JP2023512450A - ANTI-TREM2 ANTIBODY AND METHOD OF USE THEREOF - Google Patents

Abstract

In one aspect, antibodies are provided that specifically bind to a human myeloid cell-expressed trigger receptor 2 (TREM2) protein. In some embodiments, the antibody reduces levels of soluble TREM2 (sTREM2). In some embodiments, the antibody enhances TREM2 activity. [Selection figure] None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on U.S. Provisional Application No. 62/960,663, filed Jan. 13, 2020, U.S. Provisional Application No. 63/070, filed Aug. 26, 2020, 728, and U.S. Provisional Patent Application No. 63/091,717, filed Oct. 14, 2020, the disclosure of which is incorporated herein by reference in its entirety for all purposes. .

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

There remains a need for therapeutic agents that modulate TREM2 activity or levels of sTREM2.

Overview In one aspect, antibodies are provided that specifically bind to human myeloid cell-expressed trigger receptor 2 (TREM2). In some embodiments, the antibody comprises a modified Fc polypeptide capable of binding to transferrin receptor protein. In any of the embodiments disclosed herein, the antibody can comprise CDRs, _VH , and/or _VL according to any of the exemplary sequences provided herein, herein Modified Fc polypeptides containing the described transferrin receptor binding mutations may further be included.

In some embodiments, antibodies that specifically bind TREM2 include:
(a) i. a CDR-H1 sequence comprising the sequence of GFTFT-α ₆ -FYMS (SEQ ID NO: 28), wherein α ₆ is D or N;
ii. VIRN-β ₅ -β ₆ -N-β ₈ -YT-β ₁₁ -β ₁₂ -YNPS-VKG (SEQ ID NO: 29) (in sequence , _β5 is K or R, _β6 is A or P, _β8 is G or A, _β11 is A or T, _β12 is G or D). CDR-H2 sequences,
iii. comprising the sequence γ ₁ -RL-γ ₄ -YGFDY (SEQ ID NO: 30), in which γ ₁ is A or T and γ ₄ is T or S CDR-H3 sequences,
iv. QSSKSXLLHS-δ ₁₀ -GKTYLN (SEQ ID NO: 31) (where δ ₁₀ is N or T) a CDR-L1 sequence comprising the sequence of
v. vi. a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); a variable region comprising a CDR-L3 sequence comprising the sequence QQFLE-φ ₆ -PFT (SEQ ID NO: 32), wherein φ ₆ is Y or F;
(b) a first Fc polypeptide modified to specifically bind to a transferrin receptor; and (c) a second Fc polypeptide.

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

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

In some embodiments, the variable region comprises:
(a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7 , CDR-L2 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, comprising the amino acid sequence of SEQ ID NO:5 CDR-H2, 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 comprising the amino acid sequence of SEQ ID NO: 18. -L3, 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, the amino acid sequence of SEQ ID NO: 7 CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, 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, SEQ ID NO:25 CDR-H2 comprising the amino acid sequence, 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 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, SEQ ID NO:7 CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and sequences CDR-L3 comprising the amino acid sequence of No. 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID No. 4, CDR-H2 comprising the amino acid sequence of SEQ ID No. 25, CDR-H3 comprising the amino acid sequence of SEQ ID No. 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, the variable region comprises a _VH sequence having at least 85% sequence identity to any one of SEQ ID NOs:2, 10, 15, 19, 21, 24, and 26.

In certain embodiments, the _VH sequence has at least 90% sequence identity to SEQ ID NO:15. In certain embodiments, the _VH sequence has at least 95% sequence identity to 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 to SEQ ID NO:24. In certain embodiments, the _VH sequence has at least 95% sequence identity to SEQ ID NO:24. In certain embodiments, the _VH sequence comprises SEQ ID NO:24.

In some embodiments of this aspect, the variable region comprises a V _L sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3, 11, 16, 20, 22, and 27 .

In certain embodiments, the V _L sequence has at least 90% sequence identity to SEQ ID NO:16. In certain embodiments, the V _L sequence has at least 95% sequence identity to SEQ ID NO:16. In certain embodiments, the _VL sequence comprises SEQ ID NO:16.

In certain embodiments, the V _L sequence has at least 90% sequence identity to SEQ ID NO:22. In certain embodiments, the V _L sequence has at least 95% sequence identity to SEQ ID NO:22. In certain embodiments, the _VL sequence comprises SEQ ID NO:22.

In certain embodiments, the V _L sequence has at least 90% sequence identity to SEQ ID NO:27. In certain embodiments, the V _L sequence has at least 95% sequence identity to SEQ ID NO:27. In certain embodiments, the _VL sequence comprises SEQ ID NO:27.

In some embodiments of this aspect, the variable region comprises:
(a) a _VH sequence comprising SEQ ID NO: 15 and a VL sequence comprising SEQ ID NO: 16, or (b) a _VH sequence comprising SEQ ID NO: 19 and a _VL sequence comprising SEQ ID NO: 20, or ₍ c) a sequence a _VH sequence comprising SEQ ID NO:21 and a _VL sequence comprising SEQ ID NO:20; or (d) a _VH sequence comprising SEQ ID NO:19 and a _VL sequence comprising SEQ ID NO:22; or (e) comprising SEQ ID NO:21 a _VH sequence and a VL sequence comprising _SEQ ID NO:22, or (f) a _VH sequence comprising SEQ ID NO:24 and a _VL sequence comprising SEQ ID NO:20, or (g) a _VH sequence comprising SEQ ID NO:26, and SEQ ID NO:20, or (h) a _VH sequence containing SEQ ID NO:24 and a _VL sequence containing SEQ ID NO:22, or (i) a _VH sequence containing _SEQ ID NO:26 and SEQ ID NO:22 or (j) a _VH sequence comprising SEQ ID NO:2 and a _VL sequence comprising SEQ ID NO:3, or (k) a _VH sequence comprising _SEQ ID NO:10 and a VL sequence comprising SEQ ID NO: ₁₁ sequences, or (l) _VH sequences comprising SEQ ID NO:24 and _VL sequences comprising SEQ ID NO:27.

In some embodiments of this aspect, the first Fc polypeptide is Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, according to EU numbering. Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; In some embodiments, the first Fc polypeptide binds to the apical domain of the transferrin receptor. In certain embodiments, the antibody has improved brain uptake compared to an antibody with a wild-type Fc dimer.

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

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

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

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

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 (HC) comprising a _VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 41, (ii) SEQ ID NO: a second heavy chain (HC) comprising a _VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 39, and (iii) two light chains each comprising a _VL comprising SEQ ID NO: 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:64, (ii) SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:42, (ii) the amino acid sequence set forth in SEQ ID NO:53 and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:65, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

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 _VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 44, (ii) SEQ ID NO: a second heavy chain (HC) comprising a _VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 39, and (iii) two light chains each comprising a _VL comprising SEQ ID NO: 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) SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:45, (ii) the amino acid sequence set forth in SEQ ID NO:53 and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:67, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

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) SEQ ID NO: _{(iii) two light chains each comprising a V L comprising} SEQ ID NO: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) SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, (ii) the amino acid sequence set forth in SEQ ID NO:53 and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

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) SEQ ID NO: a second heavy chain (HC) comprising a _VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 61, and (iii) two light chains each comprising a _VL comprising SEQ ID NO: 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) SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:84, and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, (ii) the amino acid sequence set forth in SEQ ID NO:52 and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69, (ii) the amino acid sequence set forth in SEQ ID NO:72. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

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 (HC) comprising a _VH comprising SEQ ID NO: 24 and a first Fc polypeptide comprising SEQ ID NO: 50, (ii) SEQ ID NO: a second heavy chain (HC) comprising a _VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 39, and (iii) two light chains each comprising a _VL comprising SEQ ID NO: 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) SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51, (ii) the amino acid sequence set forth in SEQ ID NO:53 and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In certain embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:71, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a _VH comprising SEQ ID NO:24 and a first heavy chain (HC) comprising a first Fc polypeptide comprising SEQ ID NO:41; (ii) a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39; A second heavy chain (HC) and (iii) two light chains each comprising a _VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:42, (ii) the amino acid sequence set forth in SEQ ID NO:53. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

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

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a _VH comprising SEQ ID NO:24 and a first heavy chain (HC) comprising a first Fc polypeptide comprising SEQ ID NO:44; (ii) a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39; A second heavy chain (HC) and (iii) two light chains each comprising a _VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:45, (ii) the amino acid sequence set forth in SEQ ID NO:53. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a VH comprising SEQ ID NO: 24 and the sequence a first heavy chain (HC) comprising a first Fc polypeptide comprising number 66; (ii) a second comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:67, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising: (i) a _VH comprising SEQ ID NO:24; a first heavy chain (HC) comprising a first Fc polypeptide comprising SEQ ID NO:47, (ii) a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39; A second heavy chain (HC), (iii) comprising two light chains each comprising a _VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, (ii) the amino acid sequence set forth in SEQ ID NO:53. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a VH comprising SEQ ID NO: 24 and the sequence a first heavy chain (HC) comprising a first Fc polypeptide comprising number 68; (ii) a second comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising: (i) a _VH comprising SEQ ID NO:24; a first heavy chain (HC) comprising a first Fc polypeptide comprising SEQ ID NO:47, (ii) a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:61; A second heavy chain (HC) and (iii) two light chains each comprising a _VL comprising SEQ ID NO:22. In some embodiments, (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, (ii) comprising the amino acid sequence set forth in SEQ ID NO:52, or a second HC consisting thereof, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a VH comprising SEQ ID NO: 24 and the sequence (ii) a VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:61; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69, (ii) the amino acid sequence set forth in SEQ ID NO:72. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a _VH comprising SEQ ID NO:24 and a first heavy chain (HC) comprising a first Fc polypeptide comprising SEQ ID NO:50; (ii) a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39; A second heavy chain (HC) and (iii) two light chains each comprising a _VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51, (ii) the amino acid sequence set forth in SEQ ID NO:53. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In another aspect, the disclosure provides an isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), the antibody comprising (i) a VH comprising SEQ ID NO: 24 and the sequence a first heavy chain (HC) comprising a first Fc polypeptide comprising number 70; (ii) a second comprising a VH comprising SEQ ID NO: 24 and a second Fc polypeptide comprising SEQ ID NO: 63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22. In some embodiments, the antibody comprises (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:71, (ii) the amino acid sequence set forth in SEQ ID NO:73. a second HC comprising or consisting of, and (iii) first and second light chains (LC) each comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54.

In some embodiments of any of the aspects delineated herein, the antibody reduces levels of soluble TREM2 protein (sTREM2). In some embodiments, the antibody enhances TREM2 activity. In some embodiments, the antibody enhances phagocytosis or enhances myeloid cell, microglia, or macrophage migration, differentiation, function, or survival. In some embodiments, the antibody enhances microglial function without increasing neuroinflammation. In some embodiments, the antibody enhances Syk phosphorylation. In some embodiments, the antibody enhances Syk phosphorylation in the presence of TREM2 ligand. In some embodiments, the antibody exhibits cross-reactivity with a cynomolgus TREM2 protein.

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

In another aspect, the disclosure provides a kit comprising an isolated antibody described herein or a pharmaceutical composition described herein and instructions for its use.

In another aspect, the disclosure provides a method of treating a neurodegenerative disease in a subject, comprising administering to the subject an isolated antibody described herein or a pharmaceutical composition described herein. Provide a method, including: In some embodiments, the neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontal with chromosome 17-linked parkinsonism Cranial dementia, arginous granule dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis/parkinson dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic Encephalopathy, Creutzfeldt-Jakob disease, Boxer dementia, diffuse neurofibrillary tangles with calcification, Down syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, glial tauopathy globulosa, Guadeloupe parkinsonism with dementia, Guadeloupe PSP, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy (HDLS) with spheroids, Huntington's disease, inclusion body myositis, multiple system atrophy, Myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle predominance dementia, Niemann-Pick disease type C, pontine pulmonary degeneration, Parkinson's disease, Pick's disease, post-encephalitic parkinsonism, prion protein brain amyloid selected from the group consisting of angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle only dementia.

In another aspect, the present disclosure provides a method of reducing levels of sTREM2 in a subject with a neurodegenerative disease, comprising administering an isolated antibody as described herein or a pharmaceutical composition as described herein. A method is provided comprising administering to

In another aspect, the disclosure provides a method of enhancing TREM2 activity in a subject with a neurodegenerative disease, comprising: A method is provided comprising administering.

In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding an antibody described herein.

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

In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:42, 53, and 54.

In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:45, 53, and 54.

In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:48, 53, and 54.

In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:48, 52, and 54.

In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:51, 53, and 54.

In another aspect, the disclosure provides vectors comprising the polynucleotides described herein.

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

In another aspect, the present disclosure provides a method of expressing an antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising a host cell as described herein for expression of said antibody. A method is provided comprising culturing under suitable conditions.

Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Titrated cell binding curves for humanized and sequence-optimized variants of a representative anti-TREM2 antibody (CL0020188) in human TREM2-Dap12 overexpressing HEK cells are shown. Dose-response binding curves of representative ATV:TREM2 and corresponding anti-TREM2 antibodies with non-transferrin binding Fc (“anti-TREM2”) to human TREM2 in HEK293 cells are shown. Dose-response binding curves of pSyk signaling activation by representative ATV:TREM2 and corresponding anti-TREM2 antibodies (TREM2 IgG) in TREM2-expressing HEK293 cells are shown. A representative dose-response curve of lipid clearance in iPSC microglia in response to treatment with ATV:TREM2 is shown. A representative dose-response curve of lipid clearance in iPSC microglia in response to treatment with ATV:TREM2 is shown. Representative images of lipid accumulation in iPSC-derived microglial cells treated with ATV:TREM2 after oleate stimulation are shown. Quantitation of lipid accumulation in treated cells is shown. FIG. 4 is a heatmap showing regulation of levels of triglycerides, acylcarnitines, and TCA cycle intermediate species in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. FIG. 10 shows bar graphs showing changes in representative triglyceride species levels in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. FIG. 10 shows bar graphs showing changes in representative acylcarnitine species levels in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. Bar graphs showing changes in levels of representative TCA cycle intermediate species levels in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. FIG. 13 shows a bar graph showing changes in levels of specific triglyceride species in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. FIG. 10 shows bar graphs showing changes in levels of specific ceramide species in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. FIG. 10 shows a bar graph showing changes in levels of specific acylcarnitine species in iPSC-derived microglial cells treated with ATV:TREM2 after myelin stimulation. Representative images of Western blots of mTOR signaling pathway targets in iPSC-derived microglia treated with ATV:TREM2. Plots showing changes in levels of mTOR signaling pathway targets in iPSC-derived microglia treated with ATV:TREM2 are shown. Plots showing changes in levels of mTOR signaling pathway targets in iPSC-derived microglia treated with ATV:TREM2 are shown. Plots showing changes in levels of mTOR signaling pathway targets in iPSC-derived microglia treated with ATV:TREM2 are shown. Plots showing changes in levels of mTOR signaling pathway targets in iPSC-derived microglia treated with ATV:TREM2 are shown. FIG. 4 is a bar graph showing changes in progranulin (PGRN) levels in iPSC-derived microglia treated with ATV:TREM2. Graph showing changes in levels of representative bis(monoacylglycero)phosphate (BMP) species in iPSC-derived microglia treated with ATV:TREM2. Representative kinetic graphs of oxygen consumption in iPSC-derived microglia treated with ATV:TREM2. Bar graph showing maximal respiratory capacity of iPSC-derived microglial cells treated with ATV:TREM2 in the presence and absence of CPT1 inhibitor. Dose-response curve of cell viability in human macrophage cells treated with anti-TREM2 antibody. FIG. 11B shows a bar graph showing the EC50 of the dose-response curve in FIG. 11A. FIG. 11B shows a bar graph showing the Emax of the dose-response curve in FIG. 11A. Heat map of relative cytokine release in human macrophage cells treated with anti-TREM2 antibody. Volcano plots showing relative changes in triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibody are shown. Volcano plots showing relative changes in triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibody are shown. Volcano plots showing relative changes in triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibody are shown. FIG. 10 shows bar graphs showing changes in levels of representative triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibody. FIG. 10 shows bar graphs showing changes in levels of representative triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibody. Plot of TREM2 levels versus antibody concentration in cell lysates of iPSC-derived microglial cells treated with anti-TREM2 antibodies. Plot of TREM2 levels versus antibody concentration in cell culture medium of iPSC-derived microglial cells treated with anti-TREM2 antibody. FIG. 4 is a plot showing the pharmacokinetic profile of anti-TREM2 antibodies administered to cynomolgus monkeys. A and B are plots showing EdU ⁺ Iba ⁺ cells per mm ² (A) and relative Iba ⁺ area (B) in the brains of TB36/hTfR KI mice treated with either ATV:TREM2 or ATV:RSV. is. A and B, EdU ⁺ Iba ⁺ cells per mm ² in the brain of TB36/hTfR KI mice treated with either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV (A). and relative Iba ⁺ area (B). Graphs show mean±SEM and p-values: one-way ANOVA with Tukey's multiple comparison test; *p≦0.05, **p≦0.01, ***p≦0.001, *** p < 0.0001. A and B, Cytokine IP-10 levels (A) and cytokine MCP-10 levels in the brain of TB36/hTfR KI mice treated with either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV. Plot showing 5 levels (B). Graphs show mean ± SEM of replicate experiments. Graphs show mean±SEM and p-values: one-way ANOVA with Tukey's multiple comparison test; **p≦0.01, ***p≦0.001, ***p≦0.0001. Plot showing glial marker CSF1R levels in the brain of TB36/hTfR KI mice treated with either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV. Graphs show mean±SEM and p-values: one-way ANOVA with Tukey's multiple comparison test; *p≦0.05, **p≦0.01, ***p≦0.001, *** p < 0.0001. FIG. 4 is a plot showing plasma PK profiles of either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV in TB36/hTfR KI mice. Plot showing the brain PK profiles of either ATV:TREM2, the corresponding TREM2 antibody, reference antibody #2, or ATV:RSV in TB36/hTfR KI mice.

DETAILED DESCRIPTION I. INTRODUCTION TREM2 is a transmembrane receptor expressed on the cell surface of microglia, dendritic cells, macrophages, and osteoclasts. Without wishing to be bound by theory, after ligand binding, TREM2 forms a signaling complex with the transmembrane adapter protein DNAX-activating protein 12 (DAP12), which is then tyrosine-phosphorylated by the protein kinase SRC. believed to be oxidized. The activated TREM2/DAP12 signaling complex is believed to mediate intracellular signaling by recruiting and phosphorylating kinases such as Syk kinase. TREM2/DAP12 signaling regulates activities such as phagocytosis, cell proliferation and survival, inflammatory cytokine secretion, and migration of cells such as microglia and macrophages. TREM2 undergoes regulated intramembrane proteolysis, in which the membrane-bound full-length TREM2 is shed from the cell by the metalloprotease ADAM10, and the membrane-retained C-terminal fragment is further degraded by gamma-secretase. and is disconnected. Altered levels of sTREM2 have been reported in patients with Alzheimer's disease or frontotemporal dementia and with mutations in TREM2. In addition, TREM2 mutations are associated with altered function such as impaired phagocytosis and reduced microglial function.

As detailed in the Examples section below, antibodies were generated that specifically bind to human TREM2 and modulate one or more downstream functions of the TREM2/DAP12 signaling complex. In certain embodiments, the antibody further comprises an Fc polypeptide that contains mutations that allow the Fc polypeptide to bind to a transferrin receptor (eg, TfR from humans). In some embodiments, the antibodies disclosed herein bind to transferrin receptor proteins (e.g., those expressed on the surface of brain endothelial cells (BECs)) via modified Fc polypeptides. and thereby cross the blood-brain barrier (BBB) more effectively than antibodies lacking TfR-binding Fc mutations. In certain embodiments, the antibodies disclosed herein have mutations in the Fc polypeptide that reduce or eliminate effector function and, for example, binding of the antibody Fc to the Fc fetal receptor (FcRn). Including mutations that increase in vivo half-life by increasing.

In some embodiments, an anti-TREM2 antibody enhances TREM2 activity (eg, enhances phagocytosis or enhances myeloid cell, microglia, or macrophage differentiation, function, migration, or survival). Accordingly, in another aspect, methods of enhancing TREM2 activity, eg, in a subject having a neurodegenerative disease, are provided.

In some embodiments, the anti-TREM2 antibody reduces shedding of sTREM2. Accordingly, in another aspect, methods are provided for reducing levels of sTREM2, eg, in a subject having a neurodegenerative disease.

II. DEFINITIONS As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include , which includes multiple referents. Thus, for example, reference to an "antibody" optionally includes combinations of two or more such molecules.

As used herein, the terms "about" and "approximately," when used to modify a specified quantity in a numerical value or range, are reasonable deviations from that numerical value and values known to those of ordinary skill in the art. Deviations such as ±20%, ±10% or ±5% are indicated to be within the intended meaning of the stated values.

As used herein, the term "TREM2 protein" refers to the myeloid cell-expressed trigger receptor 2 protein encoded by the gene TREM2. As used herein, a "TREM2 protein" is any vertebrate, including, but not limited to, humans, non-human primates (e.g., cynomolgus monkeys), rodents (e.g., mice, rats). ), and other mammalian native (ie, wild-type) TREM2 proteins. In some embodiments, the TREM2 protein is a human TREM2 protein having the sequence identified in UniprotKB Accession No. Q9NZC2 (SEQ ID NO: 1).

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

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

As used herein, the term "anti-TREM2 antigen-binding portion" refers to an antigen-binding segment or whole that specifically binds to a TREM2 protein (eg, human TREM2). The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and are one that retains the ability to specifically bind to an antigen (e.g., TREM2 protein) via its variable region. It refers to fragments of the above antibodies. Examples of antigen-binding fragments include Fab fragments (a monovalent fragment consisting of the V _L , V _H , CL and CH1 domains), F(ab′) ₂ fragments (from two Fab fragments linked by a disulfide bridge at the hinge region). composed bivalent fragment), single-chain Fv (scFv), disulfide-bonded Fv (dsFv), complementarity determining regions (CDRs), _VL (light chain variable region), and _VH (heavy chain variable region) include, but are not limited to.

The term "variable region" or "variable domain" is derived from a germline variable (V) gene, a diversity (D) gene, or a joining (J) gene (and from constant (Cμ and Cδ) gene segments). (without) refers to the domain in an antibody heavy or light chain that confers antigen-binding specificity on an antibody. Antibody variable regions typically comprise four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain interrupting the four framework regions constructed by the light and heavy chain variable regions. CDRs are primarily responsible for antibody binding to epitopes on antigens. The CDRs of each chain are usually numbered sequentially from the N-terminus as CDR1, CDR2 and CDR3 and are also usually identified by the chain on which the particular CDR is located. Thus, the V _H CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain in which it is found, while the V _L CDR1 or CDR-L1 is the CDR1 derived from the variable region of the antibody light chain in which it is found. is.

The “framework regions” or “FRs” of different light or heavy chains are relatively conserved within species. The framework region of an antibody, ie, the combination of the constituent light and heavy chain framework regions, functions to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences for human heavy and light chain variable region genes can be found at the "VBASE2" germline variable gene sequence database for human and mouse sequences.

The amino acid sequences of the CDRs and framework regions are determined using various well-known definitions in the art, e.g. can be determined. In some embodiments, CDRs are determined according to the Contact definition. MacCallum et al. , J. Mol. Biol. , 262:732-745 (1996). In some embodiments, CDRs are determined by a combination of Kabat, Chothia, and/or Contact CDR definitions.

The term "epitope" refers to the segment or region of an antigen to which the CDRs of an antibody specifically bind, and can be a few amino acids or a portion of a few amino acids, such as 5 or 6 or more, such as 20. more than one amino acid, or a portion of those amino acids. For example, if the target is a protein, the epitope may consist of contiguous amino acids (e.g., linear epitopes) or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., discontinuous or conformational epitopes). can be In some embodiments, the epitope is phosphorylated on one amino acid (eg, on a serine or threonine residue).

As used herein, the phrase "recognizes an epitope," when used in reference to an anti-TREM2 antibody, means that the antibody CDRs recognize the antigen (i.e., the TREM2 protein) and the antigen at or containing the epitope thereof. interacting at or specifically binding to a portion of

A "monoclonal antibody" refers to an antibody produced by a single clone or cell line of cells and consisting or consisting essentially of antibody molecules identical to their primary amino acid sequence.

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

A "chimeric antibody" has an altered, substituted, or exchanged constant region or portion thereof such that the antigen binding site (i.e., variable region, CDRs, or portion thereof) is linked to constant regions of different or altered classes, effector functions, and/or species, or variable regions or portions thereof having different or altered antigen specificities (e.g., CDRs and Refers to an antibody molecule that has been altered, substituted, or replaced by a variable region with a framework region). In some embodiments, chimeric antibodies are monoclonal antibodies comprising variable regions from one source or species (e.g., mouse) and constant regions from another source or species (e.g., human). be. Methods for making chimeric antibodies have been described in the art.

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

A "human antibody" or "fully human antibody" is an antibody that has human heavy and light chain sequences typically derived from human germline genes. In some embodiments, antibodies are produced by human cells, by non-human animals utilizing human antibody repertoires (e.g., transgenic mice genetically engineered to express human antibody sequences), or by phage display platforms. be done.

The term "specifically binds" refers to an epitope or target in a sample with a higher affinity and a higher affinity than binding to another epitope or a non-target compound (e.g., a structurally distinct antigen). Refers to a molecule (eg, an antibody or antigen-binding portion thereof) that binds an epitope or target with high avidity and/or for a longer duration. In some embodiments, an antibody (or antigen-binding portion thereof) that specifically binds to an epitope or target has at least 5-fold higher affinity than other epitopes or non-target compounds, e.g., at least 5-fold, 10-fold , 100-fold, 1,000-fold, or 10,000-fold or greater affinity to an epitope or target. As used herein, the terms “specific binding to”, “specifically binds to” or “specific for” a particular epitope or target refer to, for example, the epitope or target that binds for example, 10 ⁻⁴ M or less, such as 10 ⁻⁵ M, 10 ⁻⁶ M, 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, or 10 It can be demonstrated by a molecule with an equilibrium dissociation constant K _D of ⁻¹² M. One skilled in the art will appreciate that an antibody that specifically binds a target (eg, a TREM2 protein) from one species may also specifically bind an ortholog of that target (eg, a TREM2 protein).

The term "binding affinity" is used herein to refer to the strength of non-covalent interactions between two molecules, eg, an antibody (or antigen-binding portion thereof) and an antigen. Thus, for example, unless indicated otherwise, or clear from context, the term can refer to a 1:1 interaction between an antibody (or antigen-binding portion thereof) and antigen. Binding affinity can be quantified by measuring the equilibrium dissociation constant (K _D ) ^, which is the ^dissociation rate _constant (k _d , time ⁻¹ ). _KD analyzes the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (e.g. Biacore™ systems), binding equilibrium exclusion methods such as KinExA®, and BioLayer interferometry (e.g. , using the ForteBio® Octet platform). As used herein, "binding affinity" refers not only to formal binding affinity as reflecting a 1:1 interaction between an antibody (or antigen-binding portion thereof) and antigen, but also to It also includes apparent affinities from which _KD values are calculated that may reflect binding.

The term "cross-react," as used herein, refers to the ability of an antibody to bind to an antigen other than the antigen with which it was raised. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from a different species than the antigen with which the antibody was raised. As a non-limiting example, an anti-TREM2 antibody described herein raised against a human TREM2 peptide exhibits cross-reactivity with TREM2 peptides or proteins from different species (eg, monkey or mouse). obtain.

As used herein, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO:62. Transferrin receptor protein 1 sequences from other species are also known (e.g. chimpanzee: accession number XP_003310238.1, rhesus monkey: NP_001244232.1, dog: NP_001003111.1, bovine: NP_001193506.1, mouse: NP_035768 .1, rat: NP_073203.1, and chicken: NP_990587.1). The term "transferrin receptor" also encompasses an exemplary reference sequence encoded by a gene at the transferrin receptor protein 1 chromosomal locus, eg, allelic variants of the human sequence. The full-length transferrin receptor protein contains 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 apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO:55.

As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of IgG antibodies, a CH3 domain polypeptide refers to the segment of amino acids around position 341 to around position 447, numbered according to the EU numbering scheme, and a CH2 domain polypeptide, when numbered according to the EU numbering scheme. , refers to the segment of amino acids from around position 231 to around position 340. CH2 and CH3 domain polypeptides can also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, according to the IMGT Scientific chart numbering (IMGT website), CH2 domain numbering is 1-110, CH3 domain numbering is The numbers are 1-107. The CH2 and CH3 domains are part of the Fc region of immunoglobulins. In the context of IgG antibodies, the Fc region refers to the amino acid segment around position 231 to around position 447 when 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 set forth in SEQ ID NO:57.

When used in the context of identifying a given amino acid residue in a polypeptide sequence, "corresponding to", "determined with reference to", or "numbered with reference to" The term refers to the residue position of a particular reference sequence when a given amino acid sequence is compared to the reference sequence with maximal alignment. Thus, for example, when optimally aligned to SEQ ID NO:38, the amino acid residues in the polypeptide are "to" the region of amino acids 111-217 of SEQ ID NO:38 when the residues align with the amino acids of SEQ ID NO:38. handle". A polypeptide aligned to a 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 native immunoglobulin heavy chain polypeptide, featuring the Ig fold as the structural domain. An Fc polypeptide contains a constant region sequence comprising at least the CH2 and/or CH3 domains, and may contain at least part of the hinge region, but not the variable region.

A "modified Fc polypeptide" has at least one mutation, e.g., substitution, deletion or insertion, as compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but has the overall Refers to Fc polypeptides that retain the Ig fold or structure.

The term "isolated" when used in reference to a nucleic acid or protein (eg, an antibody) indicates that the nucleic acid or protein is essentially free of other cellular components with which it is associated in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (eg, polyacrylamide gel electrophoresis) or chromatography (eg, high performance liquid chromatography). In some embodiments, an 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 naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code, as well as those to which those amino acids have been later modified, eg, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Natural α-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile ), Arginine (Arg), Lysine (Lys), Leucine (Leu), Methionine (Met), Asparagine (Asn), Proline (Pro), Glutamine (Gln), Serine (Ser), Threonine (Thr), Valine (Val ), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of natural α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu ), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser) , D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. "Amino acid analogue" means a compound having the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen, carboxyl group, amino group, and an alpha carbon bonded to the R group, e.g., homoserine, norleucine, methionine sulfoxide, It refers to methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to chemical compounds that have structures that differ from the general chemical structure of amino acids, but that function in a manner similar to naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as naturally occurring and non-naturally occurring amino acid polymers. Amino acid polymers can comprise all L-amino acids, all D-amino acids, or a mixture of L-amino acids and D-amino acids.

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

The terms "polynucleotide" and "nucleic acid" refer interchangeably to chains of nucleotides of any length, and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogues thereof, or any substrate that can be incorporated into a strand by a DNA or RNA polymerase. A polynucleotide may comprise 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 single- and double-stranded DNA and single-stranded DNA. Hybrid molecules having a mixture of RNA and double-stranded RNA are included.

The terms "conservative substitution", "conservative mutation" refer to changes that result in the replacement of one amino acid by another amino acid that can be classified as having similar characteristics. Examples of classifications of conservative amino acid groups thus defined include Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), as well as 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 specified. For example, groups of charged or polar amino acids are a "positively charged subgroup" made up of Lys, Arg and His, a "negatively charged subgroup" made up of Glu and Asp, and a "negatively charged subgroup" made up of Asn and Gln. can be subdivided into subgroups, including the "polar subgroup" In another example, the aromatic or cyclic group is subdivided into subgroups including the "nitrogen ring subgroup" consisting of Pro, His and Trp, and the "phenyl subgroup" consisting of Phe and Tyr. can be done. In yet another example, the aliphatic groups are subgroups such as the "aliphatic non-polar subgroup" consisting of Val, Leu, Gly and Ala, and the "aliphatic nonpolar subgroup" consisting of Met, Ser, Thr and Cys. can be subdivided into a family of "slightly polar subgroups". Examples of classes of conservative mutations include, but are not limited to, amino acid substitutions of amino acids within the aforementioned subgroups, such as, but not limited to, Lys for Arg or vice versa, such that a positive charge can be maintained; Glu instead of Asp or vice versa, such that _a such as Gln instead of Asn or vice versa. In some embodiments, hydrophobic amino acids are substituted for naturally-occurring hydrophobic amino acids, eg, in the active site, to retain their hydrophobicity.

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

For polypeptide sequence comparison, typically one amino acid sequence acts as a reference sequence, to which candidate sequences are compared. To obtain maximal alignment, the alignment can be performed using a variety of methods available to those skilled in the art, such as visual alignment or publicly available software using known algorithms. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for alignment to obtain maximal alignment can be determined by one skilled in the art. Sequence comparisons of polypeptide sequences for the purposes of this application use the BLASTP algorithm standard protein BLAST to align two protein sequences using default parameters.

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

As used herein, the terms "treat," "treatment," and the like generally mean obtaining a desired pharmacological and/or physiological effect. "Treat" or "treatment" means alleviation, amelioration, improvement in patient survival, increase in survival time or survival rate, reduction of symptoms or making the disease more tolerable by the patient, degeneration or decline. Neurodegenerative diseases (e.g., Alzheimer's disease or other neurological diseases as described herein), including all objective and subjective parameters such as slowing or improving the physical or mental well-being of the patient. It can refer to any indication of success in treating or ameliorating a degenerative disease). Treatment or amelioration of symptoms may be based on objective or subjective parameters. The effect of treatment can be compared to an untreated individual or pool of individuals, or to the same patient at different time points before or during treatment.

The term "pharmaceutically acceptable excipient" means an inactive pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals such as, but not limited to, buffers, carriers, or preservatives. point to

As used herein, a “therapeutic amount” or “therapeutically effective amount” of an agent (e.g., an antibody described herein) treats or reduces the severity of symptoms of a disease in a subject, It is the amount of drug that alleviates or reduces. A "therapeutic amount" of an agent (e.g., an antibody described herein) is defined as improving patient survival, increasing survival time or survival rate, reducing symptoms, damage, disease or condition (e.g., neurodegenerative disease). ) can be made more tolerable, the rate of degeneration or decline slowed, or the physical or mental well-being of the patient can be improved.

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

The terms "control" or "control value" refer to a reference or baseline value. Appropriate controls can be determined by one skilled in the art. In some cases, a control value may be determined relative to a baseline within the same subject or experiment, e.g., a measurement of sTREM2 taken prior to treatment with an anti-TREM2 antibody is equivalent to treatment of sTREM2 levels in the same subject. It can be a control value for a post-measurement value. In other cases, the control value is relative to a control subject (e.g., healthy or diseased controls) or a population of control subjects (e.g., healthy or diseased controls, e.g., 10, 20, 50, 100, 200, A population of 500, 1000 or more control subjects), e.g., measured sTREM2 levels in subjects, either at baseline or after treatment, compared to healthy control values can be

III. Anti-TREM2 Antibodies In one aspect, antibodies are provided that specifically bind to a TREM2 protein. In some embodiments, the antibody specifically binds to human TREM2 protein. In some embodiments, the anti-TREM2 antibody is selective for TREM2 over other TREM-like receptors (eg, TREM1).

In some embodiments, the anti-TREM2 antibody is an antibody comprising one or more complementarity determining region (CDR) sequences, heavy chain variable region sequences, and/or light chain variable region sequences disclosed herein. be. In some embodiments, an anti-TREM2 antibody comprises one or more CDR sequences, heavy chain variable region sequences, and/or light chain variable region sequences disclosed herein, an antibody that enhances one or more functional properties, e.g., TREM2 activity (e.g., enhances phagocytosis or enhances migration, differentiation, function or survival of cells such as myeloid cells, microglia, or macrophages), or Further included are antibodies that decrease the level of sTREM2. In some embodiments, an anti-TREM2 antibody comprises an Fc polypeptide with one or more modifications described herein.

In some embodiments, the anti-TREM2 antibody is a chimeric antibody. In some embodiments, the anti-TREM2 antibody is a humanized and/or affinity matured antibody.

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

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 to any one of the amino acid sequences of SEQ ID NOs: 4 and 12, or up to two amino acid substitutions to any one of SEQ ID NOs: 4 and 12; a heavy chain CDR1 (CDR-H1) sequence having
(b) has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 13 and 25, or to the amino acid sequence of any one of SEQ ID NOs: 5, 13 and 25; a heavy chain CDR2 (CDR-H2) sequence having up to two amino acid substitutions in
(c) has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 6, 14 and 17, or to the amino acid sequence of any one of SEQ ID NOs: 6, 14 and 17; a heavy chain CDR3 (CDR-H3) sequence having up to two amino acid substitutions in
(d) have at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 7 and 23, or up to two amino acid substitutions to the amino acid sequence of any one of SEQ ID NOs: 7 and 23; a light chain CDR1 (CDR-L1) sequence having
(e) having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NO:8 or having up to two amino acid substitutions to any one of SEQ ID NO:8; and (f) at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs:9 and 18, or any one of SEQ ID NOs:9 and 18 A light chain CDR3 (CDR-L3) sequence with up to two amino acid substitutions relative to the amino acid sequence.

In some embodiments, the anti-TREM2 antibody comprises two, three, four, five or all six of (a)-(f). In some embodiments, the anti-TREM2 antibody comprises (a) CDR-H1, (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, a CDR with up to 2 amino acid substitutions has 1 amino acid substitution relative to the reference sequence. In some embodiments, a CDR with up to 2 amino acid substitutions has 2 amino acid substitutions relative to the reference sequence. In some embodiments, 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) a CDR-H1 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 4 and 12;
(b) a CDR-H2 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 5, 13 and 25;
(c) a CDR-H3 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 6, 14 and 17;
(d) a CDR-L1 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 7 and 23;
(e) a CDR-L2 sequence comprising the amino acid sequence of any one of SEQ ID NO:8; and (f) a CDR-L3 sequence comprising 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)-(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 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7 , CDR-L2 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, comprising the amino acid sequence of SEQ ID NO:5 CDR-H2, 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 comprising the amino acid sequence of SEQ ID NO: 18. -L3, 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, the amino acid sequence of SEQ ID NO: 7 CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, 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, SEQ ID NO:25 CDR-H2 comprising the amino acid sequence, 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 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, SEQ ID NO:7 CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and sequences CDR-L3 comprising the amino acid sequence of No. 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID No. 4, CDR-H2 comprising the amino acid sequence of SEQ ID No. 25, CDR-H3 comprising the amino acid sequence of SEQ ID No. 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, the anti-TREM2 antibody is at least 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. 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 is at least 85%, 90%, 91%, 92%, 93%, Light chain variable regions comprising amino acid sequences having 94%, 95%, 96%, 97%, 98% or 99% sequence identity are included. 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 is at least 85%, 90%, 91%, 92%, 93 A heavy chain variable region comprising an amino acid sequence having %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and SEQ ID NOs: 3, 11, 16, 20, 22, and 27 amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of A light chain variable region comprising In some embodiments, the anti-TREM2 is 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 SEQ ID NOs: 3, 11, 16, A light chain variable region comprising any one of 20, 22 and 27 amino acid sequences.

In some embodiments, the anti-TREM2 antibody comprises:
(a) a _VH sequence having at least 85% sequence identity to SEQ ID NO:2 and a _VL sequence having at least 85% sequence identity to SEQ ID NO:3; or (b) to SEQ ID NO:10. and a V _L sequence having at least 85% sequence identity to SEQ ID NO:11, or (c) at least 85% sequence identity _to SEQ ID NO:15. and (d) a _VH sequence having at least 85% sequence identity to SEQ ID NO: 19 _, and a V _L sequence having at least 85% sequence identity to SEQ ID NO: 19. , and a V _L sequence having at least 85% sequence identity to SEQ ID NO:20, or (e) a _VH sequence having at least 85% sequence identity to SEQ ID NO:21, and to SEQ ID NO:20 or (f) a _VH sequence having at least 85% sequence identity to SEQ ID NO:19 and at _least 85% sequence identity to SEQ ID NO:22. or (g) a V _H sequence having at least 85% sequence identity to SEQ ID NO _: 21 and a V _L sequence having at least 85% sequence identity to SEQ ID NO:22; or (h) a _VH sequence having at least 85% sequence identity to SEQ ID NO:24 and a _VL sequence having at least 85% sequence identity to SEQ ID NO:20, or (i) SEQ ID NO:26 and a V _L sequence having at least 85% sequence identity to SEQ ID NO:20, or (j) at least ₈₅ % sequence identity to SEQ ID NO:24 a _VH sequence having sequence identity and a _VL sequence having at least 85% sequence identity to SEQ ID NO:22; or (k) a VH sequence having at least 85% sequence identity to SEQ ID NO:26 _. and a V _L sequence having at least 85% sequence identity to SEQ ID NO:22, or (l) a _VH sequence having at least 85% sequence identity to SEQ ID NO:24, and SEQ ID NO:27. A _VL sequence that has at least 85% sequence identity to.

In some embodiments, an 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 (eg, CDRs) to antibodies from the same (or similar) germline. In some embodiments, consensus sequences can be generated from antibodies containing sequences of the same (or similar) length and/or sequences with at least one very similar CDR (e.g., very similar CDR3) . In some embodiments, such sequences in the antibodies contain conserved amino acids or motifs (i.e., where changes in the sequence can alter protein function) and/or regions where mutations are present in the sequence (i.e. , where sequence variation is unlikely to significantly affect protein function) can be aligned and compared. Alternatively, the consensus sequence aligns heavy or light chain sequences (e.g., CDRs) to antibodies that bind the same or similar (e.g., overlapping) epitopes to conserved amino acids or motifs (i.e., , where changes in sequence can alter protein function) and regions where mutations are present in alignments of sequences (i.e., where sequence mutations are unlikely to significantly affect protein function). obtain. In some embodiments, one or more consensus sequences can be identified for antibodies that recognize the same or similar epitopes as the anti-TREM2 antibodies disclosed herein. Exemplary consensus sequences include SEQ ID NOs:28-32. In the consensus sequences of SEQ ID NOs:28-32, capital letters represent amino acid residues that are fully conserved among the aligned sequences (e.g., aligned CDR sequences), while capital letters "X" or Greek letters (e.g., , “α”, “β”, “γ”, “δ”, “ε”, or “φ”) represent amino acid residues that are not completely conserved among the aligned sequences. When selecting an amino acid for insertion at a position marked by an "X" or Greek letter, in some embodiments the amino acid may be selected from those found at the corresponding position in the aligned sequences. will be understood.

Variants of Clones CL0020188, CL0020306, CL0020307, and CL0020188 In some embodiments, the anti-TREM2 antibody comprises:
(a) a CDR-H1 sequence comprising the sequence of GFTFT-α ₆ -FYMS (SEQ ID NO: 28), where α ₆ is D or N;
(b) VIRN-β ₅ -β ₆ -N-β ₈ -YT-β ₁₁ -β ₁₂ -YNPSVKG (SEQ ID NO: 29) (wherein _β5 is K or R, _β6 is A or P, _β8 is G or A, _β11 is A or T, and _β12 is G or D) a CDR-H2 sequence comprising a sequence;
(c) γ ₁ -RL-γ ₄ -YGFDDY (SEQ ID NO: 30) (where γ ₁ is A or T and γ ₄ is T or S) a CDR-H3 sequence comprising a sequence;
(d) QSSKSLHLHS-δ ₁₀ -GKTYLN (SEQ ID NO: 31) (where δ ₁₀ is N or T a CDR-L1 sequence comprising the sequence of
(e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); and (f) QQFLE-φ ₆ -PFT (SEQ ID NO: 32) (where ₆ is Y or F).

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

In some embodiments, the anti-TREM2 antibody 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 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 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 amino acid sequence of any one of SEQ ID NOs:3, 11, 16, 20, 22, and 27.

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

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

In some embodiments, the anti-TREM2 antibody comprises heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 17, respectively, and has at least 85% sequence identity to SEQ ID NO:15 (e.g., , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody comprises light chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 18, respectively, and has at least 85% sequence identity to SEQ ID NO:16 (e.g. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the anti-TREM2 antibody is an antibody described herein (eg, heavy chain CDRs 1-3 and light chain comprising the amino acid sequences of SEQ ID NOS: 4, 5, 17, 7, 8, and 18, respectively) antibody comprising CDRs 1-3, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16) for binding.

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

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises amino acids having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24. A heavy chain variable region containing sequence is included. 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 amino acids having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:22. A light chain variable region containing sequence is included. 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 amino acids having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24. A heavy chain variable region comprising a sequence and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:22 including. 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 comprising the amino acid sequence of SEQ ID NO:22.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOS: 4, 25, and 17, respectively, and at least 85% sequence relative to SEQ ID NO:24 Heavy chain variable regions with identity (eg, at least 90%, 95%, or 97% sequence identity) are included. In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises light chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOS:23, 8, and 18, respectively, and at least 85% sequence relative to SEQ ID NO:22 Light chain variable regions with identity (eg, at least 90%, 95%, or 97% sequence identity) are included.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion is an antibody described herein (eg, a heavy chain CDR1 to SEQ ID NO: 4, 25, 17, 23, 8, and 18 amino acid sequences, respectively). 3 and light chain CDRs 1-3, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22).

In some embodiments, the anti-TREM2 antibody or antigen-binding portion 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, an amino acid sequence of SEQ ID NO:17 A CDR-H3 sequence, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises amino acids having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24. A heavy chain variable region containing sequence is included. 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 amino acids having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:27. A light chain variable region containing sequence is included. 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 amino acids having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24. A heavy chain variable region comprising a sequence and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:27 including. 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 comprising the amino acid sequence of SEQ ID NO:27.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOS: 4, 25, and 17, respectively, and at least 85% sequence relative to SEQ ID NO:24 Heavy chain variable regions with identity (eg, at least 90%, 95%, or 97% sequence identity) are included. In some embodiments, the anti-TREM2 antibody or antigen-binding portion comprises light chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 9, respectively, and at least 85% sequence relative to SEQ ID NO:27 Light chain variable regions with identity (eg, at least 90%, 95%, or 97% sequence identity) are included.

In some embodiments, the anti-TREM2 antibody or antigen-binding portion is an antibody described herein (eg, a heavy chain CDR1 to SEQ ID NO: 4, 25, 17, 7, 8, and 9 amino acid sequences, respectively) 3 and light chain CDRs 1-3, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27).

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

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

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

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

In some embodiments, the anti-TREM2 antibody comprises heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively, and has at least 85% sequence identity to SEQ ID NO:2 (e.g., , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody comprises light chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 9, respectively, and has at least 85% sequence identity to SEQ ID NO:3 (e.g., , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the anti-TREM2 antibody is an antibody described herein (eg, heavy chain CDRs 1-3 and light chain comprising the amino acid sequences of SEQ ID NOS: 4, 5, 6, 7, 8, and 9, respectively) antibody comprising CDRs 1-3, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:3) for binding.

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

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

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

In some embodiments, the anti-TREM2 antibody comprises heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively, and has at least 85% sequence identity to SEQ ID NO: 10 (e.g. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the anti-TREM2 antibody comprises light chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 9, respectively, and has at least 85% sequence identity to SEQ ID NO:11 (e.g. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the anti-TREM2 antibody is an antibody described herein (eg, heavy chain CDRs 1-3 and light chain comprising the amino acid sequences of SEQ ID NOS: 12, 13, 14, 7, 8, and 9, respectively) It is an antibody that competes for binding with an antibody comprising CDRs 1-3, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11).

Binding Properties of Anti-TREM2 Antibodies In some embodiments, antibodies described herein that specifically bind to a TREM2 protein are used in cells (e.g., primary cells or cell lines that endogenously express TREM2, such as human macrophages). or a primary cell or cell line that has been engineered to express TREM2, eg, as described in the Examples section below). In some embodiments, an antibody that specifically binds a TREM2 protein described herein binds to a purified or recombinant TREM2 protein or portion thereof, or a chimeric protein comprising TREM2 or a portion thereof ( For example, an Fc fusion protein comprising TREM2 or an Fc fusion protein comprising the ectodomain of TREM2).

In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with one or more other TREM2 proteins from another species. In some embodiments, an antibody that specifically binds to a human TREM2 protein exhibits cross-reactivity with cynomolgus monkey (“cyno”) TREM2 protein. In some embodiments, an antibody that specifically binds to human TREM2 protein exhibits cross-reactivity with mouse TREM2 protein. In some embodiments, the anti-TREM2 antibody exhibits 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. These methods include solid phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), binding equilibrium exclusion methods (e.g., KinExA ( ®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, Calif.)), and Western blot analysis; It is not limited to these. In some embodiments, ELISA is used to determine binding affinity and/or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, binding equilibrium exclusion methods are used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and/or cross-reactivity.

Epitopes Recognized by Anti-TREM2 Antibodies In some embodiments, the anti-TREM2 antibodies recognize the same or substantially the same epitopes of human TREM2 as the epitopes recognized by the antibody clones described herein. As used herein, the term "substantially the same" when used in reference to the epitope recognized by the antibody clones described herein and the epitope recognized by the antibody clones described herein. are identical, within or nearly identical to the epitope (e.g., have at least 90% sequence identity to the epitope, or have one, two or have 3 amino acid substitutions, e.g. It means that the anti-TREM2 antibody recognizes the epitope.

In some embodiments, the anti-TREM2 antibody is the same or substantially the same epitope recognized by an antibody clone selected from the group consisting of clone CL0020306, clone CL0020188, clone CL0020307, and variants thereof. Recognize epitopes.

In some embodiments, the anti-TREM2 antibody binds human TREM2 at an epitope within the stalk region of TREM2. In some embodiments, the anti-TREM2 antibody recognizes an epitope of human TREM2 comprising, within, or consisting of residues 129-172 or residues 131-169 of SEQ ID NO:1. In some embodiments, the anti-TREM2 antibody has an epitope of human TREM2 comprising, within, or consisting of residues 129-148 of SEQ ID NO:1 (e.g., 143-148 of SEQ ID NO:1). recognize. In some embodiments, an anti-TREM2 antibody activates TREM2/DAP12 signaling (e.g., by inducing phosphorylation of a kinase such as Syk) and binds human TREM2 at an epitope within the stalk region of TREM2. is an agonist. In some embodiments, an anti-TREM2 antibody binds human TREM2 at an epitope within the stalk region of TREM2 and inhibits cleavage of TREM2 by a protease (eg, ADAM17).

Functional Properties of Anti-TREM2 Antibodies In some embodiments, an anti-TREM2 antibody (e.g., an antibody having one or more of the disclosed CDR sequences, heavy chain variable region sequences, and/or light chain variable region sequences) comprises: Functions in one or more TREM2 activities disclosed herein. For example, in some embodiments, an anti-TREM2 antibody modulates the level of sTREM2 protein (e.g., the level of sTREM2 shed from the cell surface into an extracellular sample) and interacts with the TREM2/DAP12 signaling complex. and/or one or more downstream signaling complexes such as phagocytosis, cell proliferation, cell survival, cell differentiation, cytokine secretion, or cell migration. regulates the activity of

In some embodiments, an anti-TREM2 antibody enhances one or more ligand-induced TREM2 activities (eg, those described herein). In some embodiments the ligand is a lipid ligand. Examples of TREM2 lipid ligands include 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), 2-arachidonoylglycerol (2-AG), 7-ketocholesterol ( 7-KC), 24(S) hydroxycholesterol (24OHC), 25(S) hydroxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), acylcarnitine (AC), alkylacylglycerophosphocholine (PAF), α- Galactosylceramide (KRN7000), bis(monoacylglycero)phosphate (BMP), cardiolipin (CL), ceramide, ceramide-1-phosphate (C1P), cholesteryl ester (CE), cholesterol phosphate (CP), diacylglycerol 34:1 (DG34:1), diacylglycerol 38:4 (DG38:4), diacylglycerol pyrophosphate (DGPP), dihydroceramide (DhCer), dihydrosphingomyelin (DhSM), ether phosphatidylcholine (PCe), free cholesterol ( FC), galactosylceramide (GalCer), galactosylsphingosine (GalSo), ganglioside GM1, ganglioside GM3, glucosylsphingosine (GlcSo), Hank's Balanced Salt Solution (HBSS), Kdo2-lipid A (KLA), lactosylceramide (LacCer), Lysoalkylacylglycerophosphocholine (LPAF), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysosphingomyelin (LSM) ), lysophosphatidylserine (LPS), N-acyl-phosphatidylethanolamine (NAPE), N-acyl-serine (NSer), phosphatidylcholine oxide (oxPC), palmitate-9-hydroxy-stearate (PAHSA), phosphatidylethanol Amine (PE), phosphatidylethanol (PEtOH), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphinganine, sphinganine-1-phosphate (Sa1P ), Sphingomyeri (SM), sphingosine, sphingosine-1-phosphate (So1P), and sulfatide.

Modulation of sTREM2 Shedding In some embodiments, an anti-TREM2 antibody alters the level of sTREM2 protein in a sample, eg, the level of sTREM2 shed from the cell surface into an extracellular sample. In some embodiments, the anti-TREM2 antibody reduces levels of sTREM2.

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

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

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

Modulation of Kinase Recruitment or Phosphorylation In some embodiments, the anti-TREM2 antibody interacts with the TREM2/DAP12 signaling complex (e.g., but not limited to Syk, ZAP70, PI3K, Erk, AKT, or GSK3b) induce phosphorylation of kinases that In some embodiments, anti-TREM2 antibodies induce phosphorylation of kinases that interact with the TREM2/DAP12 signaling complex without blocking binding of the native TREM2 ligand. In some embodiments, anti-TREM2 antibodies enhance 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 phosphorylation of Syk. In some embodiments, the anti-TREM2 antibody reduces the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody relative to control values by at least 10%, at least 20%, at least 30%, at least 40%, An increase of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more induces or enhances phosphorylation of Syk. In some embodiments, the anti-TREM2 antibody increases the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody as compared to control values by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, A 7-fold, 8-fold, 9-fold, or 10-fold or greater increase induces phosphorylation of Syk. In some embodiments, the control value is an untreated sample (e.g., a sample comprising TREM2-expressing cells that has not been treated with an anti-TREM2 antibody, or a sample from a subject that has not been treated with an anti-TREM2 antibody), or a TREM2 ligand Levels of Syk phosphorylation in samples treated with but not anti-TREM2 antibody or treated with the appropriate non-TREM2 binding antibody.

Immunoassays are used in some embodiments to detect and/or quantify phosphorylation (eg, Syk phosphorylation) in a sample. In some embodiments, the immunoassay is enzyme immunoassay (EIA), enzyme multiplex immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immune tissue Chemical method (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemiluminescence immunoassay ( ECL). In some embodiments, phosphorylation is detected and/or quantified using an immunoassay that utilizes an amplified luminescence proximity homogeneous assay (AlphaLISA®, PerkinElmer Inc.). be done.

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

Modulation of Phagocytosis In some embodiments, anti-TREM2 antibodies enhance phagocytosis of dead cell debris, tissue debris, amyloid beta particles, or foreign bodies. In some embodiments, the anti-TREM2 antibody enhances phagocytosis without blocking binding of the native TREM2 ligand. In some embodiments, anti-TREM2 antibodies enhance phagocytosis induced by TREM2 ligands (eg, lipid ligands). In some embodiments, the anti-TREM2 antibody reduces the level of phagocytosis in a sample treated with the anti-TREM2 antibody by at least 10%, at least 20%, at least 30%, at least 40%, at least An increase of 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more enhances phagocytosis. In some embodiments, the anti-TREM2 antibody increases the level of phagocytosis in a sample treated with the anti-TREM2 antibody by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold compared to control values. A fold, eight-fold, nine-fold, or ten-fold increase or more enhances phagocytosis. In some embodiments, the control value is the rate of phagocytosis in an untreated sample, a sample treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with a suitable non-TREM2 binding antibody. level.

In some embodiments, phagocytosis is measured using a phagocytosis assay with a labeled substrate. Phagocytosis assays are known in the art. In some embodiments, the phagocytosis assay is performed on a sample comprising cells that endogenously express TREM2, eg, human macrophages or microglia. In some embodiments, a phagocytosis assay is performed on a sample containing cells engineered to express TREM2. In some embodiments, phagocytosis is measured using the Human Macrophage Phagocytosis Assay described in the Examples section below.

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

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

In some embodiments, cell migration is measured using a chemotaxis assay. Chemotaxis assays are known in the art. In some embodiments, a cell migration assay (eg, a chemotaxis assay) is performed on a sample comprising cells endogenously expressing TREM2, eg, human macrophages. In some embodiments, cell migration assays (eg, chemotaxis assays) are performed on samples containing cells engineered to express TREM2. In some embodiments, cell migration is measured using the human macrophage chemotaxis assay described in the Examples section below.

In some embodiments, cell survival is measured using a cell viability assay. Cell viability assays are known in the art. In some embodiments, a cell survival assay (eg, cell viability assay) is performed on a sample comprising cells endogenously expressing TREM2, eg, human macrophages. In some embodiments, cell viability assays (eg, cell viability assays) are performed on samples containing cells engineered to express TREM2. In some embodiments, cell survival is measured using the human macrophage viability assay described in the Examples section below.

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

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

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

In some embodiments, anti-TREM2 antibodies enhance microglial function without increasing neuroinflammation. Levels of neuroinflammation measure levels of cytokines (eg, inflammatory cytokines) such as, but not limited to, TNF-α, IL-1β, IL-6, IL-1ra, TGFβ, IL-15, or IFN-γ can be determined by In some embodiments, cytokine levels are measured by immunoassays, e.g., enzyme immunoassay (EIA), enzyme multiplex immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay ( MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemistry Measured using a luminescent immunoassay (ECL).

IV. Fc Polypeptide Mutations of Proteins Having Anti-TREM2 Antigen-Binding Portions In some embodiments, an anti-TREM2 antibody comprises two Fc polypeptides, one or both of which are independently selected modifications (e.g., mutations) , respectively, or may be a wild-type Fc polypeptide, eg, a human IgG1 Fc polypeptide. Non-limiting examples of mutations that can be introduced into one or both Fc polypeptides include, for example, the BBB receptor of the Fc polypeptide (or antibody comprising it), e.g. For example, mutations that allow binding to human or cynomolgus monkey TfR, such as those that can be expressed on brain endothelial cells, increase serum stability, modulate effector function, affect glycosylation, immunity in humans. Mutations to reduce virulence and/or to effect heterodimerization of the Fc polypeptide knob and hole are included.

Transferrin Receptor Binding Mutations In some embodiments, an anti-TREM2 antibody comprises an Fc polypeptide comprising a modification (eg, amino acid substitution) that permits binding of the Fc polypeptide to a TfR protein. Briefly, binding to a TfR protein expressed on, for example, brain endothelial cells (e.g., the apical domain thereof) is, in some embodiments, a modified Fc polypeptide of the present disclosure or an antibody comprising the same It may allow crossing the blood-brain barrier via receptor-mediated transcytosis. In certain embodiments, receptor-mediated transcytosis can enhance or improve the ability of proteins, including Fc polypeptides, to reside in the brain (i.e., on the luminal side of the blood-brain barrier), which may allow improved binding to TREM2 in the CNS and other functions such as clearance, neutralization, or immunodepletion of targets.

Exemplary TfR-binding amino acid modifications to Fc (e.g., CH2 and/or CH3 portions, fragments, or domains), as well as Fc polypeptides and portions thereof comprising amino acid modifications, are described in PCT Patent Publication No. WO2018/152326A1. ing. These amino acid modifications, TfR binding Fc polypeptide sequences and TfR binding Fc polypeptides, and techniques for making and testing them, are incorporated herein by reference. One or two Fc polypeptides of the Fc dimers of the present disclosure can be engineered to contain modifications that allow binding to TfR. In certain embodiments, one Fc polypeptide of the Fc dimer comprises a modification that allows binding to TfR, and the other Fc polypeptide does not.

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 TxxExxxF (SEQ ID NO:59) motifs.

In some embodiments, the modified Fc polypeptide comprises wild-type amino acid residues at positions 380, 389, 390, and 415, according to EU numbering, wherein the wild-type amino acid residues correspond to It is found in the position where

In some embodiments, the anti-TREM2 antibody comprises the following amino acids according to EU numbering: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; 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; include.

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

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

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

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

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

Additional examples of modified Fc polypeptides are listed in Table 8.

Mutations that Facilitate Heterodimerization of Fc Polypeptides In some embodiments, the Fc polypeptides present in the anti-TREM2 antibodies disclosed herein promote heterodimer formation and homodimerization. Contains knob and hole mutations that prevent mer formation. In general, this modification introduces protrusions (“knobs”) at the interface of the first polypeptide and corresponding voids (“holes”) at the interface of the second polypeptide, resulting in heterodimerization. Protrusions can be positioned within the cavity such that body formation is promoted and homodimer formation is prevented. Protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (eg, tyrosine or tryptophan). Complementary depressions of identical or similar size to the protrusions are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller side chains (eg, alanine or threonine). In some embodiments, such additional mutations are at positions in the Fc polypeptide that do not adversely affect (eg, inhibit) binding of the Fc polypeptide to a BBB receptor, eg, TfR.

In one exemplary embodiment of the knobhole approach to dimerization, one of the Fc polypeptides present in the proteins described herein, position 366 (numbered according to the EU numbering scheme) is a native threonine contains tryptophan instead of The other Fc polypeptide in the dimer has a valine in place of the native tyrosine at position 407 (numbering according to the EU numbering scheme). The other Fc polypeptide has a substitution of serine for the native threonine at position 366 (numbering according to the EU numbering scheme) and a substitution for alanine for the native leucine at position 368 (numbering according to the EU numbering scheme) It can further include substitutions that are Thus, one of the Fc polypeptides of an anti-TREM2 protein of the present disclosure has a knob mutation of T366W and the other Fc polypeptide has a Y407V mutation, typically with T366S and L368A hole mutations.

In some embodiments, one or both Fc polypeptides may be engineered to contain other modifications for heterodimerization, such as the CH3-CH3 interface, which is naturally charged. There is electrostatic manipulation or hydrophobic patch modification of contact residues within.

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

Fc Effector Function In some embodiments, one or both Fc polypeptides of the anti-TREM2 proteins disclosed herein are expressed on effector cells with modifications that reduce effector function, i.e., mediate effector function. may contain modifications that reduce its ability to induce a particular biological function upon binding to the Fc receptor. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), Includes, but is not limited to, downregulation of cell surface receptors (eg, B cell receptors), and B cell activation. Effector functions can vary by antibody class. For example, native human IgG1 and IgG3 antibodies can elicit ADCC and CDC activity upon binding to appropriate Fc receptors present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 ADCP function can be triggered upon binding to appropriate Fc receptors present on immune system cells.

In some embodiments, one or both Fc polypeptides may contain modifications that modulate effector function.

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

Additional Fc polypeptide mutations that modulate effector function include the following: at position 329, proline is between glycine, arginine, serine, or proline 329 of Fc and tryptophan residues Trp87 and Trp110 of FcγRIII. mutations that are substituted with amino acid residues that are large enough to disrupt the Fc/Fcγ receptor interface that forms at, but are not limited to. Additional exemplary substitutions include S228P, E233P, L235E, N297A, N297D, N297G, and P331S according to the EU numbering scheme. Multiple substitutions may be present, for example L234A and L235A of the human IgG1 Fc region, L234A, L235A and P329G of the human IgG1 Fc region, L234A, L235A and P329S of the human IgG1 Fc region according to the EU numbering scheme. , human IgG4 Fc region S228P 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 S228P and L236E of the human IgG4 Fc region. In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, e.g., substitutions at positions 298, 333, and/or 334 according to the EU numbering scheme .

FcRn Binding Sites and Mutations that Extend Serum Half-Life In certain aspects, an Fc polypeptide (eg, a modified Fc polypeptide) present in an anti-TREM2 protein of the present disclosure can comprise an FcRn binding site. In some embodiments, the FcRn binding site is in the Fc polypeptide or fragment thereof.

In some embodiments, the FcRn binding site comprises a native 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 native FcRn binding site is an IgG binding site, eg, a human IgG binding site. In some embodiments, the FcRn binding site comprises modifications that alter FcRn binding.

In some embodiments, the FcRn binding site has one or more mutated, e.g., substituted, amino acid residues, wherein the mutation(s) increases or reduces serum half-life. (i.e., no less than 25% reduction in serum half-life when assayed under the same conditions compared to an equivalent Fc polypeptide with the wild-type residue at the mutated position) ). In some embodiments, the FcRn binding site has one or more amino acid residues substituted at positions 251-256, 428, and 433-436 according to the EU numbering scheme.

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

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

V. Preparation of Antibodies In some embodiments, antibodies are prepared by immunizing animal(s) (eg, mice, rabbits, or rats) with an antigen or mixture of antigens to induce an antibody response. In some embodiments, the antigen or antigen mixture is administered in conjunction with an adjuvant (eg, Freund's adjuvant). After the initial immunization, one or more subsequent booster injections of antigen(s) may be administered to improve antibody production. After immunization, antigen-specific B cells are recovered, eg, from spleen and/or lymphoid tissue. To generate monoclonal antibodies, B cells are fused with myeloma cells and subsequently screened for antigen specificity. Methods of preparing antibodies are also described in the Examples section below.

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. can. Gene libraries encoding heavy and light chains of monoclonal antibodies are also made from hybridomas or plasma cells. Alternatively, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to an antigen of choice. Antibodies can also be bispecific. That is, it can recognize two different antigens. Antibodies can also be heterologous conjugates, eg, 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, eg, a hybridoma or CHO cell expression system. Many such systems are widely available from commercial sources. In embodiments where the antibody comprises both V _H and V _L regions, the V _H and V _L regions are expressed using a single vector, for example in a dicistronic expression unit or under the control of different promoters. can be In other embodiments, the _VH and _VL regions may be expressed using separate vectors. The _VH or _VL regions described herein may optionally include a methionine at the N-terminus.

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

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

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

Human antibodies can also be produced using phage display or yeast display technology. In phage display, repertoires of variable heavy and variable light chain genes are amplified and expressed in phage display vectors. In some embodiments, antibody libraries are natural repertoires amplified from human sources. In some embodiments, the antibody library is a synthetic library made by cloning and recombining heavy and light chain sequences to create a large pool of antibodies with varying antigenic specificities. Phage typically display antibody fragments (eg, Fab or scFv fragments), which 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 generated. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab') ₂ fragments. According to another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

In some embodiments, the antibody or antibody fragment is conjugated (pegylated) to another molecule, e.g., polyethylene glycol, or conjugated to serum albumin, to provide increased half-life in vivo. gated.

VI. Nucleic Acids, Vectors and Host Cells In some embodiments, the anti-TREM2 antibodies disclosed herein are prepared using recombinant methods. Thus, in some aspects, the disclosure provides any anti-TREM2 antibody described herein (e.g., any one of the CDRs, heavy chain variable regions and light chain variable regions described herein). vectors containing such nucleic acids; and nucleic acids that are used to replicate the antibody-encoding nucleic acids and/or to express the antibodies are introduced. a host cell.

In some embodiments, the polynucleotide (e.g., isolated polynucleotide) is an antibody described herein (e.g., those described in the section above under the heading "Anti-TREM2 Antibody Sequences") comprising a nucleotide sequence that encodes In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes one or more amino acid sequences (eg, CDR sequences, heavy chain sequences or light chain sequences) disclosed in Table 8 below. In some embodiments, the polynucleotide has 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) Includes nucleotide sequences that encode amino acid sequences. In some embodiments, a polynucleotide described herein is operably linked to a heterologous nucleic acid, eg, a heterologous promoter.

Suitable vectors containing polynucleotides encoding an antibody of the disclosure, or fragments thereof, include cloning vectors and expression vectors. Although the cloning vector chosen may vary depending on the host cell intended to be used, useful cloning vectors will generally have the ability to replicate themselves, may have a single target for a particular restriction endonuclease, and/or Alternatively, it may carry a marker gene that can be used to select clones containing the vector. Examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (eg pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttles such as pSA3 and pAT28. vectors. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

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

Suitable host cells for cloning or expressing the polynucleotides or vectors described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell is prokaryotic. In some embodiments, the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells. In some embodiments, the host cells are human cells, eg, human embryonic kidney (HEK) cells.

In another aspect, methods of making the anti-TREM2 antibodies described herein are provided. In some embodiments, the methods involve culturing a host cell described herein (e.g., a host cell expressing a polynucleotide or vector described herein) under conditions suitable for expression of the antibody. Including. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

VII. Methods of Treatment Using Anti-TREM2 Antibodies In another aspect, methods of treatment using the anti-TREM2 antibodies disclosed herein (eg, the anti-TREM2 antibodies described in Section III above) are provided. In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, methods of modulating one or more TREM2 activities (eg, in a subject with a neurodegenerative disease) are provided.

In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, the neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontal with chromosome 17-linked parkinsonism Cranial dementia, arginous granule dementia, amyotrophic lateral sclerosis, Guam amyotrophic lateral sclerosis/parkinson dementia complex (ALS-PDC), corticobasal degeneration, chronic traumatic Encephalopathy, Creutzfeldt-Jakob disease, Boxer dementia, diffuse neurofibrillary tangles with calcification, Down syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, glial tauopathy globulosa, Guadeloupe parkinsonism with dementia, Guadeloupe PSP, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy (HDLS) with spheroids, Huntington's disease, inclusion body myositis, multiple system atrophy, Myotonic dystrophy, Nasu-Hakola disease, neurofibrillary tangle predominance dementia, Niemann-Pick disease type C, pontine pulmonary degeneration, Parkinson's disease, Pick's disease, post-encephalitic parkinsonism, prion protein brain amyloid selected from the group consisting of angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and neurofibrillary senile dementia. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neurodegenerative disease is Nasu-Hakola disease. In some embodiments, the neurodegenerative disease is frontotemporal dementia. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the method comprises an isolated antibody or antigen-binding fragment thereof (e.g., an anti-TREM2 antibody described herein) that specifically binds to a human TREM2 protein, or an anti-TREM2 antibody described herein. administering to the subject a pharmaceutical composition comprising an anti-TREM2 antibody of

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

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

In some embodiments, the subject to be treated is a human, such as an adult human or a pediatric human.

In some embodiments, methods of reducing plaque accumulation in a subject with a neurodegenerative disease are provided. In some embodiments, the method comprises administering an antibody or pharmaceutical composition described herein to the subject. In some embodiments, the subject has Alzheimer's disease. In some embodiments, the subject is an animal model of neurodegenerative disease (eg, 5XFAD or APP/PS1 mouse model). In some embodiments, plaque accumulation is measured by amyloid plaque imaging and/or tau imaging, eg, using positron emission tomography (PET) scanning. In some embodiments, the administration of the anti-TREM2 antibody is at least 20%, at least 30%, at least 40% compared to the baseline value (e.g., the level of plaque accumulation in the subject prior to administration of the anti-TREM2 antibody) , reduces plaque accumulation by 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 subject in a therapeutically effective amount or dosage. However, dosages may vary according to several factors, including the chosen route of administration, formulation of the composition, patient response, severity of the condition, body weight of the subject and the judgment of the prescribing physician. Dosages may be increased or decreased over time as needed by individual patients. In certain cases, the patient is initially given a low dose and then titrated up to an effective dose tolerated by the patient. Determination of an effective amount is well within the capabilities of those skilled in the art.

The routes of administration of the anti-TREM2 antibodies described herein include oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intralesional, rectal, intrabronchial, nasal, intranasal. It may be mucosal, intestinal, ocular or aural, or any other method known in the art. In some embodiments, antibodies are administered orally, intravenously, or intraperitoneally.

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

VIII. Pharmaceutical Compositions and Kits In another aspect, pharmaceutical compositions and kits comprising antibodies that specifically bind to human TREM2 protein are provided. In some embodiments, the pharmaceutical compositions and kits are for use in treating neurodegenerative diseases. In some embodiments, pharmaceutical compositions and kits are for use in modulating (eg, enhancing or inhibiting) one or more TREM2 activities, eg, Syk phosphorylation. In some embodiments, the pharmaceutical compositions and kits are for use in modulating (eg, decreasing) sTREM2 levels.

Pharmaceutical Compositions In some embodiments, pharmaceutical compositions comprising an anti-TREM2 antibody or antigen-binding fragment thereof are provided. In some embodiments, the anti-TREM2 antibody is an antibody or antigen-binding fragment thereof described in Section III, supra.

In some embodiments, pharmaceutical compositions comprise an anti-TREM2 antibody described herein and further comprise 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. A pharmaceutically acceptable carrier is one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). may include (s). Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce clearance or hydrolysis of the active agent. , or excipients or other stabilizers and/or buffers. Other pharmaceutically acceptable carriers and their formulation are well known in the art.

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

For oral administration, anti-TREM2 antibodies can be formulated by combining them with pharmaceutically acceptable carriers well known in the art. With such carriers the compounds can be formulated into tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, solutions, gels, syrups, for oral ingestion by the patient to be treated. It becomes possible to formulate as a slurry, suspension and the like. Pharmaceutical preparations for oral use are prepared by mixing the compound with solid excipients, optionally grinding the resulting mixture, processing the mixture into granules and, if desired, after adding suitable excipients into tablets. Or it can be obtained by obtaining a dragee core. Suitable excipients include fillers such as sugars including, for example, lactose, sucrose, mannitol, or sorbitol; Cellulose preparations such as sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as the 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, eg, by bolus injection or continuous infusion. For injection, by dissolving, suspending or emulsifying the compound(s) in aqueous or non-aqueous solvents such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; If necessary, the preparation can be formulated with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, compounds can be formulated in aqueous solutions, for example, in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. can be done.

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

Dosages and desired drug concentrations of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. Determination of the appropriate dose or route of administration is well within the skill of those in the art. Suitable dosages are also described in Section VII above.

Kits In some embodiments, kits containing anti-TREM2 antibodies are provided. In some embodiments, the anti-TREM2 antibody is an antibody or antigen-binding fragment thereof described in Section III, supra.

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

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

In some embodiments, the kit includes instructional materials (e.g., for the therapeutic methods described in Section VI above) that include instructions (i.e., protocols) for the practice of the methods described herein. instructions for using the kit). While the instructional material typically consists of written or printed materials, it is not limited to such. Any medium capable of storing such instructions and capable of communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (eg, magnetic discs, magnetic tapes, magnetic cartridges, magnetic chips), optical media (eg, CD-ROM), and the like. Such media may include addresses to Internet sites that provide such instructional materials.

IX. EXAMPLES The present disclosure will be described in more detail by means of specific examples. The following examples are presented for illustrative purposes only and are not intended to limit the disclosure in any way.

Example 1. Generation and initial characterization of anti-TREM2 antibodies Recombinant expression and purification of human TREM2 ECD fused with mouse Fc The ectodomain (residues 19-172) of human TREM2 (UniProtKB ID-Q9NZC2) was added to the N-terminal region with mouse IgG kappa including a secretion signal from chain V-III, amino acids 1-20 (UniProtKB ID-P01661), and a mouse Fc tag (with GGGGS (SEQ ID NO: 34) between TREM2 ECD and Fc) in the C-terminal region, Subcloned into the pRK vector.

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

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

Recombinant expression and purification of His-tagged TREM2 ECD The ectodomain (residues 19-172) of TREM2 (UniProtKB-Q9NZC2) was added to the N-terminal region with mouse Ig kappa chain V-III, amino acids 1-20 (UniProtKB ID-P01661 ) and a 6X-His tag (SEQ ID NO: 35) in the C-terminal region were subcloned into the pRK vector. Inserts were confirmed by sequencing and maxiprep plasmid purification was performed.

Purified plasmids were transfected into Expi293F™ cells (Thermo Fisher) using the Expi293F™ Expression System Kit according to the manufacturer's instructions. Transfected cells were incubated in an orbital shaker (Infors HT Multitron) at 37° C., 125 rpm in a humidified atmosphere of 6% CO ₂ . Sixteen hours after transfection, ExpiFectamine™ 293 Transfection Enhancers 1 and 2 were added to the cells and medium supernatants were 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 Nalgene™ Rapid-Flow™ disposable filter units with 0.4 micron pore size (Thermo Fisher). HisPur™ Ni-NTA Resin (Thermo Fisher) was washed with MQ water and equilibrated with load buffer (20 mM Tris, 150 mM NaCl, and 10 mM imidazole at pH 8.0). Affinity purification was performed using the gravity flow method. Collected medium was loaded onto the resin and non-specifically bound proteins were washed with loading buffer supplemented with 50 mM and 100 mM imidazole. Bound His-tagged TREM2 ectodomain was eluted with 20 mM Tris, 150 mM NaCl, and 200 mM imidazole at pH 8.0. The eluted protein was concentrated using an Amicon 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 onto a HiLoad Superdex 200 16/600 (GE Healthcare Life Sciences) column equilibrated with 1×PBS and eluted and fractionated using 1×PBS as the running buffer. Eluted fractions were analyzed by electrophoresis on polyacrylamide (PAGE) gels under denaturing and native conditions. Eluted fractions were further characterized by analytical size exclusion chromatography and intact protein mass spectrometry. Using results from PAGE and analytical characterization, the highly glycosylated protein fractions were pooled, aliquoted and stored at -80°C.

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

Single cell suspensions were analyzed to determine the binding properties of secreted antibodies. Antibody-secreting cells were loaded into microfluidic devices and isolated in nanoliter volume reaction chambers, allowing detection of secreted antibodies using fluorescence and brightfield image-based microscopic assays (e.g., US Patent See No. 9,188,593). Binding assays were performed, including detection of antibody binding to antigen-coated microbeads, detection of soluble fluorescently labeled antigen binding to antibody immobilized on the beads, and detection of antibody binding to cell surface expressed antigens. Cell surface expressed antigens included both recombinant and native antigens displayed on the cell surface.

Using image analysis, chambers exhibiting a positive fluorescent signal indicating the presence of single cells producing antibodies with the desired properties are identified and the contents of the chambers are harvested and plated in 384-well plates. dissolved (see, eg, US Pat. No. 10,087,408). Single cell lysates were then subjected to RT-PCR to amplify heavy and light chain variable region sequences. The resulting amplicons were then sequenced to determine the cDNA sequences of the paired heavy and light chain variable regions from the selected single cells. The sequences obtained were manually inspected and analyzed to determine sequence diversity and somatic hypermutation. Sequences were selected for expression based on screening data and sequence diversity. The expressed antibodies were tested to confirm antigen binding specificity.

Example 2. Sequence Optimization and Humanization of Anti-TREM2 Antibodies Exemplary anti-TREM2 antibodies were sequence optimized and humanized prior to characterization for binding kinetics and binding specificity.

Sequence optimization was performed for residues amenable to chemical modification (e.g. asparagine deamidation motif (NG), aspartate isomerization motif (DS)) as well as residues likely to be oxidized (tryptophan (W) and This was done by searching within the CDR sequences of methionine (M)) and by amino acid substitutions by conservative and germline residues to eliminate such sequence trends. The humanized, sequence-optimized anti-TREM2 antibody variants were then analyzed for binding kinetics using Biacore and titrated cell binding to HEK293-H6 cells (see Example 5 for a representative protocol). (see ).

Example 3. Generation of Anti-TREM2 Antibodies with Modified Fc Polypeptides (“ATV:TREM2”) The Fd (V _H +CH1) regions (SEQ ID NOs: 22 and 24) of the humanized affinity-matured anti-TREM2 antibodies were combined with the human transferrin receptor ( (CH3C.35.23.1.1, CH3C.35.23.3, CH3C.35.23.3 cisLALA, or CH3C.35.24 ) or an expression vector containing a sequence (CH3C.35.21) encoding an Fc polypeptide that binds to the cynomolgus transferrin receptor. The sequence encoding the Fc polypeptide has a "knob" (T366W) mutation that prevents homodimerization and promotes heterodimerization with the Fc polypeptide, including a "hole" (T366S/L368A/Y407V) mutation contained. The Fd region was also cloned into the corresponding "hole" vector containing the sequence encoding the Fc polypeptide with the hole mutation but lacking the TfR binding mutation. The coding sequences (both Fd-knob-Fc and Fd-hole-Fc constructs) also contain the 'LALA'(L234A; L235A) mutation in the hinge region that reduces effector function (Wines et al., J. Immunol. 164:5313-5318 (2000) and "LS"(M428L; N434S) mutations in the FcCH3 region that increase binding to FcRn (e.g., Zalevsky et al., Nat. Biotech. 28(2):157-159 ( 2010)), the final encoded heavy chain sequence expressed by the vector is listed in Table 1.

(Table 1) ATV: TREM2 sequences

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

Binding of anti-TREM2 protein to human transferrin receptor was determined as follows. Anti-human Fabs were immobilized on CM5 chips to capture anti-TREM2 protein. Full-length human TfR or human TfR apical domain was flowed over the chip (180 sec association time) in serial dilutions (eg, concentrations of 1-1,000 nM) and then dissociated. Fitting was performed using a 1:1 binding model.

Example 4. Characterization of Anti-TREM2 Antibodies The following section describes various assays that were performed to assess the binding and functional properties of the generated anti-TREM2 antibodies.

Affinity Measurements by Biacore Kinetics Measurements Affinities of anti-TREM2 antibodies were measured for human and cynomolgus monkey TREM2 ECD using surface plasmon resonance (Biacore™ 8K instrument). Anti-TREM2 antibodies were captured on a Biacore Series S CM5 sensor chip (GE Healthcare Life Sciences, Cat. No. 29149604) using the Human Fab Capture Kit (GE Healthcare Life Sciences, Cat. No. 28958325). Three-fold serial dilutions of recombinant human TREM2 or recombinant cynomolgus TREM2 were injected at a flow rate of 30 μL/min. Antibody binding was monitored for 300 seconds followed by antibody dissociation over 600 seconds in HBS-EP+running buffer (GE Healthcare Life Sciences, Catalog No. BR100669). Binding responses were corrected by subtracting RU values from empty flow cells. A 1:1 Languir model of joint fitting of k _on and k _off was used for kinetic analysis. _KD binding values were calculated from k _on and k _off .

Evaluation of TREM2 Binding in TREM2-Expressing HEK Cells The binding properties of anti-TREM2 antibodies were evaluated in HEK293 cells expressing human TREM2 as follows.

HEK293 cell lines stably expressing human TREM2/DAP12 were generated by transfecting cells with vectors expressing wild-type human TREM2 and DAP12, and with vectors expressing DAP12 only, respectively. Stable expressing clones were selected and cell surface TREM2 expression was assessed by flow cytometry. An APC-conjugated rat anti-human/mouse-TREM2 monoclonal antibody (R&D, catalog number MAB17291) was used to detect surface TREM2 expression. A clone showing the highest wild-type TREM2 expression level was selected and named "HEK293-H6". Clones stably expressing DAP12 were analyzed by Western blot and the selected clone was named "HEK293-DAP12#1".

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

Activation of TREM2-Dependent pSyk Signaling Activation of TREM2-dependent pSyk signaling was measured in human macrophage cells or in HEK293-H6 cells using the Perkin-Elmer commercial AlphaLisa assay.

For all experiments involving the use of lipid vesicles containing 70% DOPC and 30% POPS, lipid vesicles were prepared within two weeks of the experiment as follows: 7 mg DOPC (1,2-dioleoyl-sn -glycero-3-phosphocholine) and 3 mg of POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) were combined in chloroform in a glass vial and stirred for 1-2 minutes under a stream of _N2 gas. Allow to dry for 2 hours or until completely dry. The lipid mixture was resuspended in 1 mL HBSS (to a final lipid concentration of approximately 10 mg/mL) and vortexed for 2-3 minutes. The lipid suspension was subsequently extruded using an Avanti mini-extruder constructed with one 100 nm pore size membrane to form small unilamellar vesicles at 10 mg/mL.

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

Human macrophage cells were prepared for the assay as follows. Human monocytes were isolated from fresh blood according to the RosetteSep human monocyte enrichment cocktail protocol (Stemcell Technologies, reference #15068). Isolated monocytes were washed with wash buffer (PBS + 2% FBS) and resuspended in 10 mL of ACK Lysis Buffer (ThermoFisher Scientific, Catalog No. A10492) to lyse red blood cells. 20 mL of wash buffer was added to stop cell lysis, samples were centrifuged, and medium (RPMI, 10% Hyclone FBS, 1% sodium pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% penicillin) was added. -streptomycin). Human monocytes were then differentiated into macrophage cells in 250 mL flasks in medium in the presence of 50 ng/mL human recombinant M-CSF (Gibco, Catalog No. PHC9501). Fresh human M-CSF was added on day 3 and human macrophages were subsequently harvested on day 5 and used for the assay.

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

Survival Assay in Human Macrophage Cells Human monocytes were isolated according to the RosetteSep Human Monocyte Enrichment Cocktail protocol (Stemcell Technologies, Catalog No. 15068). Isolated monocytes were washed with wash buffer (PBS + 2% FBS) and resuspended in 10 mL of ACK lysing solution (ThermoFisher Scientific, Catalog No. A10492) to lyse red blood cells. Lysis was stopped by adding 20 mL of wash buffer. The cell suspension was centrifuged and washed once with medium (RPMI 1640+10% FBS+penicillin/streptomycin). Cells were resuspended in medium at a density of 10 ⁶ cells μL/mL and used in the viability assays described below.

The day before the assay, 96-well plates were pre-coated with anti-TREM2 antibody or isotype control at titrations (45 μL/well, 12 total steps) and incubated overnight at 4°C. After overnight incubation, pre-coated plates were washed twice with PBS and then incubated with human monocytes (10 ⁵ cells/ wells) were loaded onto the plate. After 5 days at 37° C., the medium was aspirated and 100 μL PBS + 100 μL Celltiter-glo medium (Promega, Cat#G7571) was added to each well. After a 10 minute incubation, the cell culture medium was transferred to a multiwell plate adapted for use with a luminometer and luminescence recorded for cell viability.

Lipid Storage Assay Prior to the assay, human pluripotent stem cells (iPSCs) were first differentiated into hematopoietic progenitor cells (HPCs) using a commercially available kit (STEMdiff Hematopoietic Kit from StemCell Technologies). HPCs were transferred to plates containing primary human astrocytes and co-cultured for 14-21 days. After identifying the floating cells in the co-culture to be predominantly (>80%) mature microglia, microglia were used in the assay.

Cells (iPSC-derived human microglia, 30,000 cells/well) were seeded in PDL-coated 96-well plates in complete serum medium. After 24 h at 37° C., the medium was supplemented with albumin oleate (10 μM or 33 μM final concentration, Sigma O3008) or purified unlabeled myelin (50 μg/mL final concentration, Safaiyan et al. (2016, Nature Neuroscience 19(8):995). (purified from wild-type C57B1/6 mouse brain (Jackson Laboratories)) using the method described in (Jackson Laboratories)). After 24 hours of lipid treatment at 37° C., the medium was replaced with medium containing anti-TREM2 antibody. For one point experiment, the concentration of anti-TREM2 antibody used was 100 nM. For a dose-response curve, media containing 100 nM anti-TREM2 antibody was serially diluted 3-fold in a total of 10 steps. RSV was used as a control. Cells were incubated at 37° C. for an additional 48 hours before either imaging cells using Bodipy staining or extracting cells for lipidomics as described below.

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

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

Lipid levels were analyzed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled with electrospray mass spectrometry (QTRAP 6500+Sciex, Framingham, Mass., USA). For each analysis, 5 μL of sample was injected onto a BEH C18 1.7 μm, 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.25 mL/min at 55° C. For positive ionization mode, mobile phase A consisted of 60:40 acetonitrile/water (v/v) containing 10 mM ammonium formate + 0.1% formic acid and mobile phase B contained 10 mM ammonium formate + 0.1% formic acid. It consisted of 90:10 isopropyl alcohol/acetonitrile (v/v). For negative ionization mode, mobile phase A consisted of 60:40 acetonitrile/water (v/v) containing 10 mM ammonium acetate and mobile phase B consisted of 90:10 isopropyl alcohol/acetonitrile (v/v) containing 10 mM ammonium acetate. /v). Gradient from 45% B to 99% B in 0.0-8.0 min, 99% B in 8.0-9.0 min, 45% B in 9.0-9.1 min, 9 .1 to 10.0 min was programmed as 45% B, and so on. Electrospray ionization was set to the following: curtain gas: 30, collision gas: moderate, ion spray voltage: 5500 (positive mode) or 4500 (negative mode), temperature: 250°C (positive mode) or 600°C ( Negative mode), run in either positive or negative ion mode applying source gas 1:50, source gas 2:60. Analyst 1.6.3 (Sciex) was used in multiple reaction monitoring mode (MRM) with the following parameters: residence time (msec) and collision energy (CE), declustering potential (DP): 80, entrance potential Data acquisition was performed using (EP): 10 (positive mode) or −10 (negative mode) and collision cell exit potential (CXP): 12.5 (positive mode) or −12.5 (negative mode). . Lipids were quantified using a mixture of non-endogenous internal standards. Lipids were identified based on their retention time and MRM properties of a commercial reference standard (Avanti Polar Lipids, Birmingham, Ala., USA).

Activation of TREM2-Dependent mTOR Signaling Wild-type iPSC-derived human microglia were cultured with anti-TREM2 antibody (final concentration 100 nM) and either DMSO or a commercially available mTOR inhibitor (Selleckchem, cat#AZD8055, final concentration 20 nM). Treated for 96 hours. Treated cells were then lysed and cell lysates prepared for Western blotting to investigate phosphorylation of key signaling targets in the mTOR pathway. Western blot primary antibodies were obtained from Cell Signaling Technologies: (1) phospho-mTOR (Ser2448), product number 5536T; (2) mTOR (7C10), product number 2983T; (3) phospho-AKT (Ser473); (3) Phospho-GSK-3beta (Ser9), Product No. 5558T; (4) Phospho-S6 Ribosomal Protein (S235/236), Product No. 4858T; (5) Phospho-4E-BP1 (Thr37/46 ), product number 2855T; (6) beta-actin, product number 58169S.

Example 5. Results The results of analysis of the binding properties of humanized and sequence-optimized variants of antibody CL0020188 are shown in Table 2 and Figures 1A-1H. The NG motifs in the CL0020188 CDR-H2 sequence (SEQ ID NO:5) and CDR-L1 sequence (SEQ ID NO:7) were modified, grafted onto human framework regions and analyzed. Table 2 shows K _D values measured by Biacore and EC ₅₀ values measured by titrated binding assays in HEK293-H6 cells. Figures 1A-1H contain representative dose-response curves of binding to TREM2 expressed by HEK293-H6 cells for humanized and sequence-optimized variants. Variants are represented by filled circles (●) and isotype controls by open circles (○).

Table 2. Binding properties of sequence optimized and humanized variants of CL0020188

As shown in Table 2, humanized and sequence-optimized clones of CL00201088 exhibited similar affinity values for hTREM2 compared to the parental antibody (K _D =9.5 nM) as measured by Biacore. . This is consistent with the cell binding results for HEK293-H6 cells shown in Table 1 and the corresponding dose-response curves shown in Figures 1A-1H. Compared to the parental antibody (EC ₅₀ =0.44 nM), the humanized, sequence-optimized clone showed comparable and sub-nanomolar affinity for TREM2 expressed in HEK293-H6 cells. Taken together, the results demonstrate comparable binding kinetics between the parental antibody and the humanized, sequence-optimized variants.

Results for the ATV:TREM2 variants described in Example 3 are summarized in Table 3 below. An exemplary cell binding curve based on binding to human TREM2-expressing HEK cells and analysis by FACS is shown in FIG.

(Table 3) ATV: Summary of TREM2 characteristics

Regarding TREM2-dependent pSyk signaling in HEK-H6 cells, ability to promote survival of human macrophage cells, and ability to modulate lipid accumulation in iPSC-derived human microglial cells (hereinafter referred to as "iPSC microglia" or "iMG"). , evaluated the antibodies. FIG. 3 shows results for an ATV:TREM2 variant (ATV:TREM2#3) and the corresponding anti-TREM2 antibody. ATV:TREM2 is able to activate pSyk signaling in HEK293-H6 cells expressing TREM2 to a significantly greater extent than the corresponding TREM2 antibody, which is due to the addition of ATV to the molecule. It shows that potency can be increased (Fig. 3). In addition, ATV:TREM2 induced macrophage survival with an _EC50 of 4.1 + 0.3 nM. Finally, the anti-TREM2 antibody demonstrated the ability to reduce lipid accumulation in myelin-treated iMG (Figures 4A and 4B), with an _IC50 for inhibition of lipid storage of 0.20 nM (97.7 + 0.3% maximal inhibition). there were.

Additional studies were performed to investigate the ability of ATV:TREM2 to reduce lipid accumulation. Figures 5A-5F and Figures 6A-6C show that a representative ATV:TREM2 variant (ATV:TREM2#3) reduces lipid accumulation while increasing fatty acid oxidation intermediates, which are associated with mitochondrial suggesting a potential role for ATV:TREM2 in enhancing function. Cells treated with oleate lipid stimulation (33 μM) followed by incubation with ATV:TREM2 (iMG) were able to reduce lipid accumulation as shown by Bodipy staining (FIGS. 5A and 5B). LCMS analysis of iMG incubated with ATV:TREM2 for 48 h after treatment with myelin for 24 h showed that ATV:TREM2 decreased triglyceride (TG) species while simultaneously reducing beta-oxidation intermediates (acylcarnitines) and TCA cycle intermediates. (FIGS. 5C-5F). FIG. 5C provides a heatmap showing all TG, acylcarnitine, and TCA cycle intermediate species that exhibited a fold change greater than 1.5-fold (p<0.05). 5D-5F, on the other hand, show representative species changes in vehicle- and myelin-stimulated iMG incubated with ATV:TREM2 or isotype control after stimulation. Figures 6A-6C show changes in specific TG, acylcarnitine, and TCA cycle intermediate species in iMG incubated with ATV:TREM2 or isotype control after myelin stimulation. As shown in Figures 6A-6C, ATV:TREM2 increased certain short-chain acylcarnitine species while reducing all species of TGs and ceramides, suggesting that ATV:TREM2 enhances mitochondrial function. It shows that you get

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

Example 6. Role of ATV:TREM2 in Lysosomal Dysfunction The role of ATV:TREM2 in lysosomal function was investigated. Progranulin (PGRN) and bis(monoacylglycero)phosphate (BMP) levels were measured in iPSC-derived microglial cells (“iMG”) treated with ATV:TREM2 variants to assess effects on lysosomal function. bottom.

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

For measurement of PGRN levels, Thermo Scientific 384-well Maxisorp plates were coated with 4 μg/mL capture antibody (R&D anti-PGRN antibody, DuoSet ELISA kit, Cat# DY2420) diluted in phosphate-buffered saline (PBS), Incubate overnight at 4°C. Sample wells were blocked with PBS containing 3% BSA for 90 minutes. Cell samples were diluted 1:10 in PBS containing 3% BSA and added to each sample well on the plate, followed by incubation at room temperature for 90 minutes. A detection antibody (R&D PGRN antibody, 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-conjugated streptavidin (R&D SA-HRP, DuoSet ELISA kit, catalog number DY2420) was diluted 1:200 and added to each sample well. Plates were incubated for 20 minutes at room temperature. After washing the sample wells with PBS, a coloring reagent (TMB substrate) was added and allowed to react _for 5 minutes, after which the reaction was stopped with 4N _H2SO4 . Absorbance was measured using a BioTek Synergy Neo2 plate reader and PGRN levels were determined by interpolation from a standard curve fitted to a 4-parameter logistic curve. As shown in Figure 8, incubation with ATV:TREM2 increased PGRN levels in both supernatants and cell lysates compared to isotype controls.

To assess the effect on BMP levels, iMG were stimulated with myelin or vehicle for 24 hours followed by incubation with ATV:TREM2#3 (100 nM) for 48 hours. Cellular lipids were extracted by addition of methanol containing internal standard mixture and liquid chromatography-mass spectrometry (LC-MS) on a Q-trap 6500 (SCIEX) similar to that described in International PCT Publication No. WO2020/112889. /MS) to measure BMP abundance. BMP species were quantified using an internal standard BMP (14:0_14:0) and identified based on their retention times and MRM properties. Quantification was performed using MultiQuant 3.02 (Sciex) after correction for isotopic overlap. BMP species were normalized to the median of all measured lipid species contents. Protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce, Rockford, Ill., USA). Representative BMP results are shown in FIG. As shown in FIG. 9, iMG treated with ATV:TREM2 after myelin stimulation had reduced levels of BMP species, suggesting a potential rescue against myelin-induced lysosomal stimulation.

As shown in Figures 8 and 9, incubation of iMG with ATV:TREM2 increased PGRN levels and modified myelin-induced BMP levels, indicating a role for ATV:TREM2 in regulating lysosomal effects. be 8 and 9, the ATV:TREM2#3 isotype control (ISO) contains the sequences shown in Table 5.

Example 7. Role of ATV:TREM2 in Mitochondrial Respiration To assess effects on mitochondrial respiration, using the Seahorse XFe96 Analyzer (Agilent) in iPSC-derived microglial cells (“iMG”) treated with ATV:TREM2 variants or isotype control, Oxygen consumption was measured using the materials and protocol of the Seahorse XF Palmitate Oxidation Stress Kit (Agilent 103693). Cells were cultured with ATV:TREM2#3 (100 nM) or isotype control for 72 hours on XF96 microplates (Agilent, catalog number 102416) prior to Seahorse experiments. 0.5 mM glucose (Agilent 103577), 1 mM glutamine (Agilent 103579), 0.5 mM L-carnitine (part of the Seahorse XF Palmitate Oxidation Stress Kit), and 1% Hyclone FBS were added 16 hours prior to Seahorse experiments. Cells were exposed to substrate-restricted medium consisting of Seahorse XF RPMI (Agilent 103576). Immediately prior to the experiment, cells were dosed with either palmitate-BSA conjugate (166 μM) or BSA control. (1) etomoxir (4 μM) (carnitine palmitoyltransferase 1 (CPT1) inhibitor) or vehicle, (2) oligomycin (1.5 μM) (ATP synthase complex V inhibitor), (3) carbonyl cyanide 4-( using sequential injections of (trifluoromethoxy)phenylhydrazone (FCCP, mitochondrial uncoupler, 1 μM) and (4) rotenone/antimycin (0.5 mM each, complex I and complex III inhibitors, respectively). The mitochondrial respiratory capacity of cells was assessed. Oxygen consumption rate during the experiment was measured by a Seahorse analyzer (Agilent). A summary of experimental conditions for assessing mitochondrial respiration is shown in Table 4.

(Table 4) Experimental conditions

A representative kinetic graph of oxygen consumption is shown in FIG. 10A and a bar graph showing the maximal respiratory capacity of the cells is shown in FIG. 10B. As shown, ATV:TREM2 increases maximal respiration to the same extent as the fatty acid substrate palmitate (PAL). This effect was diminished in the presence of CPT1 inhibitors. The results indicate that ATV:TREM2 increases maximal mitochondrial respiration, and this effect appears to be conferred by increased capacity for fatty acid oxidation.

Example 8. ATV:TREM2 Property Comparison The properties of ATV:TREM2#1 and ATV:TREM2#3 were compared with those of reference antibodies that bind to TREM2, described in WO2019/028292. The heavy and light chain sequences of Reference Antibody #1 (“Ref. Ab. #1”) are represented by SEQ ID NOs:74 and 75, respectively. The heavy and light chain sequences of Reference Antibody #2 ("Ref. Ab. #2") are represented by SEQ ID NOs:76 and 75, respectively. The isotype control heavy and light chain sequences for each anti-TREM2 antibody are shown in Table 5.

(Table 5) Isotype control sequences

ATV:TREM2 is more potent in vitro and induces less inflammation (less cytokine release) than the reference Ab.
Anti-TREM2 antibodies were evaluated using the human macrophage cell survival assay described in Example 4. FIG. 11A shows cell viability dose-response curves with anti-TREM2 antibodies, where “ISO” refers to ATV:TREM2#3 isotype control (Table 5). The corresponding efficacy (EC50) and maximal response (Emax) values determined from the dose-response curves are shown in Figures 11B and 11C. Each mark represents a single value obtained from human cell donors (n=3). The results shown in FIGS. 11A-11C demonstrate that ATV:TREM2#3 is more potent in promoting human macrophage survival in vitro than the reference antibodies #1 and #2.

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

Taken together, ATV:TREM2#3, reference antibody #1, and reference antibody #2 are able to promote survival and proliferation of human macrophages (FIG. 11A). However, ATV:TREM2#3 showed greater potency on cell survival and reduced the overall cytokine signature compared to reference antibodies #1 and #2 (FIGS. 11B, 11C, and 12).

ATV:TREM2 can reduce triglyceride species levels after stimulation.
Anti-TREM2 antibodies were assessed by a lipid storage assay (using 10 μM oleate stimulation) as described in Example 4. Results for ATV:TREM2#3, reference antibody #1, and reference antibody #2 are shown in Figures 13A-13E.

Figures 13A-13C show volcano plots with p<0.05 cutoff and >1.5-fold fold change for triglyceride species in iPSC-derived microglial cells ("iMG") quantified by LCMS. Data were normalized to isotype controls for each antibody. As shown in Figures 13A-13C, ATV:TREM2#3 is able to modulate triglyceride (TG) species after oleate administration, whereas the reference antibody does not show altered levels of TG species after oleate stimulation. did not change significantly. Figures 13D and 13E show bar graphs of representative TG species measurements, with data normalized to the ATV:TREM2#3 isotype control.

ATV:TREM2 can regulate TREM2 levels in vitro.
To assess how anti-TREM2 antibodies affect TREM2 levels in iPSC-derived microglial cells (“iMGs”), iMGs were incubated with ATV:TREM2#3 or reference antibody #1 at various concentrations for 72 h. , followed by measurement of TREM2 levels in iMG cell lysates and cell culture media. TREM2 was measured as follows. Briefly, MSD small spot streptavidin plates (Meso Scale Discovery) were coated with biotinylated goat anti-hTREM2 polyclonal antibody (R&D Systems, BAF1828) for 1 hour at room temperature. Plates were then blocked with MSD Block A buffer (Meso Scale Discovery) for 1 hour at room temperature. After samples and standards were prepared/diluted in assay buffer (25% MSD Block A buffer in TBST) and blocked, 30 μL of samples and standards were loaded onto the plate. After incubation for 1 hour at room temperature, plates were washed with TBST and allowed to bind primary antibody (ATV: TREM2#3) for 1 hour at room temperature. Diluted sulfo-tagged goat anti-human IgG (Southern Biotech, 2049-01) was then added to the plate and incubated for 1 hour at room temperature. After washing with TBST, the MSD plates were developed using 2x MSD reading buffer T and subsequently detected using an MSD Sector plate reader. MSD values were converted to absolute quantification of TREM2 by standard curve fitting using Meso Scale Discovery software (Discovery Workbench).

Figures 14A and 14B show plots of TREM2 levels versus antibody concentration in iMG cell lysates and cell culture media after incubation with anti-TREM2 antibodies. TREM2 levels for each antibody were normalized to its own isotype control. In Figures 14A and 14B, "ISO" represents the ATV:TREM2#3 isotype control. As shown in FIG. 14A, total TREM2 levels increased with increasing amounts of antibody in cell lysates of iMG treated with ATV:TREM2#3 and reference antibody #1. In contrast, levels of soluble TREM2 decreased with increasing amounts of antibody in the cell culture medium of antibody-treated cells.

ATV:TREM2 exhibits a superior pharmacokinetic profile in non-human primates.
The pharmacokinetic (PK) properties of anti-TREM2 antibodies were investigated in non-human primates. Briefly, young adult/adult male cynomolgus monkeys ranging in age from 36 to 53 months were administered a single dose of 30 mg/kg of test article intravenously (Table 6, n=5 per cohort). CSF samples were collected at 24, 168, and 336 hours after dosing.

The results are shown in Table 6 and FIG. Table 6 shows the PK values of anti-TREM2 antibodies in non-human primate cerebrospinal fluid (CSF). ATV: The PK values of the TREM2 variants indicate that these antibodies are more potent than the TREM2 Ab (no transferrin receptor binding capacity; heavy and light chain sequences represented by SEQ ID NOs:83 and 54, respectively) and reference antibody #1 in CSF. have higher exposure compared to

(Table 6) CSF pharmacokinetic profiles of anti-TREM2 antibodies in non-human primates

Figure 15 shows the PK profile of anti-TREM2 antibody in the CSF of administered non-human primates. As shown in Figure 15 and Table 6, ATV:TREM2 shows at least a 2-fold increase in CSF PK compared to reference antibody #1.

Example 9. Characterization of ATV:TREM2 in Humanized TREM2 Mice To investigate the in vivo effects of ATV:TREM2, BAC transgenic mice expressing human TREM2 were generated. These mice were bred with human transferrin receptor knock-in mice described in US Pat. No. 10,143,187 to allow characterization of the ATV:TREM2 molecules described herein.

Generation of a Human TREM2 BAC Transgenic Model ATV: To investigate the in vivo effects of TREM2, several cells expressing human TREM2 were generated by using the modified BAC DNA CTD-2210D2 (ThermoFisher Scientific; Catalog No. 96012). A transgenic mouse strain was generated. In the original BAC DNA CTD-2210D2, the human TREM2 coding region and its regulatory elements are flanked by two other TREM-like genes, TREML1 and TREML2. To avoid interference from these TREM-like genes, their expression was silenced by deleting exon 1 from TREML1 and exon 3 from TREML2. This engineered BAC CTD-2210D2 DNA construct was injected into the pronuclei of fertilized mouse ova of C57BL/6J mice. Two independent founder lines (termed TB36 and TB45) were obtained and shown to have germline transmission.

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

Human TREML1 and TREML2 were not detected in either TB36 or TB45, indicating successful deletion of these genes. qPCR analysis shows that TB36 and TB45 have two and one copy of the human TREM2 transgene, respectively, corresponding to higher human TREM2 expression in TB36 compared to TB45 in brain and peripheral tissues. In vitro survival assays showed that human TREM2 agonistic antibodies elicited a stronger response in strain TB36 than in strain TB45.

Based on this ex vivo and in vitro characterization, TB36 was selected for the following breeding and in vivo studies. Hemizygous TB36 mice were backcrossed to C57BL/6J three more times and then bred to hTfR KI mice (described in US Pat. No. 10,143,187) to human TREM2 BAC hemizygosity; Sexual mice were generated for in vivo studies.

Pharmacokinetic and Pharmacodynamic Responses by ATV:TREM2 in TB36/hTfR KI Mice Isotype control (ATV:RSV) (100 mg/kg) was administered intravenously to TB36/hTfR KI mice on day 0 and mice were sacrificed on day 1 or 4 post-dosing for ex vivo analysis. At the time of sacrifice, animals were anesthetized by intraperitoneal injection of 2.5% Avertin. Terminal blood was collected from cardiac puncture into EDTA tubes, gently inverted (10 times), and then centrifuged at 15,350 g for 7 minutes at 4°C. Plasma (upper layer) was transferred to 1.5 ml Eppendorf tubes and stored at -80°C until measurement. After blood collection, CSF samples were collected by pre-drawn glass capillary tubes from the cisterna magna, then transferred to 0.5 mL Protein LoBind Eppendorf tubes and centrifuged at 12,700 rpm for 7 minutes at 4°C. Supernatants of CSF samples were snap frozen on dry ice and stored at -80°C until measurement. Animals were then perfused with cold PBS and the brain dissected to separate the two hemispheres. The right hemibrain was immersion-fixed in 4% paraformaldehyde for 24 hours at 4°C, then transferred to phosphate-buffered saline (PBS) solution containing 0.1% sodium azide, treated with 30% sucrose and subjected to sectioning. Stored until ready. The left hemibrain was cut in two and snap frozen into two tubes each for PK measurements and linkage/cytokine analysis of other targets.

Plasma and brain levels of human IgG were assessed on days 1 and 4 after dosing. It is shown in Table 7 below.

Table 7. Plasma and brain pharmacokinetic profiles in TB36/hTfR-KI mice

Effect of ATV:TREM2 on Microglial Proliferation. #3 or ATV: mice were dosed intraperitoneally on days 0, 1, 2 and 3 after treatment with RSV.

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

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

ATV:TREM2 transiently increases brain cytokine levels in TB36/hTfR KI mice.
To determine the effect of ATV:TREM2 on cytokine (e.g., chemokine) levels, terminal plasma and brain lysates (Cell Cytokine levels were measured in Signaling lysis buffer #9803). Treatment with ATV:TREM2#3 did not alter cytokine levels measured in plasma, but several cytokine levels (eg IP-10 and MCP-5) measured in the brain 24 hours after dosing. increased sharply and levels of these cytokines returned to baseline levels 96 hours after dosing. ATV:TREM2#3 at 3 mg/kg had the greatest effect on brain cytokine levels (Figures 18A and 18B).

ATV:TREM2 increases brain CSF1R levels in TB36/hTfR KI mice.
To determine the effect of ATV:TREM2 on glial marker CSF1R levels, a commercially available ELISA kit (Abcam ab240681) was used in TB36/hTfR KI mouse brain lysates (prepared with Cell Signaling lysis buffer #9803). CSF1R protein levels were measured. Increasing doses of ATV:TREM2#3 were found to increase CSF1R protein levels at 1 and 4 days post-treatment (Figure 19).

Plasma PK Profile of ATV:TREM2 A proportional increase in plasma PK was observed at doses ranging from 1 mg/kg to 100 mg/kg of ATV:TREM2#3. Plasma concentrations at 24 hours were not significantly different for ATV:TREM2#3, reference antibody #2, and TREM2 Ab matched at a dose of 30 mg/kg. Reference antibody #2 appeared to have lower clearance compared to ATV:TREM2#3 and TREM2 Abs at the same dose (Figure 20).

ATV:TREM2 Brain PK Profile At 24 hours, brain concentrations of 10 mg/kg ATV:TREM2#3 were similar to 30 mg/kg TREM2 Ab and 1-3 mg/kg ATV:TREM2#3 brain Internal concentrations were similar to reference antibody #2 at 30 mg/kg. Greater than proportional increases were observed at doses of 3 mg/kg to 10 mg/kg of ATV:TREM2#3, and proportional increases were observed at other doses. At 96 hours, brain concentrations of 10 mg/kg ATV:TREM2#3 were similar to 30 mg/kg TREM2 Ab and reference antibody #2 (Figure 21). Overall, brain uptake of ATV:TREM2#3 is more effective than TREM2 Ab and reference antibody #2.

(Table 8) Abbreviated Sequence Listing

Claims (102)

1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(a) i. a CDR-H1 sequence comprising the sequence of GFTFT-α ₆ -FYMS (SEQ ID NO: 28), wherein α ₆ is D or N;
ii. VIRN-β ₅ -β ₆ -N-β ₈ -YT-β ₁₁ -β ₁₂ -YNPS-VKG (SEQ ID NO: 29) (in sequence , _β5 is K or R, _β6 is A or P, _β8 is G or A, _β11 is A or T, _β12 is G or D). CDR-H2 sequences,
iii. comprising the sequence γ ₁ -RL-γ ₄ -YGFDY (SEQ ID NO: 30), in which γ ₁ is A or T and γ ₄ is T or S CDR-H3 sequences,
iv. QSSKSXLLHS-δ ₁₀ -GKTYLN (SEQ ID NO: 31) (where δ ₁₀ is N or T) a CDR-L1 sequence comprising the sequence of
v. vi. a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID NO: 8); a variable region comprising a CDR-L3 sequence comprising the sequence QQFLE-φ ₆ -PFT (SEQ ID NO: 32) (where φ ₆ is Y or F);
(b) a first Fc polypeptide modified to specifically bind to the transferrin receptor; and (c) a second Fc polypeptide. 2. The antibody of claim 1, wherein said CDR-H1 sequences are selected from SEQ ID NO:4 or 12. 3. The antibody of claim 1 or 2, wherein said CDR-H2 sequences are selected from SEQ ID NOs:5, 13, or 25. 4. The antibody of any one of claims 1-3, wherein the CDR-H3 sequence is selected from SEQ ID NOs:6, 14, or 17. The antibody of any one of claims 1-4, wherein the CDR-L1 sequence is selected from SEQ ID NO:7 or 23. The antibody of any one of claims 1-5, wherein said CDR-L3 sequence is selected from SEQ ID NO:9 or 18. The variable region is
(a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, CDR-L1 containing the amino acid sequence of SEQ ID NO: 7 , CDR-L2 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, comprising the amino acid sequence of SEQ ID NO:5 CDR-H2, 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 comprising the amino acid sequence of SEQ ID NO: 18. -L3, 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, the amino acid sequence of SEQ ID NO: 7 CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, 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, SEQ ID NO:25 CDR-H2 comprising the amino acid sequence, 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 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, SEQ ID NO:7 CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and sequences CDR-L3 comprising the amino acid sequence of No. 9, or (g) CDR-H1 comprising the amino acid sequence of SEQ ID No. 4, CDR-H2 comprising the amino acid sequence of SEQ ID No. 25, CDR-H3 comprising the amino acid sequence of SEQ ID No. 17 , CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9.
The antibody of any one of claims 1-6, comprising 8. The method of claims 1-7, wherein said variable region comprises a _VH sequence having at least 85% sequence identity to any one of SEQ ID NOs: 2, 10, 15, 19, 21, 24, and 26. The antibody of any one of claims 1 to 3. 9. The antibody of claim 8, wherein said _VH sequence has at least 90% sequence identity to SEQ ID NO:15. 10. The antibody of claim 9, wherein said _VH sequence has at least 95% sequence identity to SEQ ID NO:15. 11. The antibody of claim 10, wherein said _VH sequence comprises SEQ ID NO:15. 9. The antibody of claim 8, wherein said _VH sequence has at least 90% sequence identity to SEQ ID NO:24. 13. The antibody of claim 12, wherein said _VH sequence has at least 95% sequence identity to SEQ ID NO:24. 14. The antibody of claim 13, wherein said _VH sequence comprises SEQ ID NO:24. 15. Any one of claims 1-14, wherein said variable region comprises a V _L sequence having at least 85% sequence identity to any one of SEQ ID NOS: 3, 11, 16, 20, 22, and 27. 2. The antibody of item 1. 16. The antibody of claim 15, wherein said _VL sequence has at least 90% sequence identity to SEQ ID NO:16. 17. The antibody of claim 16, wherein said _VL sequence has at least 95% sequence identity to SEQ ID NO:16. 18. The antibody of claim 17, wherein said _VL sequence comprises SEQ ID NO:16. 16. The antibody of claim 15, wherein said _VL sequence has at least 90% sequence identity to SEQ ID NO:22. 20. The antibody of claim 19, wherein said _VL sequence has at least 95% sequence identity to SEQ ID NO:22. 21. The antibody of claim 20, wherein said _VL sequence comprises SEQ ID NO:22. 16. The antibody of claim 15, wherein said _VL sequence has at least 90% sequence identity to SEQ ID NO:27. 23. The antibody of claim 22, wherein said _VL sequence has at least 95% sequence identity to SEQ ID NO:27. 24. The antibody of claim 23, wherein said _VL sequence comprises SEQ ID NO:27. The variable region is
(a) a _VH sequence comprising SEQ ID NO: 15 and a VL sequence comprising SEQ ID NO: 16, or (b) a _VH sequence comprising SEQ ID NO: 19 and a _VL sequence comprising SEQ ID NO: 20, or ₍ c) a sequence a _VH sequence comprising SEQ ID NO:21 and a _VL sequence comprising SEQ ID NO:20; or (d) a _VH sequence comprising SEQ ID NO:19 and a _VL sequence comprising SEQ ID NO:22; or (e) comprising SEQ ID NO:21 a _VH sequence and a VL sequence comprising _SEQ ID NO:22, or (f) a _VH sequence comprising SEQ ID NO:24 and a _VL sequence comprising SEQ ID NO:20, or (g) a _VH sequence comprising SEQ ID NO:26, and SEQ ID NO:20, or (h) a _VH sequence containing SEQ ID NO:24 and a _VL sequence containing SEQ ID NO:22, or (i) a _VH sequence containing _SEQ ID NO:26 and SEQ ID NO:22 or (j) a _VH sequence comprising SEQ ID NO:2 and a _VL sequence comprising SEQ ID NO:3, or (k) a _VH sequence comprising _SEQ ID NO:10 and a VL sequence comprising SEQ ID NO: ₁₁ or (l) a V _H sequence comprising SEQ ID NO:24 and a V _L sequence comprising SEQ ID NO:27. wherein said 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 at position 389, according to EU numbering , Ala, or Val, 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. Antibodies as described in. 27. The antibody of claim 26, wherein said first Fc polypeptide binds to the apical domain of said transferrin receptor. 28. The antibody of claim 26 or 27, which has improved brain uptake compared to an antibody with wild-type Fc dimers. 29. Any one of claims 1-28, wherein said first Fc polypeptide has a T366W substitution and said second Fc polypeptide has a T366S, L368A, and Y407V substitution, according to EU numbering. antibody. 29. Any one of claims 1-28, wherein said first Fc polypeptide has T366S, L368A and Y407V substitutions and said second Fc polypeptide has T366W substitutions according to EU numbering. antibody. The antibody of any one of claims 1-30, wherein said first Fc polypeptide and/or said second Fc polypeptide comprise modifications that reduce effector function. 32. The antibody of claim 31, wherein said modifications that reduce effector function comprise substitutions that are Ala at position 234 and Ala at position 235 according to EU numbering. said first Fc polypeptide and/or said second Fc polypeptide,
33. The antibody of any one of claims 1-32, comprising an amino acid modification to the native Fc sequence that increases serum half-life. 34. The antibody of claim 33, wherein said amino acid modifications comprise substitutions that are Leu at position 428 and Ser at position 434 according to EU numbering. 35. Any one of claims 1-34, wherein said first Fc polypeptide comprises the sequence of SEQ ID NO:41 or SEQ ID NO:64 and said second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO:63 Antibodies as described in. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:41;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively 36. The antibody of claim 35, comprising two light chains. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:64;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively 36. The antibody of claim 35, comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:42;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
37. The antibody of claim 35 or 36, comprising (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:65;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
38. The antibody of claim 35 or 37, comprising 35. Any one of claims 1-34, wherein said first Fc polypeptide comprises the sequence of SEQ ID NO:44 or SEQ ID NO:66 and said second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO:63 Antibodies as described in. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:44;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively 41. The antibody of claim 40, comprising two light chains. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:66;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively 41. The antibody of claim 40, comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:45;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
42. The antibody of claim 40 or 41, comprising (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:67;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
43. The antibody of claim 40 or 42, comprising 35. Any one of claims 1-34, wherein said first Fc polypeptide comprises the sequence of SEQ ID NO:47 or SEQ ID NO:68 and said second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO:63 Antibodies as described in. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:47;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively 46. The antibody of claim 45, comprising two light chains. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:68;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively 46. The antibody of claim 45, comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
47. The antibody of claim 45 or 46, comprising (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
48. The antibody of claim 45 or 47, comprising 35. Any one of claims 1-34, wherein said first Fc polypeptide comprises the sequence of SEQ ID NO:47 or SEQ ID NO:68 and said second Fc polypeptide comprises the sequence of SEQ ID NO:61 or SEQ ID NO:84 Antibodies as described in. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:47;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:61, and (iii) a _VL comprising SEQ ID NO:22, respectively 51. The antibody of claim 50, comprising two light chains. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:68;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:84, and (iii) a _VL comprising SEQ ID NO:22, respectively 51. The antibody of claim 50, comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:52; chain (LC)
52. The antibody of claim 50 or 51, comprising (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:72; chain (LC)
53. The antibody of claim 50 or 52, comprising 35. Any one of claims 1-34, wherein said first Fc polypeptide comprises the sequence of SEQ ID NO:50 or SEQ ID NO:70 and said second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO:63 Antibodies as described in. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:50;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively 56. The antibody of claim 55, comprising two light chains. (i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said first Fc polypeptide comprising SEQ ID NO:70;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and said second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively 56. The antibody of claim 55, comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
57. The antibody of claim 55 or 56, comprising (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:71;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
58. The antibody of claim 55 or 57, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:41;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:42;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
61. The antibody of claim 60, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:64;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:65;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
63. The antibody of claim 62, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:44;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:45;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
65. The antibody of claim 64, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:66;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:67;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
67. The antibody of claim 66, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(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) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
69. The antibody of claim 68, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:68;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
71. The antibody of claim 70, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(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) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:61, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:52; chain (LC)
73. The antibody of claim 72, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:68;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:61, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:72; chain (LC)
75. The antibody of claim 74, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:50;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:39, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53; chain (LC)
77. The antibody of claim 76, comprising 1. An isolated antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising:
(i) a first heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a first Fc polypeptide comprising SEQ ID NO:70;
(ii) a second heavy chain (HC) comprising a _VH comprising SEQ ID NO:24 and a second Fc polypeptide comprising SEQ ID NO:63, and (iii) a _VL comprising SEQ ID NO:22, respectively. said antibody comprising two light chains. (i) a first heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:71;
(ii) a second HC comprising or consisting of the amino acid sequence set forth in SEQ ID NO:73; chain (LC)
79. The antibody of claim 78, comprising 80. The antibody of any one of claims 1-79, which decreases the level of soluble TREM2 protein (sTREM2). 81. The antibody of any one of claims 1-80, which enhances TREM2 activity. 82. The antibody of any one of claims 1-81, which enhances phagocytosis or enhances myeloid cell, microglia, or macrophage migration, differentiation, function, or survival. 83. The antibody of any one of claims 1-82, which enhances microglial function without increasing neuroinflammation. 84. The antibody of any one of claims 1-83, which enhances Syk phosphorylation. 85. The antibody of claim 84, which enhances Syk phosphorylation in the presence of TREM2 ligand. 86. The antibody of any one of claims 1-85, which exhibits cross-reactivity with cynomolgus TREM2 protein. A pharmaceutical composition comprising the isolated antibody of any one of claims 1-86 and a pharmaceutically acceptable carrier. the isolated antibody of any one of claims 1-86 or the pharmaceutical composition of claim 87;
A kit, including instructions for its use and . A method of treating a neurodegenerative disease in a subject comprising administering to said subject the isolated antibody of any one of claims 1-86 or the pharmaceutical composition of claim 87 , said method. The neurodegenerative disease is Alzheimer's disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Aggranular Dementia, Amyotrophic Lateral Sclerosis, Guam Amyotrophic Lateral Sclerosis/Parkinson's Dementia Complex (ALS-PDC), Corticobasal Degeneration, Chronic Traumatic Encephalopathy, Creutzfeldt-Jakob boxer's dementia, diffuse neurofibrillary tangles with calcification, Down's syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, glial tauopathy, cognition Guadeloupe parkinsonism, Guadeloupe PSP, Hallervorden-Spatz disease, hereditary diffuse leukoencephalopathy (HDLS) with spheroids, Huntington's disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Nasu・Hakola disease, neurofibrillary tangle predominance dementia, Niemann-Pick disease type C, pontine nigroderma degeneration, Parkinson's disease, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy, progressive cortex 90. The method of claim 89, selected from the group consisting of inferior gliosis, subacute sclerosing panencephalitis, and tangle only dementia. A method of reducing levels of sTREM2 in a subject having a neurodegenerative disease, comprising administering to said subject the isolated antibody of any one of claims 1-86 or the pharmaceutical composition of claim 87. The above method, comprising administering. A method of enhancing TREM2 activity in a subject having a neurodegenerative disease, comprising administering to said subject the isolated antibody of any one of claims 1-86 or the pharmaceutical composition of claim 87. The above method, comprising: An isolated polynucleotide comprising a nucleotide sequence encoding the antibody of any one of claims 1-86. 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 a nucleotide sequence encoding SEQ ID NOS: 42, 53, and 54. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOS: 45, 53, and 54. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:48, 53, and 54. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:48, 52, and 54. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOs:51, 53, and 54. A vector comprising the polynucleotide of any one of claims 93-99. A host cell comprising a polynucleotide according to any one of claims 93-99 or a vector according to claim 100. 102. A method of expressing an antibody that specifically binds to human myeloid cell-expressed trigger receptor 2 (TREM2), comprising culturing the host cell of claim 101 under conditions suitable for expression of said antibody. The method above.

Images