83 and 54 respectively) and the reference antibody No. 1 The exposure of other antibodies in CSF is higher. Table 6. CSF pharmacokinetic profile of anti-TREM2 antibodies in non-human primates Inspection Dose (mg/kg) C _max (nM) T _max (hr) AUC _{0- Last} (nM*hr) ATV No. 1: TREM2 30 3.17 twenty four 822.59 ATV No. 3: TREM2 30 1.49 twenty four 461.79 ISO* 30 3.23 twenty four 618.90 TREM2 Ab 30 0.98 twenty four 280.38 Reference antibody No. 1 30 0.70 twenty four 166.69 * No. 1 ATV: Isotype control of TREM2

Figure 15 illustrates the PK profile of anti-TREM2 antibodies in the CSF of administered non-human primates. As illustrated in Figure 15 and Table 6, ATV:TREM2 showed that the CSF PK was at least twice that of the reference antibody No. 1. Example 9. ATV: TREM2 properties in humanized TREM2 mice

To study the effect of ATV:TREM2 in vivo, we produced BAC gene transgenic mice that express human TREM2. These mice were crossed with the human transferrin receptor knock-in mice described in US Patent No. 10,143,187 to allow the characterization of the ATV:TREM2 molecules described herein. Generation of human TREM2 BAC gene transfer model

To study the effect of ATV:TREM2 in vivo, we used modified BAC DNA CTD-2210D2 (ThermoFisher Scientific; catalog number 96012) to generate several gene-transgenic mouse strains expressing human TREM2. 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. In order to avoid the interference of these TREM-like genes, we eliminated TREML1 exon 1 and TREML2 exon 3 to eliminate their performance. The engineered BAC CTD-2210D2 DNA construct was injected into the pronucleus of mouse fertilized eggs from C57BL/6J mice. Two independent ancestor lines (called TB36 and TB45) were obtained, and they showed germline transmission.

In order to characterize and compare the two gene transgenic lines, hemizygous transgenic animals and wild-type non-gene transgenic same-feet control animals were used for analysis. The qPCR analysis of tail genomic DNA was used to determine the copy number of human TREM2. Measure human TREM2 mRNA and protein levels in brain, liver, lung and spleen by qRT-PCR and MSD analysis. The human TREML1 and TREML2 mRNA in brain sorted microglia were analyzed by qRT-PCR. The surface TREM2 expression in bone marrow-derived macrophages (BMDM) was quantified by FACS. The human TREM2 function was evaluated by in vitro BMDM survival analysis.

Human TREML1 and TREML2 were not detected in TB36 or TB45, indicating that these genes were successfully deleted. The qPCR analysis showed that there were two and one human TREM2 transgenic gene copies in TB36 and TB45, respectively. Compared with TB45, TB36 showed higher performance of human TREM2 in the brain and peripheral tissues. In vitro survival analysis showed that the human TREM2 agonist antibody triggered a stronger response in the TB36 strain than in the TB45 strain.

Based on this in vitro and in vitro characterization, we chose TB36 for the following breeding and in vivo studies. Hemizygous TB36 mice were further backcrossed with C57BL/6J for three rounds, and then hTfR KI mice (described in U.S. Patent No. 10,143,187) were mated to produce human TREM2 BAC hemizygotes; hTfR KI homozygous mice were used for In vivo research. ATV: Pharmacokinetics and pharmacodynamics of TREM2 in TB36/hTfR KI mice

To determine whether ATV:TREM2 can trigger a microglial cell response in vivo, a single dose of No. 3 ATV:TREM2 or the corresponding isotype control (ATV:RSV) (100 mg/kg) was administered intravenously on day 0 TB36/hTfR KI mice, and the mice were sacrificed on day 1 or 4 after administration for ex vivo analysis. At the time of sacrifice, the animals were anesthetized by intraperitoneal injection of 2.5% Avertin. The peripheral blood was collected in an EDTA tube via cardiac puncture and slowly inverted (10 times), and then centrifuged at 15,350 g for 7 minutes at 4°C. The plasma (top layer) was transferred to a 1.5-ml Eppendorf tube and stored at -80°C until measurement. After blood collection, the CSF sample was collected from the cisterna magna by a pre-drawn glass capillary, and then transferred to a 0.5 mL Protein LoBind Eppendorf tube for centrifugation at 12,700 rpm at 4°C for 7 minutes. The CSF sample supernatant was quickly frozen on dry ice and stored at -80°C until measurement. Then the animals were perfused with cold PBS, the brain was dissected, and the two hemispheres were separated. The right hemibrain was immersed and fixed in 4% paraformaldehyde at 4°C for 24 hours, and then transferred to a phosphate buffered saline (PBS) solution containing 0.1% sodium azide for storage until ready for 30 % Sucrose processing and slicing. The left hemibrain was cut into two pieces and quickly frozen in two tubes for PK measurement and other target engagement/cytokine analysis.

The plasma and brain levels of human IgG were evaluated at 1 and 4 days after administration and are shown in Table 7 below. Table 7. Plasma and brain pharmacokinetic profile in TB36/hTfR-KI mice TB36 * hTfR-KI plasma brain ATV:RSV [hIgG] uM ATV:188-14 [hIgG] uM ATV:RSV [hIgG] nM ATV:188-14 [hIgG] nM 24 hours 2.96 ± 0.17 1.78 ± 0.073 34.1 ± 3.2 24.4 ± 1.6 96 hours 0.401 ± 0.074 0.29 ± 0.013 13.8 ± 1.8 6.67 ± 1.0 ATV: The effect of TREM2 on the proliferation of microglia

After treatment with No. 3 ATV:TREM2 or ATV:RSV on day 0, four doses of 5-ethynyl-2´-deoxyuridine on day 0, day 1, day 2 and day 3 (EdU, 80 mg/kg) (thymidine analog that can be incorporated into newly synthesized DNA) was administered to mice intraperitoneally.

In order to detect proliferative microglia (EdU+Iba1+), the Click-iT EdU imaging kit (ThermoFisher Scientific, C10637) was used to process the brain sections on the 4th day after administration, and then Iba1 immunostaining was performed. EdU-Iba1 staining showed that ATV:TREM2 significantly increased the number of newborn microglial cells (EdU+ Iba1+; Figure 16A) and the total microglial cell coverage area (Iba1+ area; Figure 16B) in the brain compared with the isotype control. This proves that ATV:TREM2 significantly increases microglial cell proliferation compared to the control. Compared with non- ATV TREM2 antibodies, ATV:TREM2 has a significantly stronger effect on the proliferation of microglial cells in a mouse model

To determine the lowest effective dose of ATV:TREM2 and compare it with non-ATV TREM2 antibodies, a single dose of No. 3 ATV:TREM2 (1, 3, 10, 30 mg/kg) or corresponding TREM2 reference antibody (" TREM2 Ab"; 30 mg/kg), reference antibody No. 2 (30 mg/kg) or isotype control (ATV: RSV; 30 mg/kg) were intravenously administered to TB36/hTfR KI mice. To measure the proliferation of microglial cells, EdU was administered IP to mice on day 0, day 1, day 2, and day 3 after antibody treatment. Mice were taken down on day 1 and day 4 for analysis. EdU-Iba1 double staining showed a dose-dependent increase in neonatal microglia from animals administered with ATV:TREM2 from 1 mg/kg to 10 mg/kg on day 4, of which 10 mg/kg ATV:TREM2 showed the largest pair The effect of microglial cell proliferation (Figure 17A and Figure 17B). In addition, the effect of 1 mg/kg ATV:TREM2 on microglial cell proliferation was slightly higher (not statistically significant) than anti-TREM2 or reference antibody No. 2, even though the administration was up to 30 times higher. ATV: TREM2 transiently increases brain interleukin levels in TB36/hTfR KI mice

To determine the effect of ATV:TREM2 on the levels of cytokines (such as chemokines), a mouse cytokine array/chemokine array 44-Plex (MD44) was used to measure the final plasma of TB36/hTfR KI mice And the level of cytokines in brain lysate (prepared by Cell Signaling lysis buffer number 9803). We found that treatment with No. 3 ATV:TREM2 did not change the level of cytokines measured in plasma, but it drastically increased the levels of some cytokines measured in the brain (such as IP-10 and IP-10) 24 hours after administration. MCP-5), and the levels of these cytokines returned to baseline levels 96 hours after administration. 3 mg/kg ATV:TREM2 had the greatest effect on brain cell interleukin levels (Figure 18A and Figure 18B). ATV: TREM2 increases brain CSF1R levels in TB36/hTfR KI mice

To determine the effect of ATV:TREM2 on the level of the glial marker CSF1R, a commercially available ELISA kit (Abcam ab240681) was used to measure the brain lysates of TB36/hTfR KI mice (prepared by Cell Signaling lysis buffer number 9803) CSF1R protein level in the middle. We found that increasing the dose of No. 3 ATV:TREM2 increased the protein level of CSF1R 1 day after treatment and 4 days after treatment (Figure 19). ATV: plasma PK profile of TREM2

When the dose of No. 3 ATV:TREM2 was in the range of 1 mg/kg to 100 mg/kg, a proportional increase in plasma PK was observed. At 24 hours, there was no significant difference in plasma concentrations of 30 mg/kg No. 3 ATV:TREM2, No. 2 reference antibody and TREM2 Ab at the matched dose. Compared with No. 3 ATV: TREM2 and TREM2 Ab at the same dose, No. 2 reference antibody appeared to have a lower clearance rate (Figure 20). ATV: Overview of the brain PK of TREM2

At 24 hours, the brain concentration of No. 3 ATV:TREM2 at 10 mg/kg is similar to that of TREM2 Ab at 30 mg/kg, and the brain concentration of No. 3 ATV:TREM2 at 1-3 mg/kg is 2 of 30 mg/kg. No. The reference antibody is similar. An out-of-proportional increase was observed at the doses of No. 3 ATV:TREM2 from 3 mg/kg to 10 mg/kg, and a proportional increase was observed at other doses. At 96 hours, the brain concentration of ATV:TREM2 No. 3 at 10 mg/kg was similar to TREM2 Ab and Reference Antibody No. 2 at 30 mg/kg (Figure 21). In short, the brain uptake efficiency of ATV:TREM2 No. 3 is higher than that of TREM2 Ab and No. 2 reference antibody. Table 8. Informal Sequence Listing SEQ ID NO sequence illustrate 1 MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISASLLACEIPFQDTLIPGAAAQDTLIPGL Human TREM2 protein 2 EVKLLDSGGGLVQAGGSLRLSCAGSGFTFTDFYMSWIRQPPGKAPEWLGVIRNKANGYTAGYNPSVKGRFTISRDNTQNILYLQMNTLRAEDTAIYYCARLSYGFDYWGQGVMVTVSS CL0020306 V _H 3 DIVMTQGALPNPVPSGESASITCQSSKSLLHSNGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISSVEAEDVGVYYCQQFLEFPFTFGSGTKLEIK CL0020306 V _L 4 GFTFTDFYMS CL0020306 CDR-H1; CL0020164 CDR-H1; CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3, CL0020188-4, CL0020188-5, CL0020188-6, CL0020188-7 and CL0020188-8 CDR-H1 5 VIRNKANGYTAGYNPSVKG CL0020306 CDR-H2; CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3 and CL0020188-4 CDR-H2 6 ARLSYGFDY CL0020306 CDR-H3 7 QSSKSLLHSNGKTYLN CL0020306 CDR-L1; CL0020164 CDR-L1; CL0020307 CDR-L1; CL0020307-1 CDR-L1; CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-5 and CL0020188-6 CDR-L1 8 WMSTRAS CL0020306 CDR-L2; CL0020307 CDR-L2; CL0020307-1 CDR-L2; CL0020164 CDR-L2; CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3, CL0020188-4, CL0020188-5, CL0020188-6, CDR-L2 of CL0020188-7 and CL0020188-8 9 QQFLEFPFT CL0020306 CDR-L3; CL0020307 CDR-L3; CL0020307-1 CDR-L3 10 EVKLLESGGGLVQPGGSLRLSCAASGFTFTNFYMSWIRQPPGRAPEWLGVIRNRPNGYTTDYNPSVKGRFTISRDNTQNILYLQMSTLRADDTAFYYCTRLTYGFDYWGQGVMVTVSS CL0020307 V _H 11 DIVMTQGALPNPVPSGESASITCQSSKSLLHSNGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISSVEAEVVGVYYCQQFLEFPFTFGSGTKLEIK CL0020307 V _L 12 GFTFTNFYMS CL0020307 CDR-H1 13 VIRNRPNGYTTDYNPSVKG CL0020307 CDR-H2 14 TRLTYGFDY CL0020307 CDR-H3 15 EVKLLDSGGGLVQAGGSLRLSCAGSGFTFTDFYMSWIRQPPGKAPEWLGVIRNKANGYTAGYNPSVKGRFTISRDNTQNILYLQMNTLRAEDTAIYYCARLTYGFDYWGQGVMVTVSS CL0020188 V _H 16 DIVMTQGALPNPVPSGESASITCQSSKSLLHSNGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISSVEAEDVGVYYCQQFLEYPFTFGSGTKLEIK CL0020188 V _L 17 ARLTYGFDY CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3, CL0020188-4, CL0020188-5, CL0020188-6, CL0020188-7 and CL0020188-8 CDR-H3; CL0020164 CDR-H3 18 QQFLEYPFT CDR-L3 of CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3, CL0020188-4, CL0020188-5, CL0020188-6, CL0020188-7 and CL0020188-8 19 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANGYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS CL0020188-1 V _H ; CL0020188-3 V _H 20 DIVMTQTPLSLPVTPGEPASISCQSSKSLLHSNGKTYLNWYLQKPGQSPQLLIYWMSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQFLEYPFTFGQGTKVEIK CL0020188-1 V _L ; CL0020188-2 V _L ; CL0020188-5 V _L ; CL0020188-6 V _L twenty one EVQLVESGGGLVQPGGSLRLSCAGSGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANGYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS CL0020188-2 V _H ; CL0020188-4 V _H twenty two DIVMTQTPLSLPVTPGEPASISCQSSKSLLHSTGKTYLNWYLQKPGQSPQLLIYWMSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQFLEYPFTFGQGTKVEIK CL0020188-3 V _L ; CL0020188-4 V _L ; CL0020188-7 V _L ; CL0020188-8 V _L twenty three QSSKSLLHSTGKTYLN CDR-L1 of variants CL0020188-3, CL0020188-4, CL0020188-7 and CL0020188-8 twenty four EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS CL0020188-5 V _H ; CL0020188-7 V _H 25 VIRNKANAYTAGYNPSVKG CDR-H2 of variants CL0020188-5, CL0020188-6, CL0020188-7 and CL0020188-8 26 EVQLVESGGGLVQPGGSLRLSCAGSGFTFTDFYMSWVRQAPGKGPEWLSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS CL0020188-6 V _H ; CL0020188-8 V _H 27 DIVMTQSPDSLAVSLGERATINCQSSKSLLHSNGKTYLNWYQQKPGQPPKLLIYWMSTRASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFLEFPFTFGQGTKVEIK CL0020307-1 V _L 28 GFTFT- α ₆ -FYMS, where α ₆ is D or N CDR-H1 consensus sequence 29 VIRN- β ₅ - β ₆ -N- β ₈ -YT- β ₁₁ - β ₁₂ -YNPSVKG, where β ₅ is K or R; β ₆ is A or P; β ₈ is G or A; β ₁₁ is A or T; and β ₁₂ is G or D CDR-H2 consensus sequence 30 γ ₁ -RL- γ ₄ -YGFDY, where γ ₁ is A or T; and γ ₄ is T or S CDR-H3 consensus sequence 31 QSSKSLLHS- δ ₁₀ -GKTYLN, where δ ₁₀ is N or T CDR-L1 consensus sequence 32 QQFLE- ϕ ₆ -PFT, where ϕ ₆ is Y or F CDR-L3 consensus sequence 33 000 34 GGGGS Linker sequence 35 HHHHHH 6X-His tags 36 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP Heavy chain constant domain 1 (CH1) 37 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Light chain constant domain (CL) 38 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWETTPPGNVSLTCLVKGFYPSDIAVEWETTPPGNVSLTCLVKGFYPSDIAVEWETTPPGQPENNYKSKTSH Wild-type human Fc sequence Position 231-447 EU index number 39 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWETTPPGNHSKVTVLSKLSKTSHYVKGFYPSDIAVEWETTPPGNHSGVFSVLSKLSKH Fc polypeptide with hole, LALA and LS mutations 40 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVWWESYVSLTCLVKGFYPSDIAVWWESYVSLTCLVKGFYPSGFSKVTLSHYVTSKLSKFSKFSKFFLSKVTHSKFSKFFTSH Pure line 35.21 41 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVWWESYVSLPGLSKLSKFSKVTKSHYVLSKEFSKVTHSKVTLSKEFSKVTHSKFSKFSKF Pure CH3C.35.21 Fc polypeptide with knob, LALA and LS mutations 42 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVWWESYGTEWSSYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPGK Anti-TREM2/CH35.21 heavy chain with club, LALA and LS mutations 43 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVTTPPVCSPGHSKEFFLSKHSKFFLSKFSKFFLSKFSKFSKFSKFYPSQQNQPREPQVYTLPPS Pure line CH3C.35.23.1.1 44 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSQQLSHYPGHSKLSKEFFLSKLSKEFFLSKFSKEFFLSKFSKEFFLSKFSKFFLSK Pure CH3C.35.23.1.1 Fc polypeptide with knob, LALA and LS mutations 45 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESFGTEWSNYKTTPPVLDSDGSFFLYSKLTVSKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPGK Anti-TREM2/CH35.23.1.1 heavy chain with club, LALA and LS mutations 46 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVTTPPVVTLDKLSHYPGHYGLSHYGLSKFSKFSKFSKFSKFSKFYTLPPSRDELTKNQVSLTCLVTSKGFYPSDIAVESYGTEWVNYGFLSKLSKLSHY Pure series CH3C.35.23.3 47 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSQQLSHYGLSHYGLSKLSHYVYTLPPSRDELTKNQVSLWCLVKVTLDKSHYGLSHYGLSKFSKLSKEFFLSKEFFLSK CH3C.35.23.3 Fc polypeptide with knob, LALA and LS mutations 48 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWVNYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPGK Anti-TREM2/CH35.23.3 heavy chain with club, LALA and LS mutations 49 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVTTPPVCSKLSKLSKEFFLSKFSKLSKFSKFSKFFLSKFSKFSKFFLSK Pure series CH3C.35.23.4 50 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSQQLSHYLTHSKLSKEFSKLSKEFFLSKLSKEFFLSKFSKFFLSKEFFLSK CH3C.35.23.4 Fc polypeptide with knob, LALA and LS mutations 51 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWSNYKTTPPVLDSDGSFFLYSKLTVSKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPGK Anti-TREM2/CH35.23.4 heavy chain with club, LALA and LS mutations 52 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK Anti-TREM2 heavy chain with hole and LS mutation 53 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK Anti-TREM2 heavy chain with hole, LALA and LS mutations 54 DIVMTQTPLSLPVTPGEPASISCQSSKSLLHSTGKTYLNWYLQKPGQSPQLLIYWMSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQFLEYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPDNATEKVQSSKASLACEGTASVVCLLNNFYPDNAKVQNSKVATTSLACE Anti-TREM2 light chain 55 NSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDVCRMVTSES Human TfR apical domain 56 GGGGSGGGGS Connector 57 DKTHTCPPCP Part of human IgG1 hinge sequence 58 YxTEWSS CH3 motif (TfR-binding) 59 TxxExxxxF CH3 motif (TfR-binding) 60 Cyno TfR 61 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWETTPPGNHSPGVTSVLKSHYVKGFYPSDIAVEWETTPPGNHSKPENNYKSFFQQH Fc polypeptide with hole and LS mutation 62 Human transferrin receptor protein 1 (TFR1) 63 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWETTPPGNHSPGVTSVLKSHYVKGFYPSDIAVEWETTPPGNHSPGVTSVLKSHYVKGFYPSDIAVEW Truncated Fc polypeptide with hole, LALA and LS mutations 64 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVWWESYVSLWCLVKGFYPSDIAVWWESYVSLPGLSHYVLSKEFSKVTLSKEVTHSGVTHSKVTLSKVTHSKVTHSKVTLSKGFYV Truncated pure CH3C.35.21 Fc polypeptide with knob, LALA and LS mutations 65 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVWWESYGTEWSSYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPG Truncated anti-TREM2/CH35.21 heavy chain with knob, LALA and LS mutations 66 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSQQLSHYPGHSKLSKEFFLSKEFFLSKEFSKEFFLSKEFSKEFFLSKEFSKEFFLSKEFSKEFFLSKEFSKEFFLSK Truncated pure CH3C.35.23.1.1 Fc polypeptide with knob, LALA and LS mutations 67 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESFGTEWSNYKTTPPVLDSDGSFFLYSKLTVSKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPG Truncated anti-TREM2/CH35.23.1.1 heavy chain with knob, LALA and LS mutations 68 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSQQLSHYGLSHYGLSHYGLSHYVYTLPPSRDELTKNQVSLWCLVKVTLSKEVSKEFFLSKEVSKEFFLSSKHYGLSHY Truncated CH3C.35.23.3 Fc polypeptide with knob, LALA and LS mutations 69 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWVNYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPG Truncated anti-TREM2/CH35.23.3 heavy chain with knob, LALA and LS mutations 70 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSQQLSHYLTHSPGVLHSKEVLSKEFFLSKEFSKEFFLSKEFSKEFFLSKEFFLSKFSKFSKFSKFFLSK Truncated CH3C.35.23.4 Fc polypeptide with knob, LALA and LS mutations 71 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWSNYKTTPPVLDSDGSFFLYSKLTVSKEEWQQGFVFSCSVLHEALHSHYTQKSLSLSPG Truncated anti-TREM2/CH35.23.4 heavy chain with knob, LALA and LS mutations 72 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG Truncated anti-TREM2 heavy chain with hole and LS mutation 73 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG Truncated anti-TREM2 heavy chain with hole, LALA and LS mutations 74 QVQLVQSGAEVKKPGASVKVSCKASGYAFSSQWMNWVRQAPGQRLEWIGRIYPGGGDTNYAGKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCARLLRNQPGESYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Reference antibody heavy chain No. 1 75 DVVMTQSPDSLAVSLGERATINCRSSQSLVHSNRYTYLHWYQQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTRVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLIYKVSLDYGSLVTASVVCQSGTLSTKVSSTKVSSTKVSSKV No. 1 and No. 2 reference antibody light chain 76 QVQLVQSGAEVKKPGASVKVSCKASGYAFSSQWMNWVRQAPGQRLEWIGRIYPGGGDTNYAGKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCARLLRNQPGESYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYTQKSLSLSPGK No. 2 reference antibody heavy chain 77 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQPPGKGLEWIGYMSYSGSTRYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Isotype control of reference antibody No. 1 and No. 2, heavy chain 78 EIVMTQSPATLSLSPGERATLSCSASSSVSYMYWYQQKPGQAPRLLIYDTSNLASGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQWSSYPPITFGQGTKVEIKRTVAAPSVFIFPPSTKDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSKDSHQSLGESLGESFYPREAKVQWKVDNALQSGNSQESVTSVTEQSLGESLG Isotype control of No. 1 and No. 2 reference antibodies, light chain 79 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMEPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVWWESYGTEWSSYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLSLSPGK ATV No. 1: Isotype control of TREM2, heavy chain with club and LALA 80 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMEPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWVNYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLSLSPGK No. 3 ATV: Isotype control of TREM2, heavy chain with club and LALA 81 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMEPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK No. 1 and No. 3 ATV: TREM isotype control, with mortar and LALA heavy chain 82 DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALLIYDTSKLASGVPSRFSGSGSG No. 1 and No. 3 ATV: isotype control of TREM2, light chain 83 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGKGLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-TREM2 heavy chain with LALA mutation 84 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWETTPPGNHSPGVTSVLKSHYVKFSKFFLSQQHSPGH Truncated Fc polypeptide with hole and LS mutation

Figures 1A to 1H include dose titration cell binding curves of humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in HEK cells overexpressing human TREM2-Dap12. Figure 2 includes the dose-response binding curves of representative ATV:TREM2 and corresponding anti-TREM2 antibodies with non-transferrin binding Fc ("anti-TREM2") and human TREM2 in HEK 293 cells. Figure 3 includes the dose-response curves of pSyk signal activation of representative ATV:TREM2 and corresponding anti-TREM2 antibodies (TREM2 IgG) in HEK 293 cells expressing TREM2. Figures 4A and 4B include dose-response curves of lipid clearance of iPSC microglia in response to representative ATV:TREM2 treatment. Figures 5A and 5B show representative images of lipid accumulation in iPSC-derived microglial cells treated with ATV:TREM2 after oleic acid challenge (Figure 5A) and the quantification of lipid accumulation in treated cells (Figure 5B) . Figure 5C is a heat map illustrating the level adjustment of triglycerides, carnitine and TCA cycle intermediate substances in iPSC-derived microglial cells treated with ATV:TREM2 after myelin attack. Figures 5D to 5F include bar graphs illustrating representative triglycerides, carnitines and TCA cycle intermediate substances in iPSC-derived microglial cells treated with ATV:TREM2 after myelin attack The level of the level changes. Figures 6A to 6C include bar graphs illustrating the changes in the levels of specific triglycerides, ceramide and carnitine substances in iPSC-derived microglial cells treated with ATV:TREM2 after myelin attack . Figure 7A includes representative images from western blots of mTOR signal pathway targets in iPSC-derived microglial cells treated with ATV:TREM2. Figures 7B to 7E include diagrams illustrating the changes in the level of mTOR signal pathway targets in iPSC-derived microglial cells treated with ATV:TREM2. Figure 8 is a bar graph illustrating the changes in the level of pregranulin (PGRN) in iPSC-derived microglial cells treated with ATV:TREM2. Figure 9 is a bar graph illustrating the changes in the level of representative bis(monoglycerol)phosphate (BMP) substances in iPSC-derived microglial cells treated with ATV:TREM2. Figure 10A is a representative kinetic graph of oxygen consumption in iPSC-derived microglia treated with ATV:TREM2. Figure 10B is a bar graph illustrating the maximum respiratory capacity of iPSC-derived microglial cells treated with ATV:TREM2 in the presence and absence of CPT1 inhibitors. Figure 11A is a dose-response curve of the cell survival rate of human macrophages treated with anti-TREM2 antibody. 11B and 11C illustrated in FIG. 11A comprises the dose - EC50 and bar E of the maximum response curves. Figure 12 is a heat map of relative cytokine release in human macrophages treated with anti-TREM2 antibody. Figures 13A to 13C include volcano graphs illustrating the relative changes of triglyceride substances in iPSC-derived microglial cells treated with anti-TREM2 antibodies. Figures 13D and 13E include bar graphs showing changes in the levels of representative triglycerides in iPSC-derived microglial cells treated with anti-TREM2 antibodies. Fig. 14A is a graph showing the change of TREM2 level with the antibody concentration in the lysate of iPSC-derived microglial cells treated with anti-TREM2 antibody. Figure 14B is a graph showing changes in TREM2 levels with antibody concentration in the cell culture medium of iPSC-derived microglial cells treated with anti-TREM2 antibody. Figure 15 is a graph illustrating the pharmacokinetic profile of anti-TREM2 antibodies administered in cynomolgus monkeys. Figures 16A and 16B are graphs showing ^{EdU +} Iba ⁺ cells/mm ² (Figure 16A) and relative Iba ⁺ area (Figure 16B) in the brain of TB36/hTfR KI mice treated with ATV:TREM2 or ATV:RSV. Figure 17A and Figure 17B ^{show EdU +} Iba ⁺ cells/mm ² (Figure 17A) and relative Iba ⁺ in the brain of TB36/hTfR KI mice treated with ATV: TREM2, corresponding TREM2 antibody, reference antibody No. 2 or ATV: RSV. Map of area (Figure 17B). The graph shows the mean ± SEM and p value: one-way ANOVA using Tukey's multiple comparison test; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Figure 18A and Figure 18B show the cytokines IP-10 (Figure 18A) and MCP-5 (Figure 18A) and MCP-5 ( Figure 18B) Leveling diagram. The graph shows the mean ± SEM across experimental replicates. The graph shows the mean ± SEM and p value: one-way ANOVA using Tukey's multiple comparison test; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Figure 19 is a graph showing the level of glial marker CSF1R in the brain of TB36/hTfR KI mice treated with ATV: TREM2, corresponding TREM2 antibody, reference antibody No. 2 or ATV: RSV. The graph shows the mean ± SEM and p value: one-way ANOVA using Tukey's multiple comparison test; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Figure 20 is a graph showing the plasma PK profile of ATV: TREM2, corresponding TREM2 antibody, reference antibody No. 2 or ATV: RSV in TB36/hTfR KI mice. Figure 21 is a graph showing the brain PK profile of ATV: TREM2, corresponding TREM2 antibody, reference antibody No. 2 or ATV: RSV in TB36/hTfR KI mice.

Claims (102)

An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (a) a variable region comprising: i. CDR-H1 sequence comprising GFTFT- α _The sequence of 6 -FYMS (SEQ ID NO: 28), where α ₆ is D or N; ii. CDR-H2 sequence, which includes VIRN- β ₅ - β ₆ -N- β ₈ -YT- β ₁₁ - β ₁₂ -The sequence of YNPSVKG (SEQ ID NO: 29), wherein β ₅ is K or R; β ₆ is A or P; β ₈ is G or A; β ₁₁ is A or T; and β ₁₂ is G or D; iii CDR-H3 sequence, which includes the sequence of γ ₁ -RL- γ ₄ -YGFDY (SEQ ID NO: 30), wherein γ ₁ is A or T; and γ ₄ is T or S; iv. CDR-L1 sequence, It includes the sequence of QSSKSLLHS- δ ₁₀ -GKTYLN (SEQ ID NO: 31), where δ ₁₀ is N or T; v. CDR-L2 sequence, which includes the sequence WMSTRAS (SEQ ID NO: 8); and vi. CDR- L3 sequence comprising the sequence of QQFLE- ϕ _6- PFT (SEQ ID NO: 32), wherein ϕ ₆ is Y or F; (b) the first Fc polypeptide modified to specifically bind to transferrin receptor; And (c) a second Fc polypeptide. The antibody of claim 1, wherein the CDR-H1 sequence is selected from SEQ ID NO: 4 or 12. The antibody of claim 1 or 2, wherein the CDR-H2 sequence is selected from SEQ ID NO: 5, 13 or 25. The antibody according to any one of claims 1 to 3, wherein the CDR-H3 sequence is selected from SEQ ID NO: 6, 14 or 17. The antibody according to any one of claims 1 to 4, wherein the CDR-L1 sequence is selected from SEQ ID NO: 7 or 23. The antibody according to any one of claims 1 to 5, wherein the CDR-L3 sequence is selected from SEQ ID NO: 9 or 18. The antibody of any one of claims 1 to 6, wherein the variable region comprises: (a) CDR-H1, including the amino acid sequence of SEQ ID NO: 4, CDR-H2, including the amino acid sequence of SEQ ID NO: 5, CDR-H3, including the amino acid sequence of SEQ ID NO: 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or (b) CDR-H1, including the amino acid sequence of SEQ ID NO: 4, CDR-H2, including the amino acid sequence of SEQ ID NO: 5, CDR-H3, including the amino acid sequence of SEQ ID NO: 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or (c) CDR-H1, including the amino acid sequence of SEQ ID NO: 4, CDR-H2, including the amino acid sequence of SEQ ID NO: 25, CDR-H3, including the amino acid sequence of SEQ ID NO: 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or (d) CDR-H1, including the amino acid sequence of SEQ ID NO: 4, CDR-H2, including the amino acid sequence of SEQ ID NO: 25, CDR-H3, including the amino acid sequence of SEQ ID NO: 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or (e) CDR-H1, including the amino acid sequence of SEQ ID NO: 4, CDR-H2, including the amino acid sequence of SEQ ID NO: 5, CDR-H3, including 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, including the amino acid sequence of SEQ ID NO: 12, CDR-H2, including the amino acid sequence of SEQ ID NO: 13, CDR-H3, including 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, including the amino acid sequence of SEQ ID NO: 4, CDR-H2, including the amino acid sequence of SEQ ID NO: 25, CDR-H3, including the amino acid sequence of SEQ ID NO: 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9. The antibody of any one of claims 1 to 7, wherein the variable region comprises a sequence with at least 85% sequence identity with any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26 V _H sequence. The antibody of claim 8, wherein the _VH sequence has at least 90% sequence identity with SEQ ID NO:15. The antibody of claim 9, wherein the _VH sequence has at least 95% sequence identity with SEQ ID NO:15. The antibody of claim 10, wherein the _VH sequence comprises SEQ ID NO:15. The antibody of claim 8, wherein the _VH sequence has at least 90% sequence identity with SEQ ID NO:24. The antibody of claim 12, wherein the _VH sequence has at least 95% sequence identity with SEQ ID NO:24. The antibody of claim 13, wherein the _VH sequence comprises SEQ ID NO:24. The antibody of any one of items 1 to 14 as a request, wherein the variable region comprises SEQ ID NO: 3,11,16,20,22 and 27 of any one having at least 85% sequence identity to the V _L sequence. The requested item of antibody 15, wherein the V _L sequence of SEQ ID NO: 16 having at least 90% of sequence identity. The requested item of antibody 16, wherein the V _L sequence of SEQ ID NO: 16 having at least 95% of sequence identity. 16: The antibody of 17, wherein the V _L sequence comprises SEQ ID NO request entry. The requested item of antibody 15, wherein the V _L sequence of SEQ ID NO: 22 having at least 90% of sequence identity. The requested item of antibody 19, wherein the V _L sequence of SEQ ID NO: 22 having at least 95% of sequence identity. 22: The antibody of claim 20, wherein the V _L sequence comprises SEQ ID NO request entry. The requested item of antibody 15, wherein the V _L sequence of SEQ ID NO: 27 having at least 90% of sequence identity. The antibody of item 22 of the request, wherein the V _L sequence of SEQ ID NO: 27 having a sequence identity of at least 95%. The antibody of item 23 of the request, wherein the V _L sequence comprises SEQ ID NO: 27. The antibody requested item 1 to 7 of any one of, wherein the variable region comprises: (a) comprising SEQ ID NO: V _H and comprising a sequence of 15 SEQ ID NO: V 16 _L of sequence; or (b) comprising SEQ ID NO: V _H sequence 19 of which comprise SEQ ID NO: 20 of the V _L sequence; or (c) comprises SEQ ID NO: V 21 of the _H sequence and comprises SEQ ID NO: 20 of the V _L sequence; or (d ) comprising SEQ ID NO: V 19 of the _H sequence and comprises SEQ ID NO: 22 of the V _L sequence; or (e) comprises SEQ ID NO: V 21 of the _H sequence and comprises SEQ ID NO: 22 of the V _L sequence; or (f) comprises SEQ ID NO: V _H sequence 24 of which comprise SEQ ID NO: V _L sequence 20; or (g) comprises SEQ ID NO: V _H sequence 26 of which comprise SEQ ID NO: 20 of the V _L sequence ; or (h) comprises SEQ ID NO: V _H sequence 24 of which comprise SEQ ID NO: 22 of the V _L sequence; or (i) comprises SEQ ID NO: V _H sequence 26 of which comprise SEQ ID NO: 22 of V _L sequence; or (j) comprises SEQ ID NO: V _H sequence two of which comprise SEQ ID NO: 3 of the V _L sequence; or (k) comprises SEQ ID NO: V _H sequence 10 of which comprise SEQ ID NO: 11 the V _L sequence; or (l) comprising SEQ ID NO: V 24 and the _H sequence comprises SEQ ID NO: V _L sequence of 27. The antibody of any one of claims 1 to 25, wherein according to EU numbering, the first Fc polypeptide comprises: Trp, Leu or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at 387; Trp at 388; Ser, Ala, or Val at 389; Ser or Asn at 390; Thr or Ser at 413; Glu or Ser at 415; Glu at 416 ; And Phe at position 421. The antibody of claim 26, wherein the first Fc polypeptide binds to the apical domain of transferrin receptor. The antibody of claim 26 or 27, wherein the antibody has improved brain uptake compared to an antibody having a wild-type Fc dimer. The antibody of any one of claims 1 to 28, wherein according to EU numbering, the first Fc polypeptide has a T366W substitution and the second Fc polypeptide has a T366S, L368A, and Y407V substitution. The antibody of any one of claims 1 to 28, wherein according to EU numbering, the first Fc polypeptide has T366S, L368A, and Y407V substitutions and the second Fc polypeptide has T366W substitutions. The antibody according to any one of claims 1 to 30, wherein the first Fc polypeptide and/or the second Fc polypeptide comprises a modification that reduces effector function. The antibody of claim 31, wherein according to the EU numbering, the effector function-reducing modification includes the substitution of Ala at position 234 and Ala at position 235. The antibody according to any one of claims 1 to 32, wherein the first Fc polypeptide and/or the second Fc polypeptide comprises an amino acid modification relative to a native Fc sequence that extends serum half-life. Such as the antibody of claim 33, wherein according to the EU numbering, the amino acid modifications include the substitution of Leu at position 428 and Ser at position 434. The antibody of any one of claims 1 to 34, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 41 or SEQ ID NO: 64, and the second Fc polypeptide comprises SEQ ID NO: 39 or SEQ ID NO : The sequence of 63. The antibody of claim 35, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 41; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. The antibody of claim 35, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 64; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 63 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. Such as the antibody of claim 35 or 36, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 42; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. Such as the antibody of claim 35 or 37, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 65; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. The antibody of any one of claims 1 to 34, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 66, and the second Fc polypeptide comprises SEQ ID NO: 39 or SEQ ID NO : The sequence of 63. The antibody of claim 40, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 44; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. The antibody of claim 40, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 66; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 63 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. Such as the antibody of claim 40 or 41, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO:45; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. Such as the antibody of claim 40 or 42, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 67; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. The antibody of any one of claims 1 to 34, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises SEQ ID NO: 39 or SEQ ID NO : The sequence of 63. The antibody of claim 45, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 47; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. The antibody of claim 45, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 68; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 63 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. Such as the antibody of claim 45 or 46, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. Such as the antibody of claim 45 or 47, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. The antibody of any one of claims 1 to 34, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 68, and the second Fc polypeptide comprises SEQ ID NO: 61 or SEQ ID NO : The sequence of 84. The antibody of claim 50, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 47; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 61 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. The antibody of claim 50, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 68; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 84 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. Such as the antibody of claim 50 or 51, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 52; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. Such as the antibody of claim 50 or 52, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 72; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. The antibody of any one of claims 1 to 34, wherein the first Fc polypeptide comprises the sequence of SEQ ID NO: 50 or SEQ ID NO: 70, and the second Fc polypeptide comprises SEQ ID NO: 39 or SEQ ID NO : The sequence of 63. The antibody of claim 55, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 50; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 39 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. The antibody of claim 55, which comprises: (i) a first heavy chain (HC) comprising the V _H comprising SEQ ID NO: 24 and the first Fc polypeptide comprising SEQ ID NO: 70; (ii) a second heavy chain (the HC), which includes comprising SEQ ID NO: V _H 24 and comprising of SEQ ID NO: 63 of the second Fc polypeptide; and (iii) two light chains, each comprising SEQ ID comprising NO: 22 of V _L. Such as the antibody of claim 55 or 56, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 51; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. Such as the antibody of claim 55 or 57, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 71; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 41; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 60, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 42; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 64; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 62, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 65; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 44; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 64, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO:45; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 66; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 66, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 67; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 47; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 68, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 68; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 70, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 47 (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 61; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 72, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 48; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 52; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 68; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 61; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 74, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 69; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 72; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 50; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 39; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 76, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 51; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO:53; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. An isolated antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, wherein the antibody comprises: (i) the first heavy chain (HC), which comprises the V comprising SEQ ID NO: 24 _H and the first Fc polypeptide comprising SEQ ID NO: 70; (ii) the second heavy chain (HC), which comprises the V _H comprising SEQ ID NO: 24 and the second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains, each comprising comprising SEQ ID NO: V _L 22 of. Such as the antibody of claim 78, which contains: (i) The first heavy chain (HC), which includes or consists of the amino acid sequence listed in SEQ ID NO: 71; (ii) The second HC, which includes or consists of the amino acid sequence listed in SEQ ID NO: 73; and (iii) The first and second light chains (LC), each of which includes or consists of the amino acid sequence listed in SEQ ID NO:54. The antibody according to any one of claims 1 to 79, wherein the antibody reduces the level of soluble TREM2 protein (sTREM2). The antibody according to any one of claims 1 to 80, wherein the antibody enhances TREM2 activity. The antibody according to any one of claims 1 to 81, wherein the antibody enhances phagocytosis or enhances the migration, differentiation, function or survival of bone marrow cells, microglia or macrophages. The antibody of any one of claims 1 to 82, wherein the antibody enhances the function of microglia without increasing nerve inflammation. The antibody of any one of claims 1 to 83, wherein the antibody enhances Syk phosphorylation. The antibody of claim 84, wherein the antibody enhances Syk phosphorylation in the presence of a TREM2 ligand. The antibody of any one of claims 1 to 85, wherein the antibody exhibits cross-reactivity with cynomolgus monkey TREM2 protein. A pharmaceutical composition comprising the isolated antibody according to any one of claims 1 to 86 and a pharmaceutically acceptable carrier. A set that contains: The isolated antibody according to any one of claims 1 to 86 or the pharmaceutical composition according to claim 87; and Its instruction manual. A method for treating a neurodegenerative disease in an individual, which comprises administering to the individual the isolated antibody according to any one of claims 1 to 86 or the pharmaceutical composition according to claim 87. Such as the method of claim 89, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, primary age-related tau disease, progressive supranuclear palsy (PSP ), frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic dementia, muscular atrophic lateral sclerosis , Guam type muscular atrophic lateral sclerosis/parkinsonism-dementia complex of Guam (amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, ALS-PDC), cortical basal nucleus degeneration, chronic traumatic encephalopathy, Kuja two Creutzfeldt-Jakob disease, boxer-type dementia, diffuse neurofibrillary tangles with calcification, Down's syndrome, familial British dementia (familial British dementia), familial Denmark Type of dementia (familial Danish dementia), Gerstmann-Straussler-Scheinker disease, globular glial tau disease, Guadeloupe Parkinson's disease with dementia Guadeloupean parkinsonism with dementia, Guadeloupe PSP, Hallevorden-Spatz disease, hereditary diffuse white matter disease with spheroids (HDLS), Huntington's disease (Huntington's disease), inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangles dominant dementia, Niemann-Pick disease type C (Niemann-Pick disease type C) , Globus pallidus-pons-nigra degeneration, Parkinson's disease, Pick's disease, Parkinson's disease after encephalitis, prion protein cerebral amyloid angiopathy, Progressive subcortical glioma, subacute sclerosing panencephalitis, and tangled-only dementia. A method for reducing the level of sTREM2 in an individual suffering from a neurodegenerative disease, which comprises administering to the individual the isolated antibody according to any one of claims 1 to 86 or the pharmaceutical composition according to claim 87. A method for enhancing TREM2 activity in an individual suffering from a neurodegenerative disease, which comprises administering to the individual the isolated antibody according to any one of claims 1 to 86 or the pharmaceutical composition according to claim 87. An isolated polynucleotide comprising a nucleotide sequence encoding an antibody according to any one of claims 1 to 86. An isolated polynucleotide comprising a nucleotide sequence encoding any one of SEQ ID NOs: 42, 45, 48, 51, 53, 54 and 61. An isolated polynucleotide comprising the nucleotide sequences encoding SEQ ID NOs: 42, 53, and 54. An isolated polynucleotide comprising the nucleotide sequences encoding SEQ ID NOs: 45, 53 and 54. An isolated polynucleotide comprising the nucleotide sequences encoding SEQ ID NOs: 48, 53 and 54. An isolated polynucleotide comprising the nucleotide sequences encoding SEQ ID NOs: 48, 52, and 54. An isolated polynucleotide comprising the nucleotide sequence encoding SEQ ID NO: 51, 53, and 54. A vector comprising the polynucleotide according to any one of claims 93 to 99. A host cell comprising the polynucleotide of any one of claims 93 to 99 or the vector of claim 100. A method for expressing an antibody that specifically binds to human trigger receptor 2 (TREM2) expressed on bone marrow cells, comprising: culturing the host cell of claim 101 under conditions suitable for expressing the antibody.

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