EP4090682A1 - Anti-trem2 antibodies and methods of use thereof - Google Patents

Anti-trem2 antibodies and methods of use thereof

Info

Publication number
EP4090682A1
EP4090682A1 EP21704639.0A EP21704639A EP4090682A1 EP 4090682 A1 EP4090682 A1 EP 4090682A1 EP 21704639 A EP21704639 A EP 21704639A EP 4090682 A1 EP4090682 A1 EP 4090682A1
Authority
EP
European Patent Office
Prior art keywords
seq
antibody
amino acid
acid sequence
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21704639.0A
Other languages
German (de)
English (en)
French (fr)
Inventor
Mark S. Dennis
Zhenyu Gu
Mihalis S. Kariolis
Cathal S. Mahon
Kathryn M. MONROE
Joshua I. Park
Rachel PROROK
Adam P. Silverman
Bettina VAN LENGERICH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Denali Therapeutics Inc
Original Assignee
Denali Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Denali Therapeutics Inc filed Critical Denali Therapeutics Inc
Priority claimed from PCT/US2021/013200 external-priority patent/WO2021146256A1/en
Publication of EP4090682A1 publication Critical patent/EP4090682A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Triggering receptor expressed on myeloid cells-2 is a transmembrane receptor that is expressed on microglia and is believed 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-Hakola disease, Parkinson’s disease, amyotrophic lateral sclerosis, and frontotemporal dementia. Additionally, altered levels of soluble TREM2 (sTREM2) have been reported in the cerebrospinal fluid of patients having Alzheimer’s disease or frontotemporal dementia who have a mutation in TREM2.
  • antibodies that specifically binds to a human triggering receptor expressed on myeloid cells 2 are provided.
  • the antibodies comprise a modified Fc polypeptide that can bind to a transferrin receptor protein.
  • the antibody can comprise CDRs, a VH, and/or a VL according to any of the exemplary sequences provided herein, and can further comprise a modified Fc polypeptide comprising transferrin receptor-binding mutations as set forth herein.
  • an antibody that specifically binds to TREM2 comprises:
  • a variable region comprising: i. a CDR-H1 sequence comprising the sequence of G-F-T-F-T-0C6-F- Y-M-S (SEQ ID NO:28), wherein oc 6 is D or N; ii. a CDR-H2 sequence comprising the sequence of V-I-R-N-Ps-Pe-N- P 8 -Y-T- n-Pi2-Y-N-P-S-V-K-G (SEQ ID NO:29), wherein b 5 is K or R; b 6 is A or P; b 8 is G or A; bii is A or T; and b ⁇ 2 is G or D; iii.
  • a CDR-H3 sequence comprising the sequence of yi-R-L-y4-Y-G-F- D-Y (SEQ ID NO:30), wherein gi is A or T; and 74 is T or S; iv. a CDR-L1 sequence comprising the sequence of Q-S-S-K-S-L-L- H-S-5io-G-K-T-Y-L-N (SEQ ID NO:31), wherein dio is N or T; v. a CDR-L2 sequence comprising the sequence WMSTRAS (SEQ ID NO: 8); and vi. a CDR-L3 sequence comprising the sequence of Q-Q-F-L-E- ⁇
  • the first Fc polypeptide and the second Fc polypeptide associate to form an Fc dimer.
  • the CDR-H1 sequence is selected from SEQ ID NOS:4 or 12.
  • the CDR-H2 sequence is selected from SEQ ID NOS:5, 13, or 25.
  • the CDR-H3 sequence is selected from SEQ ID NOS:6, 14, or 17.
  • the CDR-L1 sequence is selected from SEQ ID NOSY or 23.
  • the CDR-L3 sequence is selected from SEQ ID NOS:9 or 18.
  • variable region comprises:
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:9; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:9; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:9.
  • variable region comprises a VH sequence that has at least 85% sequence identity to any one of SEQ ID NOS:2, 10, 15, 19, 21, 24, and 26.
  • 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. [0011] 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.
  • variable region comprises a VL sequence has at least 85% sequence identity to any one of SEQ ID NOS:3, 11, 16, 20, 22, and 27.
  • the VL sequence has at least 90% sequence identity to SEQ ID NO: 16. In certain embodiments, the VL sequence has at least 95% sequence identity to SEQ ID NO: 16. In certain embodiments, the VL sequence comprises SEQ ID NO: 16.
  • the VL sequence has at least 90% sequence identity to SEQ ID NO:22. In certain embodiments, the VL sequence has at least 95% sequence identity to SEQ ID NO:22. In certain embodiments, the VL sequence comprises SEQ ID NO:22.
  • the VL sequence has at least 90% sequence identity to SEQ ID NO:27. In certain embodiments, the VL sequence has at least 95% sequence identity to SEQ ID NO:27. In certain embodiments, the VL sequence comprises SEQ ID NO:27.
  • variable region comprises:
  • the first Fc polypeptide comprises: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, or Val at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and Phe at position 421, according to EU numbering.
  • the first Fc polypeptide binds to the apical domain of the transferrin receptor.
  • the antibody has improved brain uptake compared to an antibody having a wild-type Fc dimer.
  • the first Fc polypeptide has a T366W substitution and the second Fc polypeptide has T366S, L368A, and Y407V substitutions, according to EU numbering.
  • the first Fc polypeptide has T366S, L368A, and Y407V substitutions and the second Fc polypeptide has a T366W substitution, according to EU numbering.
  • the first Fc polypeptide and/or the second Fc polypeptide comprises a modification that reduces effector function.
  • the modification that reduces effector function comprises the substitutions of Ala at position 234 and Ala at position 235, according to EU numbering.
  • the first Fc polypeptide and/or the second Fc polypeptide comprises amino acid modifications relative to the native Fc sequence that extend serum half- life.
  • the amino acid modifications comprise substitutions of Leu at position 428 and Ser at position 434, according to EU numbering.
  • the first Fc polypeptide comprises the sequence of SEQ ID NO:41 or SEQ ID NO:64
  • the second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO: 63
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:41; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:64; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:64
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:42; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:65; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the first Fc polypeptide comprises the sequence of SEQ ID NO:44 or SEQ ID NO:66
  • the second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO: 63
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:44; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:66; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:66
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:45; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:67; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the first Fc polypeptide comprises the sequence of SEQ ID NO:47 or SEQ ID NO:68
  • the second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO: 63
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:47; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:48; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:69; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the first Fc polypeptide comprises the sequence of SEQ ID NO:47 or SEQ ID NO:68
  • the second Fc polypeptide comprises the sequence of SEQ ID NO:61 or SEQ ID NO:84
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:47; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:61; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • the anibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:84; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:84
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:48; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:52; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:69; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the first Fc polypeptide comprises the sequence of SEQ ID NO:50 or SEQ ID NO:70
  • the second Fc polypeptide comprises the sequence of SEQ ID NO:39 or SEQ ID NO: 63
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:50; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:70; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:70
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:51; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:71; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:41; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:41
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:42; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:64; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:64
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:65; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:44; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:44
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:45; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:66; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:66
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:67; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:47; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:47
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:48; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:69; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:47 (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:61; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:47
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:61
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:48;
  • a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 52;
  • a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:61; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:68
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:61
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:69; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:72; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:50; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:50
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:39
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:51; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:53; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the disclosure provides an isolated antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antibody comprises: (i) a first heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:70; (ii) a second heavy chain (HC) comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63; and (iii) two light chains each comprising a VL comprising SEQ ID NO:22.
  • HC first heavy chain
  • HC comprising a VH comprising SEQ ID NO:24 and the first Fc polypeptide comprising SEQ ID NO:70
  • a second heavy chain comprising a VH comprising SEQ ID NO:24 and the second Fc polypeptide comprising SEQ ID NO:63
  • two light chains each comprising a VL comprising SEQ ID NO:22.
  • the antibody comprises: (i) a first heavy chain (HC) that comprises or consists of the amino acid sequence set forth in SEQ ID NO:71; (ii) a second HC that comprises or consists of the amino acid sequence set forth in SEQ ID NO:73; and (iii) a first and a second light chain (LC) that each comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.
  • HC first heavy chain
  • LC first and a second light chain
  • the antibody decreases levels of soluble TREM2 protein (sTREM2).
  • sTREM2 soluble TREM2 protein
  • the antibody enhances TREM2 activity.
  • the antibody enhances phagocytosis or enhances the migration, differentiation, function, or survival of myeloid cells, microglia, or macrophages.
  • the antibody enhances microglia function without increasing neuroinflammation.
  • the antibody enhances Syk phosphorylation.
  • the antibody enhances Syk phosphorylation in the presence of a TREM2 ligand.
  • the antibody exhibits cross-reactivity with a cynomolgus TREM2 protein.
  • the disclosure provides a pharmaceutical composition comprising the isolated antibody described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides a kit comprising: the isolated antibody described herein or the pharmaceutical composition described herein; and instructions for use thereof.
  • the disclosure provides a method of treating a neurodegenerative disease in a subject, comprising administering to the subject the isolated antibody described herein or the pharmaceutical composition described herein.
  • the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis/parkinsonism- dementia complex of Guam (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, Guadeloupe
  • the disclosure provides a method of decreasing levels of sTREM2 in a subject having a neurodegenerative disease, comprising administering to the subject the isolated antibody described herein or the pharmaceutical composition described herein.
  • the disclosure provides a method of enhancing TREM2 activity in a subject having a neurodegenerative disease, comprising administering to the subject the isolated antibody described herein or the pharmaceutical composition described herein.
  • the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding the antibody described herein.
  • 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.
  • the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOS:42, 53, and 54.
  • the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOS:45, 53, and 54.
  • the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOS:48, 53, and 54.
  • the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOS:48, 52, and 54. [0049] In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NOS:51, 53, and 54.
  • the disclosure provides a vector comprising the polynucleotide described herein.
  • the disclosure provides a host cell comprising the polynucleotide described herein or the vector described herein.
  • the disclosure provides a method of expressing an antibody that specifically binds to a human triggering receptor expressed on myeloid cells 2 (TREM2), comprising: culturing the host cell described herein in under conditions suitable for expression of the antibody.
  • TERT2 human triggering receptor expressed on myeloid cells 2
  • FIGS. 1A-1H include dose-titrated cell binding curves in human TREM2-Dapl2 overexpressing HEK cells for humanized and sequence optimized variants of a representative anti-TREM2 antibody (CL0020188).
  • FIG. 2 includes dose-response binding curves to human TREM2 in HEK 293 cells for a representative ATV:TREM2 and corresponding anti-TREM2 antibody with non- transferrin-binding Fc (“anti-TREM2”).
  • FIG. 3 includes dose-response curves of pSyk signal activation by a representative ATV:TREM2 and corresponding anti-TREM2 antibody (TREM2 IgG) in TREM2-expressing HEK 293 cells.
  • FIGS. 4A and 4B include dose-response curves of lipid clearance in iPSC microglia in response to treatment with a representative ATV:TREM2.
  • FIGS. 5A and 5B show representative images of lipid accumulation in iPSC- derived microglial cells treated with ATV:TREM2 after oleic acid challenge (FIG. 5 A) with quantification of lipid accumulation in treated cells (FIG. 5B).
  • FIG. 5C is a heat map illustrating the modulation of levels of triglyceride, acyl carnitine, and TCA cycle intermediate species in iPSC-derived microglial cells treated with ATV:TREM2 after myelin challenge.
  • FIGS. 5D-5F include bar graphs illustrating the change in levels of representative triglyceride, acyl carnitine, and TCA cycle intermediate species levels in iPSC-derived microglial cells treated with ATV:TREM2 after myelin challenge.
  • FIGS. 6A-6C include bar graphs illustrating the change in levels of specific triglyceride, ceramide, and acyl carnitine species in iPSC-derived microglial cells treated with ATV:TREM2 after myelin challenge.
  • FIG. 7A includes representative images from a Western blot of mTOR signal pathway targets in iPSC-derived microglia treated with ATV:TREM2.
  • FIGS. 7B-7E include plots illustrating the change in levels of mTOR signal pathway targets in iPSC-derived microglia treated with ATV:TREM2.
  • FIG. 8 is a bar graph illustrating the change in levels of progranulin (PGRN) in iPSC-derived microglia treated with ATV:TREM2.
  • FIG. 9 is a bar graph illustrating the change in levels of a representative bis(monoacylglycero)phosphate (BMP) species in iPSC-derived microglia treated with ATV:TREM2.
  • BMP bis(monoacylglycero)phosphate
  • FIG. 10A is a representative kinetic graph of oxygen consumption in iPSC-derived microglial cells treated with ATV:TREM2.
  • FIG. 10B is a bar graph illustrating the maximal respiratory capacity of iPSC- derived microglial cells treated with ATV:TREM2 in the presence and absence of a CPT1 inhibitor.
  • FIG. 11A is a dose-response curve for cell viability in human macrophage cells treated with anti-TREM2 antibodies.
  • FIGS. 11B and 11C include bar graphs illustrating EC50 and Emax for the dose- response curves in FIG. 11 A.
  • FIG. 12 is a heat map of relative cytokine release in human macrophage cells treated with anti-TREM2 antibodies.
  • FIGS. 13A-13C include volcano plots that illustrate relative changes in triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibodies.
  • FIGS. 13D and 13E include bar graphs showing the change in levels of representative triglyceride species in iPSC-derived microglial cells treated with anti-TREM2 antibodies.
  • FIG. 14A is a plot of TREM2 level as a function of antibody concentration in the cell lysate of iPSC-derived microglial cells treated with anti-TREM2 antibodies.
  • FIG. 14B is a plot of TREM2 level as a function of antibody concentration in the cell culture media of iPSC-derived microglial cells treated with anti-TREM2 antibodies.
  • FIG. 15 is a plot illustrating the pharmacokinetic profile of anti-TREM2 antibodies dosed in cynomolgus monkeys.
  • FIGS. 16A and 16B are plots showing EdU + Iba + cells per mm 2 (FIG. 16A) and relative Iba + area (FIG. 16B) in brains of TB36/hTfR KI mice treated with either ATV:TREM2 or ATV:RSV.
  • FIGS. 17A and 17B are plots showing EdU + Iba + cells per mm 2 (FIG. 17A) and relative Iba + area (FIG. 17B) in brains of TB36/hTfR KI mice treated with either ATV:TREM2, a corresponding TREM2 antibody, reference antibody #2, or ATV:RSV.
  • Graphs display mean ⁇ SEM and p values: oneway ANOVA with Tukey’s multiple comparison test; * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIGS. 18A and 18B are plots showing cytokines IP- 10 (FIG. 18 A) and MCP-5 (FIG. 18B) levels in brains of TB36/hTfR KI mice treated with either ATV:TREM2, a corresponding TREM2 antibody, reference antibody #2, or ATV:RSV.
  • Graphs display mean values across experimental replicates ⁇ SEM.
  • Graphs display mean ⁇ SEM and p values: one-way ANOVA with Tukey’s multiple comparison test; ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 19 is a plot showing glial marker CSF1R level in brains of TB36/hTfR KI mice treated with either ATV:TREM2, a corresponding TREM2 antibody, reference antibody #2, or ATV:RSV.
  • Graphs display 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. 20 is a plot showing the plasma PK profile of either ATV:TREM2, a corresponding TREM2 antibody, reference antibody #2, or ATV:RSV in TB36/hTfR KI mice.
  • FIG. 21 is a plot showing the brain PK profile of either ATV:TREM2, a corresponding TREM2 antibody, reference antibody #2, or ATV:RSV in TB36/hTfR KI mice.
  • TREM2 is a transmembrane receptor that is expressed on the cell surface of microglia, dendritic cells, macrophages, and osteoclasts. Without being bound to a particular theory, it is believed that upon ligand binding, TREM2 forms a signaling complex with a transmembrane adapter protein, DNAX-activating protein 12 (DAP12), which in turn is tyrosine phosphorylated by the protein kinase SRC. It is believed that the activated TREM2/DAP12 signaling complex mediates intracellular signaling by recruiting and phosphorylating kinases such as Syk kinase.
  • DAP12 DNAX-activating protein 12
  • TREM2/DAP12 signaling modulates activities such as phagocytosis, cell growth and survival, pro-inflammatory cytokine secretion, and the migration of cells such as microglia and macrophages.
  • TREM2 undergoes regulated intramembrane proteolysis, in which the membrane-associated full-length TREM2 is cleaved by the metalloprotease AD AMI 0 into a sTREM2 portion that is shed from the cell and a membrane-retained C-terminal fragment that is further degraded by a gamma-secretase. Altered levels of sTREM2 have been reported in patients having Alzheimer’s disease or frontotemporal dementia and having a mutation in TREM2. Additionally, mutations in TREM2 are associated with altered functions such as impaired phagocytosis and reduced microglial function.
  • antibodies have been generated that specifically bind to human TREM2 and that modulate one or more downstream functions of the TREM2/DAP12 signaling complex.
  • the antibodies further comprise an Fc polypeptide containing mutations that permit binding of the Fc polypeptide to a transferrin receptor (TfR from, e.g., a human).
  • TfR transferrin receptor
  • the antibodies disclosed herein are able to bind, through the modified Fc polypeptide, to a transferrin receptor protein (e.g, expressed on the surface of a brain endothelial cell (BECs)) and can thereby cross the blood-brain barrier (BBB) more effectively than antibodies lacking the TfR-binding Fc mutations.
  • BECs brain endothelial cell
  • the antibodies disclosed herein comprise mutations in an Fc polypeptide that reduce or eliminate effector function and mutations that increase in vivo half-life, e.g, by increasing binding of antibody Fc to Fc neonatal receptor (FcRn).
  • the anti-TREM2 antibodies enhance TREM2 activity, e.g., enhance phagocytosis or enhance the differentiation, function, migration, or survival of myeloid cells, microglia, or macrophages.
  • methods of enhancing TREM2 activity e.g, in a subject having a neurodegenerative disease, are provided.
  • the anti-TREM2 antibodies reduce shedding of sTREM2.
  • methods of decreasing levels of sTREM2, e.g, in a subject having a neurodegenerative disease are provided.
  • TREM2 protein refers to a triggering receptor expressed on myeloid cells 2 protein that is encoded by the gene TREM2.
  • a “TREM2 protein” refers to a native (i.e., wild-type) TREM2 protein of any vertebrate, such as but not limited to human, non-human primates (e.g, cynomolgus monkey), rodents (e.g, mice, rat), and other mammals.
  • a TREM2 protein is a human TREM2 protein having the sequence identified in UniprotKB accession number Q9NZC2 (SEQ ID NO:l).
  • anti-TREM2 antibody refers to an antibody that specifically binds to a TREM2 protein (e.g, human TREM2).
  • antibody refers to a protein with an immunoglobulin fold that specifically binds to an antigen via its variable regions.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • antibody also includes antibody fragments that retain binding specificity via its variable regions, including but not limited to Fab, F(ab’)2, Fv, scFv, and bivalent scFv.
  • 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, IgG, IgM, IgA, IgD and IgE, respectively.
  • anti-TREM2 antigen binding portion refers to an antigen binding segment or entity that specifically binds to a TREM2 protein (e.g, human TREM2).
  • TREM2 protein e.g, human TREM2
  • antigen-binding portion and antigen-binding fragment are used interchangeably herein and refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen (e.g, a TREM2 protein) via its variable region.
  • antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains), F(ab’)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), single chain Fv (scFv), disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), a VL (light chain variable region), and a VH (heavy chain variable region).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab’)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • scFv single chain Fv
  • dsFv disulfide-linked Fv
  • CDRs complementarity determining regions
  • VL light chain variable region
  • VH heavy chain variable region
  • variable region refers to a domain in an antibody heavy chain or light chain that is derived from a germline Variable (V) gene, Diversity (D) gene, or Joining (J) gene (and not derived from a Constant (Cp and C5) gene segment), and that gives an antibody its specificity for binding to an antigen.
  • V germline Variable
  • D Diversity
  • J Joining
  • an antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity determining regions.”
  • CDR complementarity determining region
  • the CDRs are primarily responsible for antibody binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a VH CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which it is found
  • a VL CDRl or CDR-L1 is the CDR1 from the variable region of the light chain of the antibody in which it is found.
  • framework regions or “FRs” of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves 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, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences.
  • CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), AbM, and observed antigen contacts (“Contact”).
  • CDRs are determined according to the Contact definition. See , MacCallum el al, ./. Mol. Biol ., 262:732-745 (1996).
  • CDRs are determined by a combination of Kabat, Chothia, and/or Contact CDR definitions.
  • epitope refers to the area or region of an antigen to which the CDRs of an antibody specifically binds and can include a few amino acids or portions of a few amino acids, e.g, 5 or 6, or more, e.g, 20 or more amino acids, or portions of those amino acids.
  • the epitope can be comprised of consecutive amino acids (e.g, a linear epitope), or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g, a discontinuous or conformational epitope).
  • the epitope is phosphorylated at one amino acid (e.g, at a serine or threonine residue).
  • the phrase “recognizes an epitope,” as used with reference to an anti-TREM2 antibody, means that the antibody CDRs interact with or specifically bind to the antigen (i.e., the TREM2 protein) at that epitope or a portion of the antigen containing that epitope.
  • a “monoclonal antibody” refers to antibodies produced by a single clone of cells or a single cell line and consisting of or consisting essentially of antibody molecules that are identical in their primary amino acid sequence.
  • 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 an antigen.
  • a “chimeric antibody” refers to an antibody molecule in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen-binding site (i.e., variable region, CDR, or portion thereof) is linked to a constant region of a different or altered class, effector function and/or species, or in which the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity (e.g ., CDR and framework regions from different species).
  • the antigen-binding site i.e., variable region, CDR, or portion thereof
  • a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g, human). Methods for producing chimeric antibodies are described in the art.
  • a “humanized antibody” is a chimeric immunoglobulin derived from a non-human source (e.g, murine) that contains minimal sequences derived from the non-human immunoglobulin outside the CDRs.
  • a humanized antibody will comprise at least one (e.g, two) antigen-binding variable domain(s), in which the CDR regions substantially correspond to those of the non-human immunoglobulin and the framework regions substantially correspond to those of a human immunoglobulin sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin sequence.
  • a “human antibody” or a “fully human antibody” is an antibody having human heavy chain and light chain sequences, typically derived from human germline genes.
  • the antibody is produced by a human cell, by a non-human animal that utilizes human antibody repertoires (e.g, transgenic mice that are genetically engineered to express human antibody sequences), or by phage display platforms.
  • the term “specifically binds” refers to a molecule (e.g, an antibody or an antigen binding portion thereof) that binds to an epitope or target with greater affinity, greater avidity, and/or greater duration to that epitope or target in a sample than it binds to another epitope or non-target compound (e.g, a structurally different antigen).
  • an antibody (or an antigen-binding portion thereof) that specifically binds to an epitope or target is an antibody (or an antigen-binding portion thereof) that binds to the epitope or target with at least 5-fold greater affinity than other epitopes or non-target compounds, e.g, at least 5- fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity.
  • telomere binding can be exhibited, for example, by a molecule having an equilibrium dissociation constant KD for the epitope or target to which it binds of, e.g, 10 4 M or smaller, e.g, 10 5 M, 10 6 M, 10 7 M, 10 8 M, 10 9 M, 10 10 M, 10 11 M, or 10 12 M.
  • a target e.g ., a TREM2 protein
  • an antibody that specifically binds to a target e.g ., a TREM2 protein
  • orthologs of that target e.g., the TREM2 protein
  • binding affinity is used herein to refer to the strength of a non-covalent interaction between two molecules, e.g, between an antibody (or an antigen-binding portion thereof) and an antigen.
  • the term may refer to 1 : 1 interactions between an antibody (or an antigen-binding portion thereof) and an antigen, unless otherwise indicated or clear from context.
  • Binding affinity may be quantified by measuring an equilibrium dissociation constant (KD), which refers to the dissociation rate constant (kd, time 1 ) divided by the association rate constant (k a , time 1 M '1 ).
  • KD equilibrium dissociation constant
  • KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g, using Surface Plasmon Resonance (SPR) methods, e.g, a BiacoreTM system; kinetic exclusion assays such as KinExA ® ; and BioLayer interferometry (e.g, using the ForteBio ® Octet platform).
  • SPR Surface Plasmon Resonance
  • Binding affinity includes not only formal binding affinities, such as those reflecting 1 : 1 interactions between an antibody (or an antigen-binding portion thereof) and an antigen, but also apparent affinities for which KD values are calculated that may reflect avid binding.
  • cross-reacts refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody was raised.
  • cross-reactivity refers to the ability of an antibody to bind to an antigen from another species than the antigen against which the antibody was raised.
  • an anti-TREM2 antibody as described herein that is raised against a human TREM2 peptide can exhibit cross-reactivity with a TREM2 peptide or protein from a different species (e.g, monkey or mouse).
  • a “transferrin receptor” or “TfR” as used herein 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; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP 990587.1).
  • transferrin receptor also encompasses allelic variants of exemplary reference sequences, e.g, human sequences, that are encoded by a gene at a transferrin receptor protein 1 chromosomal locus.
  • Full length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain.
  • the extracellular domain is characterized by three domains: a protease- like domain, a helical domain, and an apical domain.
  • the apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO: 55.
  • CEB domain and CH2 domain refer to immunoglobulin constant region domain polypeptides.
  • a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme
  • a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme.
  • CH2 and CH3 domain polypeptides may also be numbered by the IMGT (ImMunoGeneTics) numbering scheme in which the CH2 domain numbering is 1-110 and the CH3 domain numbering is 1-107, according to the IMGT Scientific chart numbering (IMGT website).
  • CH2 and CH3 domains are part of the Fc region of an immunoglobulin.
  • an Fc region refers to the segment of amino acids from about position 231 to about position 447 as numbered according to the EU numbering scheme.
  • the term “Fc region” may also include at least a part of a hinge region of an antibody.
  • An exemplary partial hinge region sequence is set forth in SEQ ID NO:57.
  • amino acid residue in a polypeptide refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence.
  • an amino acid residue in a polypeptide “corresponds to” an amino acid in the region of SEQ ID NO:38 from amino acids 111-217 when the residue aligns with the amino acid in SEQ ID NO:38 when optimally aligned to SEQ ID NO:38.
  • the polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.
  • Fc polypeptide refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide that is characterized by an Ig fold as a structural domain.
  • An Fc polypeptide contains constant region sequences including at least the CH2 domain and/or the CH3 domain and may contain at least part of the hinge region, but does not contain a variable region.
  • a “modified Fc polypeptide” refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion or insertion, as compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.
  • nucleic acid or protein e.g ., antibody
  • nucleic acid or protein e.g ., antibody
  • purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g, high performance liquid chromatography).
  • an isolated nucleic acid or protein e.g, antibody
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g, hydroxyproline, g- carboxyglutamate, and O-phosphoserine.
  • Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (lie), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.
  • Stereoisomers of a naturally occurring a-amino acids include, without limitation, D- alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D- phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D- lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D- tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.
  • amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino 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.
  • polypeptide and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.
  • protein refers to either a polypeptide or a dimer ( i.e ., two) or multimer ⁇ i.e., three or more) of single chain polypeptides.
  • the single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.
  • polynucleotide and “nucleic acid” interchangeably refer to chains of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.
  • 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 hybrid molecules having mixtures of single- and double-stranded DNA and RNA.
  • conservative amino acid groups refer to an alteration that results in the substitution of an amino acid with another amino acid that can be categorized as having a similar feature.
  • categories of conservative amino acid groups defined in this manner can include: a “charged/polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gin (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R), and His (Histidine or H); an “aromatic group” including Phe (Phenylalanine or F), Tyr (Tyrosine or Y), Trp (Tryptophan or W), and (Histidine or H); and an “aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Val (Valine or V), Leu (Leucine or L), lie (Isoleucine or I), Met (Methionine or M), Ser
  • subgroups can also be identified.
  • the group of charged or polar amino acids can be sub-divided into sub-groups including: a “positively-charged sub-group” comprising Lys, Arg and His; a “negatively-charged sub-group” comprising Glu and Asp; and a “polar sub-group” comprising Asn and Gin.
  • the aromatic or cyclic group can be sub divided into sub-groups including: a “nitrogen ring sub-group” comprising Pro, His and Trp; and a “phenyl sub-group” comprising Phe and Tyr.
  • the aliphatic group can be sub-divided into sub-groups, e.g., an “aliphatic non-polar sub-group” comprising Val, Leu, Gly, and Ala; and an “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys.
  • Examples of categories of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained.
  • hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acid, e.g, in the active site, to preserve hydrophobicity.
  • nucleic or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • sequence comparison of polypeptides typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g, visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.
  • subject refers to a mammal, including but not limited to humans, non-human primates, rodents (e.g, rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species.
  • rodents e.g, rats, mice, and guinea pigs
  • rabbits cows, pigs, horses, and other mammalian species.
  • the subject, individual, or patient is a human.
  • Treating is used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • Treating may refer to any indicia of success in the treatment or amelioration of a neurodegenerative disease (e.g, Alzheimer’s disease or another neurodegenerative disease described herein), including any objective or subjective parameter such as abatement, remission, improvement in patient survival, increase in survival time or rate, diminishing of symptoms or making the disease more tolerable to the patient, slowing in the rate of degeneration or decline, or improving a patient’s physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters.
  • the effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.
  • pharmaceutically acceptable excipient refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to a buffer, carrier, or preservative.
  • a “therapeutic amount” or “therapeutically effective amount” of an agent is an amount of the agent that treats, alleviates, abates, or reduces the severity of symptoms of a disease in a subject.
  • a “therapeutic amount” of an agent may improve patient survival, increase survival time or rate, diminish symptoms, make an injury, disease, or condition (e.g, a neurodegenerative disease) more tolerable, slow the rate of degeneration or decline, or improve a patient’s physical or mental well-being.
  • administer refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, an antibody as described herein is administered intravenously.
  • control refers to a reference value or baseline value. Appropriate controls can be determined by one skilled in the art. In some instances, control values can 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 can be a control value for a post-treatment measurement of sTREM2 levels in the same subject.
  • control value can be determined relative to a control subject (e.g ., a healthy control or a disease control) or an average value in a population of control subjects (e.g., healthy controls or disease controls, e.g, a population of 10, 20, 50, 100, 200, 500, 1000 control subjects or more), e.g, a measurement of a subject’s level of sTREM2 either at baseline or after treatment can be compared to a healthy control value.
  • a control subject e.g ., a healthy control or a disease control
  • an average value in a population of control subjects e.g., healthy controls or disease controls, e.g, a population of 10, 20, 50, 100, 200, 500, 1000 control subjects or more
  • a measurement of a subject’s level of sTREM2 either at baseline or after treatment can be compared to a healthy control value.
  • antibodies that specifically bind to a TREM2 protein are provided.
  • the antibody specifically binds to a human TREM2 protein.
  • an anti-TREM2 antibody is selective for TREM2 over other TREM-like receptors (e.g, TREM1).
  • an anti-TREM2 antibody is an antibody that comprises one or more complementarity determining region (CDR), heavy chain variable region, and/or light chain variable region sequences as disclosed herein.
  • an anti- TREM2 antibody comprises one or more CDR, heavy chain variable region, and/or light chain variable region sequences as disclosed herein and further comprises one or more functional characteristics as disclosed herein, e.g, an antibody that enhances TREM2 activity (e.g, enhances phagocytosis, or enhances the migration, differentiation, function, or survival of a cell such as a myeloid cell, microglia, or macrophage) or an antibody that decreases levels of sTREM2.
  • the anti-TREM2 antibody comprises Fc polypeptides that comprise one or more modifications as described herein.
  • the anti-TREM2 antibody is a chimeric antibody. In some embodiments, the anti-TREM2 antibody is a humanized and/or affinity matured antibody.
  • a heavy chain sequence, or a portion thereof, and/or a light chain sequence, or a portion thereof is derived from an anti-TREM2 antibody described herein (e.g, Clone CL0020306, Clone CL0020188, or Clone CL0020307).
  • the CDR, heavy chain variable region, and light chain variable region amino acid sequences of these clones is set forth in Table 8.
  • an anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of:
  • CDR-H1 heavy chain CDR1 sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOS:4 and 12, or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOS:4 and 12;
  • CDR-H2 heavy chain CDR2 sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOS:5, 13, and 25, or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOS: 5, 13, and 25;
  • CDR-H3 heavy chain CDR3 sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOS:6, 14, and 17, or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOS:6, 14, and 17;
  • CDR-L1 light chain CDR1 sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOS:7 and 23, or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOS:7 and 23;
  • CDR-L2 light chain CDR2 sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:8;
  • CDR-L3 light chain CDR3 sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOS:9 and 18, or having up to two amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOS:9 and 18.
  • an anti-TREM2 antibody comprises two, three, four, five, or all six of (a)-(f). In some embodiments, an anti-TREM2 antibody comprises the CDR-H1 of (a), the CDR-H2 of (b), and the CDR-H3 of (c). In some embodiments, an anti-TREM2 antibody comprises the CDR-L1 of (d), the CDR-L2 of (e), and the CDR-L3 of (f). In some embodiments, a CDR having up to two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR having up to two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.
  • an anti-TREM2 antibody comprises one or more CDRs selected from the group consisting of:
  • an anti-TREM2 antibody comprises two, three, four, five, or all six of (a)-(f). In some embodiments, an anti-TREM2 antibody comprises the CDR-H1 of (a), the CDR-H2 of (b), and the CDR-H3 of (c). In some embodiments, an anti-TREM2 antibody comprises the CDR-L1 of (d), the CDR-L2 of (e), and the CDR-L3 of (f).
  • an anti-TREM2 antibody comprises:
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR- H2 comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:9; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:9; or
  • a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:9.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:2, 10, 15, 19, 21, 24, and 26.
  • an 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.
  • an anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:3, 11, 16, 20, 22, and 27.
  • an 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.
  • an anti-TREM2 antibody comprises: a heavy chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:2, 10, 15, 19, 21, 24, and 26, a light chain variable region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:3, 11, 16, 20, 22, and 27.
  • an anti-TREM2 comprises: a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOS:2, 10, 15, 19, 21, 24, and 26, and a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOS:3, 11, 16, 20, 22, and 27.
  • an anti-TREM2 antibody comprises:
  • VH sequence that has at least 85% sequence identity to SEQ ID NO:21 and a VL sequence has at least 85% sequence identity to SEQ ID NO:20;
  • an anti-TREM2 antibody comprises one or more sequences that are encompassed by a consensus sequence disclosed herein.
  • consensus sequences can be identified by aligning heavy chain or light chain sequences (e.g ., CDRs) for antibodies that are from the same (or similar) germlines.
  • consensus sequences may be generated from antibodies that contain sequences that are of the same (or similar) length and/or have at least one highly similar CDR (e.g., a highly similar CDR3).
  • sequences in these antibodies may be aligned and compared to identify conserved amino acids or motifs (i.e., where alteration in sequences may alter protein function) and/or regions where variation occurs the sequences (i.e., where variation of sequence is not likely to significantly affect protein function).
  • consensus sequences can be identified by aligning heavy chain or light chain sequences (e.g, CDRs) for antibodies that bind to the same or similar (e.g, overlapping) epitopes to determine conserved amino acids or motifs (i.e., where alteration in sequences may alter protein function) and regions where variation occurs in alignment of sequences (i.e., where variation of sequence is not likely to significantly affect protein function).
  • one or more consensus sequences can be identified for antibodies that recognize the same or similar epitope as an anti-TREM2 antibody as disclosed herein.
  • Exemplary consensus sequences include SEQ ID NOS:28-32.
  • the capitalized letter represents an amino acid residue that is absolutely conserved among the aligned sequences (e.g, aligned CDR sequences), while an “X” or a Greek letter (e.g, “a,” “b,” “g,” “d,” “e,” or “f”) represents an amino acid residue that is not absolutely conserved among the aligned sequences.
  • amino acid when selecting an amino acid to insert at a position marked by an “X” or by a Greek letter, in some embodiments the amino acid is selected from those amino acids found at the corresponding position in the aligned sequences.
  • an anti-TREM2 antibody comprises:
  • a CDR-H2 sequence comprising the sequence of n-I-II-N-b ⁇ -b ⁇ -N-bk- Y-T-Pii-Pi2-Y-N-P-S-V-K-G (SEQ ID NO:29), wherein b 5 is K or R; b 6 is A or P; b 8 is G or A; bii is A or T; and b ⁇ 2 is G or D;
  • a CDR-H3 sequence comprising the sequence of yi-R-L-y4-Y-G-F-D-Y (SEQ ID NO:30), wherein gi is A or T; and j is T or S;
  • a CDR-L1 sequence comprising the sequence of Q-S-S-K-S-L-L-H-S- dio-G-K-T-Y-L-N (SEQ ID NO:31), wherein dio is N or T;
  • an anti-TREM2 antibody comprises a CDR-H1 sequence that is selected from SEQ ID NOS:4 and 12. In some embodiments, an anti-TREM2 antibody comprises a CDR-H2 sequence that is selected from SEQ ID NOS:5, 13, and 25. In some embodiments, an anti-TREM2 antibody comprises a CDR-H3 sequence that is selected from SEQ ID NOS:6, 14, and 17. In some embodiments, an anti-TREM2 antibody comprises a CDR-L1 sequence that is selected from SEQ ID NOSY and 23. In some embodiments, an anti-TREM2 antibody comprises a CDR-L3 sequence is selected from SEQ ID NOS:9 and 18.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOSY, 10, 15, 19, 21, 24, and 26.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOSY, 10, 15, 19, 21, 24, and 26.
  • an anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of SEQ ID NOS:3, 11, 16, 20, 22, and 27.
  • an 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.
  • an anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 18.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15.
  • an anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16.
  • an anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
  • an anti-TREM2 antibody comprises a heavy chain variable region that comprises a heavy chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 5, and 17, respectively, and that has at least 85% sequence identity (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15.
  • an anti-TREM2 antibody comprises a light chain variable region that comprises a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:7, 8, and 18, respectively, and that has at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16.
  • an anti-TREM2 antibody competes for binding with an antibody as described herein (e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 5, 17, 7, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16).
  • an antibody as described herein e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 5, 17, 7, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16).
  • an anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 18.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24. In some embodiments, an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24.
  • an anti-TREM2 antibody or antigen binding portion comprises a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:22.
  • an anti-TREM2 antibody or antigen binding portion comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:22.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24 and a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g ., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:22.
  • an 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.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region that comprises a heavy chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 25, and 17, respectively, and that has at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24.
  • an anti-TREM2 antibody or antigen binding portion comprises a light chain variable region that comprises a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:23, 8, and 18, respectively, and that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:22.
  • an anti-TREM2 antibody or antigen binding portion competes for binding with an antibody as described herein (e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 25, 17, 23, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22).
  • an antibody as described herein e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 25, 17, 23, 8, and 18, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22).
  • an 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, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 17, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:9.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24. In some embodiments, an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24.
  • an anti-TREM2 antibody or antigen binding portion comprises a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g ., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:27.
  • an anti-TREM2 antibody or antigen binding portion comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:27.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24 and a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:27.
  • an 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.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region that comprises a heavy chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 25, and 17, respectively, and that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:24.
  • an anti-TREM2 antibody or antigen binding portion comprises a light chain variable region that comprises a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:7, 8, and 9, respectively, and that has at least 85% sequence identity (e.g, at least 90%, 95%, or 97% sequence identity) to SEQ ID NO:27.
  • an anti-TREM2 antibody or antigen binding portion is an antibody that competes for binding with an antibody as described herein (e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 25, 17, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27).
  • an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 25, 17, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27).
  • an anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO:6, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:9.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.
  • an anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • an anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2 and a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • an 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.
  • an anti-TREM2 antibody comprises a heavy chain variable region that comprises a heavy chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 5, and 6, respectively, and that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2.
  • an anti-TREM2 antibody comprises a light chain variable region that comprises a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:7, 8, and 9, respectively, and that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • an anti-TREM2 antibody competes for binding with an antibody as described herein (e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 5, 6, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:3).
  • an antibody as described herein e.g, an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:4, 5, 6, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:3.
  • an anti-TREM2 antibody comprises a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO:9.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10.
  • an anti-TREM2 antibody or antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10.
  • an anti-TREM2 antibody comprises a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11.
  • an anti-TREM2 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10 and a light chain variable region comprising an amino acid sequence that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11.
  • an anti-TREM2 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.
  • an anti-TREM2 antibody comprises a heavy chain variable region that comprises a heavy chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS: 12, 13, and 14, respectively, and that has at least 85% sequence identity (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10.
  • an anti-TREM2 antibody comprises a light chain variable region that comprises a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS:7, 8, and 9, respectively, and that has at least 85% sequence identity (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 11.
  • an anti-TREM2 antibody is an antibody that competes for binding with an antibody as described herein (e.g., an antibody comprising a heavy chain CDRl-3 and a light chain CDRl-3 comprising the amino acid sequences of SEQ ID NOS: 12, 13, 14, 7, 8, and 9, respectively, or an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11).
  • an antibody as described herein that specifically binds to a TREM2 protein binds to TREM2 that is expressed on a cell (e.g, a primary cell or cell line that endogenously expresses TREM2, such as human macrophages, or a primary cell or cell line that has been engineered to express TREM2, e.g, as described in the Examples section below).
  • a cell e.g, a primary cell or cell line that endogenously expresses TREM2, such as human macrophages, or a primary cell or cell line that has been engineered to express TREM2, e.g, as described in the Examples section below.
  • an antibody that specifically binds to a TREM2 protein as described herein binds to purified or recombinant TREM2 protein of a portion thereof, or to a chimeric protein comprising TREM2 or a portion thereof (e.g, an Fc-fusion protein comprising TREM2 or an Fc-fusion protein comprising the ecto-domain of TREM2).
  • an antibody that specifically binds to human TREM2 protein exhibits cross-reactivity with one or more other TREM2 proteins of another species.
  • an antibody that specifically binds to human TREM2 protein exhibits cross-reactivity with a cynomolgus monkey (“cyno”) TREM2 protein.
  • an antibody that specifically binds to human TREM2 protein exhibits cross-reactivity with a mouse TREM2 protein.
  • an 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, but are not limited to, solid-phase binding assays (e.g, ELISA assay), immunoprecipitation, surface plasm on resonance (e.g, Biacore TM (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g, KinExA ® ), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g, Octet TM (ForteBio, Inc., Menlo Park, CA)), and western blot analysis.
  • ELISA is used to determine binding affinity and/or cross-reactivity.
  • SPR surface plasmon resonance
  • kinetic exclusion assays are used to determine binding affinity, binding kinetics, and/or cross-reactivity.
  • BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and/or cross-reactivity.
  • an anti-TREM2 antibody recognizes an epitope of human TREM2 that is the same or substantially the same as the epitope recognized by an antibody clone as described herein.
  • the term “substantially the same,” as used with reference to an epitope recognized by an antibody clone as described herein, means that the anti-TREM2 antibody recognizes an epitope that is identical, within, or nearly identical to (e.g, has at least 90% sequence identity to, or has one, two, or three amino acid substitutions, e.g, conservative substitutions, relative to), or has substantial overlap with (e.g, at least 50%, 60%, 70%, 80%, 90%, or 95% overlap with) the epitope recognized by the antibody clone as described herein.
  • an anti-TREM2 antibody recognizes an epitope of human TREM2 that is the same or substantially the same as the epitope recognized by an antibody clone selected from the group consisting of Clone CL0020306, Clone CL0020188, Clone CL0020307, and variants of the same.
  • an anti-TREM2 antibody binds to human TREM2 at an epitope within the stalk region of TREM2. In some embodiments, an 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:l. In some embodiments, an anti-TREM2 antibody recognizes an epitope of human TREM2 comprising, within, or consisting of residues 129- 148 of SEQ ID NO:l (e.g, 143-148 of SEQ ID NO:l).
  • an anti- TREM2 antibody is an agonist that activates TREM2/DAP12 signaling (e.g, by inducing phosphorylation of a kinase such as Syk) and binds to human TREM2 at an epitope within the stalk region of TREM2.
  • an anti-TREM2 antibody binds to human TREM2 at an epitope within the stalk region of TREM2 and inhibits cleavage of TREM2 by a protease (e.g, ADAM 17).
  • an anti-TREM2 antibody functions in one or more TREM2 activities as disclosed herein.
  • an anti-TREM2 antibody modulates levels of sTREM2 protein (e.g, levels of sTREM2 that are shed from the cell surface into an extracellular sample), modulates recruitment or phosphorylation of a kinase that interacts with a TREM2/DAP12 signaling complex (e.g, Syk kinase), and/or modulates one or more activities downstream of the signaling complex, such as phagocytosis, cell growth, cell survival, cell differentiation, cytokine secretion, or cell migration.
  • an anti-TREM2 antibody enhances one or more TREM2 activities (e.g, those described herein) that are induced by a ligand.
  • the ligand is a lipid ligand.
  • TREM2 lipid ligands include, but are not limited to, 1 -palmitoyl-2-(5 ’ -oxo-valeroyl)-sn-glycero-3 -phosphocholine (POVPC), 2-
  • Lysophosphatidylinositol LSM
  • Lysophosphatidylserine LPS
  • N-Acyl-phosphatidylethanolamine NAPE
  • N-Acyl-Serine N-Acyl-Serine
  • Oxidized phosphatidylcholine oxPC
  • Palmitic-acid-9-hydroxy-stearic-acid PAHSA
  • Phosphatidylethanolamine PE
  • Phosphatidylethanol PEtOH
  • Phosphatidic acid PA
  • Phosphatidylcholine PC
  • Phosphatidylglycerol PG
  • Phosphatidylinositol PI
  • Phosphatidylserine PS
  • Sphinganine Sphinganine-1 -phosphate
  • SalP Sphingomyelin
  • Sphingosine Sphingosine-1 -phosphate
  • an anti-TREM2 antibody alters levels of sTREM2 protein in a sample, e.g., levels of sTREM2 that are shed from the cell surface into an extracellular sample. In some embodiments, an anti-TREM2 antibody decreases levels of sTREM2.
  • an anti-TREM2 antibody decreases levels of sTREM2 if the amount of sTREM2 in a treated sample is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value. In some embodiments, an anti-TREM2 antibody decreases levels of sTREM2 if the amount of sTREM2 in a treated sample is decreased by at least 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more as compared to a control value.
  • control value is the amount of sTREM2 in an untreated sample (e.g, a supernatant from a TREM2-expressing cell that has not been treated with an anti-TREM2 antibody, or a sample from a subject that has not been treated with an anti-TREM2 antibody) or a sample treated with an appropriate non-TREM2-binding antibody.
  • an untreated sample e.g, a supernatant from a TREM2-expressing cell that has not been treated with an anti-TREM2 antibody, or a sample from a subject that has not been treated with an anti-TREM2 antibody
  • sTREM2 shedding is measured using a sample that comprises a fluid, e.g, blood, plasma, serum, urine, or cerebrospinal fluid.
  • the sample comprises cerebrospinal fluid.
  • the sample comprises supernatant from cell cultures (e.g, supernatant from a primary cell or cell line that endogenously expresses TREM2, such as human macrophages, or a primary cell or cell line that has been engineered to express TREM2, e.g, as described in the Examples section below).
  • the level of sTREM2 in a 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 multiplied immunoassay (EMIA), enzyme- linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassays (CEIA), radioimmunoassays (RIA), immunofluorescence, chemiluminescence immunoassays (CL), and electrochemiluminescence immunoassays (ECL).
  • EIA enzyme immunoassays
  • EMIA enzyme multiplied immunoassay
  • ELISA enzyme- linked immunosorbent assay
  • MEIA microparticle enzyme immunoassay
  • IHC immunohistochemistry
  • IHC immunocytochemistry
  • CEIA capillary electrophoresis immunoassays
  • an anti-TREM2 antibody induces phosphorylation of a kinase that interacts with the TREM2/DAP12 signaling complex (such as, but not limited to, Syk, ZAP70, PI3K, Erk, AKT, or GSK3b). In some embodiments, an anti-TREM2 antibody induces phosphorylation of a kinase that interacts with the TREM2/DAP12 signaling complex without blocking binding of a native TREM2 ligand.
  • a kinase that interacts with the TREM2/DAP12 signaling complex such as, but not limited to, Syk, ZAP70, PI3K, Erk, AKT, or GSK3b.
  • an anti-TREM2 antibody enhances phosphorylation of a kinase that interacts with the TREM2/DAP12 signaling complex that is induced by a TREM2 ligand ( e.g ., a lipid ligand).
  • a TREM2 ligand e.g ., a lipid ligand.
  • an anti-TREM2 antibody induces or enhances phosphorylation of Syk.
  • an anti-TREM2 antibody induces or enhances phosphorylation of Syk if the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value.
  • an anti-TREM2 antibody induces phosphorylation of Syk if the level of Syk phosphorylation in a sample treated with the anti-TREM2 antibody is increased by at least 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to a control value.
  • control value is the level of Syk phosphorylation in an untreated sample (e.g., a sample comprising a TREM2-expressing cell that has not been treated with an anti-TREM2 antibody, or a sample from a subject that has not been treated with an anti-TREM2 antibody), or a sample that has been treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with an appropriate non-TREM2 -binding antibody.
  • an untreated sample e.g., a sample comprising a TREM2-expressing cell that has not been treated with an anti-TREM2 antibody, or a sample from a subject that has not been treated with an anti-TREM2 antibody
  • an immunoassay is used.
  • the immunoassay is an enzyme immunoassay (EIA), enzyme multiplied immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemiluminescence immunoassay (ECL).
  • EIA enzyme immunoassay
  • EMIA enzyme multiplied immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • MEIA microparticle enzyme immunoassay
  • IHC immunohistochemistry
  • immunocytochemistry immunocytochemistry
  • CEIA capillary electrophoresis immunoassay
  • RIA radioimmunoassay
  • RIA immunofluorescence
  • CL chemiluminescence immunoassay
  • phosphorylation is detected and/or quantified using an immunoassay that utilizes an amplified luminescent proximity homogenous assay (AlphaLISA®, PerkinElmer Inc.).
  • phosphorylation is measured using a sample that comprises one or more cells, e.g., one or more TREM2-expressing cells (e.g. , a primary cell or cell line that endogenously expresses TREM2, such as human macrophages or iPSC-derived microglia, or a primary cell or cell line that has been engineered to express TREM2, e.g, as described in the Examples section below).
  • the sample comprises a fluid, e.g, blood, plasma, serum, urine, or cerebrospinal fluid.
  • the sample comprises tissue (e.g, lung, brain, kidney, spleen, nervous tissue, or skeletal muscle) or cells from such tissue.
  • the sample comprises endogenous fluid, tissue, or cells (e.g, from a human or non-human subject).
  • an anti-TREM2 antibody enhances phagocytosis of dead cell debris, tissue debris, amyloid beta particles, or foreign material. In some embodiments, an anti-TREM2 antibody enhances phagocytosis without blocking binding of a native TREM2 ligand. In some embodiments, an anti-TREM2 antibody enhances phagocytosis that is induced by a TREM2 ligand (e.g, a lipid ligand).
  • a TREM2 ligand e.g, a lipid ligand
  • an anti-TREM2 antibody enhances phagocytosis if the level of phagocytosis in a sample treated with the anti- TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value.
  • an anti-TREM2 antibody enhances phagocytosis if the level of phagocytosis in a sample treated with the anti-TREM2 antibody is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to a control value.
  • control value is the level of phagocytosis in an untreated sample, a sample that has been treated with a TREM2 ligand but not an anti- TREM2 antibody, or a sample treated with an appropriate non-TREM2 -binding antibody.
  • phagocytosis is measured using a phagocytosis assay with a labeled substrate.
  • Phagocytosis assays are known in the art.
  • the phagocytosis assay is performed on a sample comprising cells that endogenously express TREM2, such as human macrophages or microglia.
  • the phagocytosis assay is performed on a sample comprising cells that have been engineered to express TREM2.
  • phagocytosis is measured using a human macrophage phagocytosis assay as described in the Examples section below. Modulation of cell differentiation, function, migration, and survival
  • an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation (e.g ., for myeloid cells, macrophages, and microglia, including iPSC-derived microglia and disease-associated microglia). Disease- associated microglia and methods of detecting disease-associated microglia are described in Keren-Shaul et al ., Cell , 2017, 169:1276-1290.
  • an anti-TREM2 antibody enhances cell migration of one or more cell types (e.g., myeloid cells, macrophages, or microglia).
  • an anti-TREM2 antibody enhances cell survival of one or more cell types (e.g, myeloid cells, macrophages, or microglia). In some embodiments, an anti-TREM2 antibody enhances cell function of one or more cell types (e.g, myeloid cells, macrophages, or microglia). In some embodiments, an anti-TREM2 antibody enhances cell differentiation of one or more cell types (e.g, myeloid cells, macrophages, or microglia). In some embodiments, an anti-TREM2 antibody enhances the migration, survival, function, and/or differentiation of myeloid cells. In some embodiments, an anti-TREM2 antibody enhances the migration, survival, function, and/or differentiation of macrophages.
  • an anti-TREM2 antibody enhances the migration, survival, function, and/or differentiation of microglia. In some embodiments, an anti-TREM2 antibody enhances microglia activation. In some embodiments, an anti-TREM2 antibody enhances the 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 a native TREM2 ligand. In some embodiments, an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation that is induced by a TREM2 ligand (e.g, a lipid ligand).
  • a TREM2 ligand e.g, a lipid ligand
  • an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of activity (e.g, migration, survival, function, or differentiation) in a sample treated with the anti-TREM2 antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more as compared to a control value.
  • the level of activity e.g, migration, survival, function, or differentiation
  • an anti-TREM2 antibody enhances cell migration, cell survival, cell function, or cell differentiation if the level of activity (e.g, migration, survival, function, or differentiation) in a sample treated with the anti-TREM2 antibody is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to a control value.
  • the level of activity e.g, migration, survival, function, or differentiation
  • control value is the level of activity (e.g, migration, survival, function, or differentiation) in an untreated sample (e.g ., a sample that has not been treated with an anti-TREM2 antibody), a sample that has been treated with a TREM2 ligand but not an anti-TREM2 antibody, or a sample treated with an appropriate non-TREM2-binding antibody.
  • an untreated sample e.g ., a sample that has not been treated with an anti-TREM2 antibody
  • TREM2 ligand e.g., TREM2 ligand but not an anti-TREM2 antibody
  • sample treated with an appropriate non-TREM2-binding antibody e.g., migration, survival, function, or differentiation
  • cell migration is measured using a chemotaxis assay.
  • Chemotaxis assays are known in the art.
  • the cell migration assay (e.g., chemotaxis assay) is performed on a sample comprising cells that endogenously express TREM2, such as human macrophages.
  • the cell migration assay (e.g, chemotaxis assay) is performed on a sample comprising cells that have been engineered to express TREM2.
  • cell migration is measured using a human macrophage chemotaxis assay as described in the Examples section below.
  • cell survival is measured using a cell viability assay.
  • Cell viability assays are known in the art.
  • the cell survival assay (e.g, cell viability assay) is performed on a sample comprising cells that endogenously express TREM2, such as human macrophages.
  • the cell survival assay (e.g, cell viability assay) is performed on a sample comprising cells that have been engineered to express TREM2.
  • cell survival is measured using a human macrophage viability assay as described in the Examples section below.
  • cell function is measured using a functional assay that is appropriate for that cell.
  • macrophage cell function is evaluated using a phagocytosis assay, e.g, as described in the Examples section below.
  • cell differentiation is measured by evaluating the ability of cells that endogenously express TREM2 to differentiate.
  • cell differentiation is measured by evaluating the ability of macrophages to differentiate from monocytes, e.g, as described in the Examples section below.
  • microglia activation is measured in vivo.
  • microglia activation is measured using TSPO-PET imaging.
  • TSPO-PET imaging methods are known in the art.
  • an anti-TREM2 antibody enhances microglia function without increasing neuroinflammation.
  • Levels of neuroinflammation can be determined by measuring levels of cytokines (e.g, inflammatory cytokines), such as but not limited to TNF- a, IL-Ib, IL-6, IL-lra, TGFp, IL-15, or IFN-g.
  • cytokines e.g, inflammatory cytokines
  • cytokine levels are measured using immunoassays, for example, an enzyme immunoassay (EIA), enzyme multiplied immunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC), immunocytochemistry, capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunofluorescence, chemiluminescence immunoassay (CL), or electrochemiluminescence immunoassay (ECL).
  • EIA enzyme immunoassay
  • EMIA enzyme multiplied immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • MEIA microparticle enzyme immunoassay
  • IHC immunohistochemistry
  • immunocytochemistry immunocytochemistry
  • CEIA capillary electrophoresis immunoassay
  • RIA radioimmunoassay
  • CCL electrochemiluminescence immunoassay
  • ECL electrochemilum
  • an anti-TREM2 antibody comprises two Fc polypeptides, one or both of which may each comprise independently selected modifications (e.g ., mutations) or may be a wild-type Fc polypeptide, e.g., a human IgGl Fc polypeptide.
  • Non-limiting examples of mutations that can be introduced into one or both Fc polypeptides include, e.g, mutations to permit binding of an Fc polypeptide (or antibody comprising the same) to a BBB-receptor, such as transferrin receptor (TfR) protein (e.g, a human or cynomolgus TfR, such as may be expressed on a brain endothelial cell), mutations to increase serum stability, to modulate effector function, to influence glycosylation, to reduce immunogenicity in humans, and/or to provide for knob and hole heterodimerization of the Fc polypeptides.
  • TfR transferrin receptor
  • an anti-TREM2 antibody includes an Fc polypeptide that comprises modifications (e.g, amino acid substitutions) that permit binding of the Fc polypeptide to a TfR protein.
  • binding to a TfR protein e.g, to the apical domain thereof
  • a brain endothelial cell can, in some embodiments, permit a modified Fc polypeptide of this disclosure or an antibody comprising the same to cross the blood-brain barrier via receptor-mediated transcytosis.
  • receptor-mediated transcytosis can enhance or improve the ability of the protein comprising the Fc polypeptide to be present in the brain (i.e., on the luminal side of the blood-brain barrier), which can allow for improved binding to TREM2 in the CNS, and other functions, e.g, clearance, neutralization, or immunodepletion of the target, or the like.
  • TfR-binding amino acid modifications to an Fc e.g, CH2 and/or CH3 portion, fragment, or domain
  • Fc polypeptides and portions thereof that comprise the amino acid modifications are described in PCT patent publication no. WO 2018/152326A1.
  • TfR-binding Fc polypeptide sequences and TfR-binding Fc polypeptides, and techniques for generating and testing the same are incorporated herein by reference.
  • One or two Fc polypeptides of an Fc dimer of the present disclosure can be engineered to comprise modifications to permit binding to TfR.
  • one Fc polypeptide of an Fc dimer comprises modifications to permit binding to TfR, and the other Fc polypeptide does not.
  • a modified Fc polypeptide comprises a YxTEWSS (SEQ ID NO:58) motif. In some embodiments, a modified Fc polypeptide comprises a TxxExxxxF (SEQ ID NO:59) motif. In some embodiments, a modified Fc polypeptide comprises a YxTEWSS (SEQ ID NO:58) and a TxxExxxxF (SEQ ID NO:59) motif.
  • a modified Fc polypeptide comprises a wild-type amino acid residue at positions 380, 389, 390, and 415, according to EU numbering, wherein the wild- type amino acid residue is found at a corresponding position in SEQ ID NO:38.
  • an anti-TREM2 antibody includes an Fc polypeptide having the following amino acids: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, or Val at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and Phe at position 421, according to EU numbering.
  • the anti-TREM2 antibody includes an Fc polypeptide having the following amino acids: Trp at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser at position 389; Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, according to EU numbering.
  • an anti-TREM2 antibody includes an Fc polypeptide having the following amino acids: Glu at position 380; Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser at position 389; Asn at position 390; Ser at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, according to EU numbering.
  • an anti-TREM2 antibody includes an Fc polypeptide having the following amino acids: 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, according to EU numbering.
  • an anti-TREM2 antibody includes an Fc polypeptide having the following amino acids: 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, according to EU numbering.
  • a modified Fc polypeptide comprises a sequence having at least 90% identity to an amino acid sequence set forth in any one of SEQ ID NOS:40, 43, 46, and 49. In some embodiments, a 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.
  • the Fc polypeptides present in an anti-TREM2 antibody as disclosed herein include knob and hole mutations to promote heterodimer formation and hinder homodimer formation.
  • the modifications introduce a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and thus hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g ., tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • additional mutations are at a position in the Fc polypeptide that does not have a negative (e.g, inhibitory) effect on binding of a Fc polypeptide to a BBB receptor, e.g, TfR.
  • position 366 (numbered according to the EU numbering scheme) of one of the Fc polypeptides present in the proteins described herein comprises a tryptophan in place of a native threonine.
  • the other Fc polypeptide in the dimer has a valine at position 407 (numbered according to the EU numbering scheme) in place of the native tyrosine.
  • the other Fc polypeptide may further comprise a substitution in which the native threonine at position 366 (numbered according to the EU numbering scheme) is substituted with a serine and a native leucine at position 368 (numbered according to the EU numbering scheme) is substituted with an alanine.
  • one of the Fc polypeptides of an anti-TREM2 protein of the disclosure has the T366W knob mutation and the other Fc polypeptide has the Y407V mutation, which is typically accompanied by the T366S and L368A hole mutations.
  • one or both Fc polypeptides may also be engineered to contain other modifications for heterodimerization, e.g., electrostatic engineering of contact residues within a CH3-CH3 interface that are naturally charged or hydrophobic patch modifications.
  • one or both Fc polypeptides present in an anti-TREM2 protein of the disclosure may comprise a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256, as numbered according to the EU numbering scheme.
  • one or both Fc polypeptides may have M252Y, S254T, and T256E substitutions.
  • one or both Fc polypeptides may have M428L and/or N434S substitutions, according to EU numbering.
  • one or both Fc polypeptides may have an N434S orN434A substitution.
  • one or both Fc polypeptides in an anti-TREM2 protein disclosed herein may comprise modifications that reduce effector function, i.e., having a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an effector cell that mediates the effector function.
  • antibody effector functions include, but are not limited to, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g, B cell receptor), and B-cell activation. Effector functions may vary with the antibody class.
  • native human IgGl and IgG3 antibodies can elicit ADCC and CDC activities upon binding to an appropriate Fc receptor present on an immune system cell; and native human IgGl, IgG2, IgG3, and IgG4 can elicit ADCP functions upon binding to the appropriate Fc receptor present on an immune cell.
  • one or both Fc polypeptides may include modifications that modulate effector function.
  • one or both Fc polypeptides may comprise modifications that reduce or eliminate effector function.
  • Illustrative Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 234 and 235, according to the EU numbering scheme.
  • one or both Fc polypeptides can comprise alanine residues at positions 234 and 235.
  • one or both Fc polypeptides may have L234A and L235A (LALA) substitutions.
  • Additional Fc polypeptide mutations that modulate an effector function include, but are not limited to, the following: position 329 may have a mutation in which proline is substituted with a glycine, arginine, serine, or an amino acid residue large enough to destroy the Fc/Fcy receptor interface that is formed between proline 329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcyRIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, N297G, and P331S, according to the EU numbering scheme.
  • L234A and L235A of a human IgGl Fc region L234A, L235A, and P329G of a human IgGl Fc region; L234A, L235A, and P329S of a human IgGl Fc region; S228P and L235E of a human IgG4 Fc region; L234A and G237A of a human IgGl Fc region; L234A, L235A, and G237A of a human IgGl Fc region; V234A and G237A of a human IgG2 Fc region; L235A, G237A, and E318A of a human IgG4 Fc region; and S228P and L236E of a human IgG4 Fc region, according to the EU numbering scheme.
  • one or both Fc polypeptides may have one or more amino acid substitutions that modulate
  • Fc polypeptides present in an anti-TREM2 protein of the disclosure, can comprise an FcRn binding site.
  • the FcRn binding site is within the Fc polypeptide or a fragment thereof.
  • the FcRn binding site comprises a native FcRn binding site. In some embodiments, the FcRn binding site does not comprise amino acid changes relative to the amino acid sequence of a native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, e.g, a human IgG binding site. In some embodiments, the FcRn binding site comprises a modification that alters FcRn binding.
  • an FcRn binding site has one or more amino acid residues that are mutated, e.g., substituted, wherein the mutation(s) increase serum half-life or do not substantially reduce serum half-life (i.e., reduce serum half-life by no more than 25% compared to a counterpart Fc polypeptide having the wild-type residues at the mutated positions when assayed under the same conditions).
  • an FcRn binding site has one or more amino acid residues that are substituted at positions 251-256, 428, and 433-436, according to the EU numbering scheme.
  • one or more residues at or near an FcRn binding site are mutated, relative to a native human IgG sequence, to extend serum half-life of the polypeptide.
  • mutations are introduced into one, two, or three of positions 252, 254, and 256.
  • the mutations are M252Y, S254T, and T256E.
  • an Fc polypeptide further comprises the mutations M252Y, S254T, and T256E.
  • one or both Fc polypeptides present in an anti-TREM2 protein of the disclosure may comprise a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256, as numbered according to the EU numbering scheme.
  • one or both Fc polypeptides may have M252Y, S254T, and T256E substitutions.
  • the mutations are M428L and/or N434S.
  • an Fc polypeptide further comprises the mutation N434S with or without M428L.
  • an Fc polypeptide comprises a mutation at one, two, or all three of positions T307, E380, and N434, according to the EU numbering scheme.
  • the mutations are T307Q and N434A.
  • an Fc polypeptide comprises mutations T307A, E380A, and N434A.
  • an Fc polypeptide comprises mutations at positions T250 and M428, according to the EU numbering scheme.
  • the Fc polypeptide comprises mutations T250Q and/or M428L. In some embodiments, an 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 the present disclosure can comprise two Fc polypeptides, wherein each of the two Fc polypeptides comprises M428L and/or N434S substitutions. In some embodiments, the Fc polypeptide comprises an N434S or N434A mutation. In some embodiments, an antibody of the present disclosure can comprise two Fc polypeptides, wherein each of the two Fc polypeptides comprises an N434S or N434A substitution. V. PREPARATION OF ANTIBODIES
  • antibodies are prepared by immunizing an animal or animals (e.g, mice, rabbits, or rats) with an antigen or a mixture of antigens for the induction of an antibody response.
  • the antigen or mixture of antigens is administered in conjugation with an adjuvant (e.g, Freund’s adjuvant).
  • an adjuvant e.g, Freund’s adjuvant
  • one or more subsequent booster injections of the antigen or antigens may be administered to improve antibody production.
  • antigen-specific B cells are harvested, e.g, from the spleen and/or lymphoid tissue.
  • the B cells are fused with myeloma cells, which are subsequently screened for antigen specificity.
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g, the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Alternatively, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to selected antigens.
  • Antibodies can also be made bispecific, i.e., able to recognize two different antigens.
  • Antibodies can also be heteroconjugates, e.g, two covalently joined antibodies, or immunotoxins.
  • Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems.
  • the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers.
  • the VH and VL regions may be expressed using a single vector, e.g, in a di-cistronic expression unit, or under the control of different promoters.
  • the VH and VL region may be expressed using separate vectors.
  • a VH or VL region as described herein may optionally comprise a methionine at the N-terminus.
  • the antibody is a chimeric antibody.
  • Methods for making chimeric antibodies are known in the art.
  • chimeric antibodies can be made in which the antigen binding region (heavy chain variable region and light chain variable region) from one species, such as a mouse, is fused to the effector region (constant domain) of another species, such as a human.
  • “class switched” chimeric antibodies can be made in which the effector region of an antibody is substituted with an effector region of a different immunoglobulin class or subclass.
  • the antibody is a humanized antibody.
  • a non human antibody is humanized in order to reduce its immunogenicity.
  • Humanized antibodies typically comprise one or more variable regions (e.g ., CDRs) or portions thereof that are non human ( e.g ., derived from a mouse variable region sequence), and possibly some framework regions or portions thereof that are non-human, and further comprise one or more constant regions that are derived from human antibody sequences.
  • 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, grafting specificity-determining residues (SDR), guided selection, and framework shuffling.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • JH antibody heavy-chain joining region
  • chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.
  • human antibodies can be produced by hybridoma-based methods, such as by using primary human B cells for generating cell lines producing human monoclonal antibodies.
  • Human antibodies can also be produced using phage display or yeast display technology.
  • phage display repertoires of variable heavy chain and variable light chain genes are amplified and expressed in phage display vectors.
  • the antibody library is a natural repertoire amplified from a human source.
  • the antibody library is a synthetic library made by cloning heavy chain and light chain sequences and recombining to generate a large pool of antibodies with different antigenic specificity. Phage typically display antibody fragments (e.g, Fab fragments or scFv fragments), which are then screened for binding to an antigen of interest.
  • antibody fragments (such as a Fab, a Fab’, a F(ab’)2, a scFv, a VH, or a VHH) are generated.
  • Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab’)2 fragments. According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.
  • an antibody or an antibody fragment is conjugated to another molecule, e.g., polyethylene glycol (PEGylation) or serum albumin, to provide an extended half-life in vivo.
  • another molecule e.g., polyethylene glycol (PEGylation) or serum albumin
  • the anti-TREM2 antibodies as disclosed herein are prepared using recombinant methods. Accordingly, in some aspects, the disclosure provides isolated nucleic acids comprising a nucleic acid sequence encoding any of the anti-TREM2 antibodies as described herein (e.g, any one or more of the CDRs, heavy chain variable regions, and light chain variable regions described herein); vectors comprising such nucleic acids; and host cells into which the nucleic acids are introduced that are used to replicate the antibody encoding nucleic acids and/or to express the antibodies.
  • isolated nucleic acids comprising a nucleic acid sequence encoding any of the anti-TREM2 antibodies as described herein (e.g, any one or more of the CDRs, heavy chain variable regions, and light chain variable regions described herein); vectors comprising such nucleic acids; and host cells into which the nucleic acids are introduced that are used to replicate the antibody encoding nucleic acids and/or to express the antibodies.
  • a polynucleotide (e.g, an isolated polynucleotide) comprises a nucleotide sequence encoding an antibody as described herein (e.g, as described in the Section above entitled “Anti-TREM2 Antibody Sequences”).
  • the polynucleotide comprises a nucleotide sequence encoding one or more amino acid sequences (e.g, CDR, heavy chain, or light chain sequences) disclosed in Table 8 below.
  • the polynucleotide comprises a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity (e.g, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to a sequence (e.g, a CDR, heavy chain, or light chain sequence) disclosed in Table 8 below.
  • a polynucleotide as described herein is operably linked to a heterologous nucleic acid, e.g, a heterologous promoter.
  • Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g, pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • plasmids and bacterial viruses e.g., pUC18, pUC19, Bluescript (e.g, pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • Bluescript e.g, pBS SK+
  • mpl8 mpl9
  • pBR322 mpl9
  • ColEl ColEl
  • pCRl pCRl
  • RP4
  • Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure.
  • the expression vector may 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 vector.
  • Suitable host cells for cloning or expressing a polynucleotide or vector as described herein include prokaryotic or eukaryotic cells.
  • the host cell is prokaryotic.
  • the host cell is eukaryotic, e.g, Chinese Hamster Ovary (CHO) cells or lymphoid cells.
  • the host cell is a human cell, e.g, a Human Embryonic Kidney (HEK) cell.
  • HEK Human Embryonic Kidney
  • an anti-TREM2 antibody as described herein includes culturing a host cell as described herein (e.g, a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expression of the antibody.
  • the antibody is subsequently recovered from the host cell (or host cell culture medium).
  • therapeutic methods using an anti-TREM2 antibody as disclosed herein are provided.
  • methods of treating a neurodegenerative disease are provided.
  • methods of modulating one or more TREM2 activities are provided.
  • methods of treating a neurodegenerative disease are provided.
  • the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam (ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadelopean PSP, Hallevorden-Spatz disease, hereditary diffuse leukoencephalopathy with spheroids (HDLS),
  • HDLS
  • 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 administering to the subject an isolated antibody or an antigen-binding fragment thereof that specifically binds to a human TREM2 protein, e.g., an anti-TREM2 antibody as described herein, or a pharmaceutical composition comprising an anti-TREM2 antibody as described herein.
  • an anti-TREM2 antibody (or antigen-binding portion or pharmaceutical composition thereof) as described herein is used in treating a neurodegenerative disease that is characterized by a mutation in TREM2.
  • the neurodegenerative disease that is characterized by a mutation in TREM2 is Alzheimer’s disease, e.g, Alzheimer’s disease that is characterized by a R47H mutation in TREM2.
  • methods of modulating one or more TREM2 activities in a subject comprises modulating levels of sTREM2; modulating recruitment or phosphorylation of a kinase that interacts with a TREM2/DAP12 signaling complex (e.g., Syk kinase); modulating phagocytosis (e.g, phagocytosis of cell debris, amyloid beta particles, etc.); modulating cell migration (e.g, migration of myeloid cells, macrophages, microglia, and disease associated microglia); and/or modulating cell differentiation (e.g, for myeloid cells, macrophages, microglia, and disease associated microglia).
  • a TREM2/DAP12 signaling complex e.g., Syk kinase
  • phagocytosis e.g, phagocytosis of cell debris, amyloid beta particles, etc.
  • modulating cell migration e.g, migration of myeloid cells, macrophages, microglia, and
  • methods of enhancing one or more TREM2 activities in a subject having a neurodegenerative disease are provided.
  • methods of decreasing levels of sTREM2 in a subject having a neurodegenerative disease are provided.
  • the method of modulating one or more TREM2 activities in a subject comprises administering to the subject an isolated antibody or an antigen-binding portion thereof that specifically binds to a human TREM2 protein, e.g, an anti-TREM2 antibody as describe herein, or a pharmaceutical composition comprising an anti-TREM2 antibody as described herein.
  • the subject to be treated is a human, e.g, a human adult or a human child.
  • methods of reducing plaque accumulation in a subject having a neurodegenerative disease comprise administering to the subject an antibody or pharmaceutical composition as described herein.
  • the subject has Alzheimer’s disease.
  • the subject is an animal model of a neurodegenerative disease (e.g, a 5XFAD or APP/PS1 mouse model).
  • plaque accumulation is measured by amyloid plaque imaging and/or Tau imaging, e.g, using positron emission tomography (PET) scanning.
  • PET positron emission tomography
  • administration of an anti-TREM2 antibody reduces plaque accumulation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a baseline value (e.g, the level of plaque accumulation in the subject pirior to administration of the anti-TREM2 antibody).
  • a baseline value e.g, the level of plaque accumulation in the subject pirior to administration of the anti-TREM2 antibody.
  • an anti-TREM2 antibody is administered to a subject at a therapeutically effective amount or dose.
  • the dosages may be varied according to several factors, including the chosen route of administration, the formulation of the composition, patient response, the severity of the condition, the subject’s weight, and the judgment of the prescribing physician.
  • the dosage can be increased or decreased over time, as required by an individual patient. In certain instances, a patient initially is given a low dose, which is then increased to an efficacious dosage tolerable to the patient. Determination of an effective amount is well within the capability of those skilled in the art.
  • the route of administration of an anti-TREM2 antibody as described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalational, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
  • the antibody is administered orally, intravenously, or intraperitoneally.
  • the anti-TREM2 antibody (and optionally another therapeutic agent) is administered to the subject over an extended period of time, e.g., for at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 days or longer.
  • compositions and kits comprising an antibody that specifically binds to a human TREM2 protein are provided.
  • the pharmaceutical compositions and kits are for use in treating a neurodegenerative disease.
  • the pharmaceutical compositions and kits are for use in modulating (e.g, enhancing or inhibiting) one or more TREM2 activities, e.g, Syk phosphorylation.
  • the pharmaceutical compositions and kits are for use in modulating (e.g, decreasing) sTREM2 levels.
  • compositions comprising an anti-TREM2 antibody or an antigen-binding fragment thereof are provided.
  • the anti-TREM2 antibody is an antibody as described in Section III above or an antigen-binding fragment thereof.
  • a pharmaceutical composition comprises an anti-TREM2 antibody as described herein and further comprises one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that does not interfere with or otherwise inhibit the activity of the active agent.
  • Various pharmaceutically acceptable excipients are well-known in the art.
  • the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration.
  • Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
  • Other pharmaceutically acceptable carriers and their formulations are well- known in the art.
  • compositions described herein can be manufactured in a manner that is known to those of skill in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • an anti-TREM2 antibody can be formulated by combining it with pharmaceutically acceptable carriers that are well known in the art.
  • Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents can be added, such as a cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • An anti-TREM2 antibody can be formulated for parenteral administration by injection, e.g, by bolus injection or continuous infusion.
  • the compound or compounds can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • compounds can be formulated in aqueous solutions, e.g., in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer.
  • Formulations for injection can be presented in unit dosage form, e.g, in ampules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • a pharmaceutical composition for use in in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, e.g, heat sterilization, steam sterilization, sterile filtration, or irradiation.
  • compositions of the disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of one in the art. Suitable dosages are also described in Section VII above.
  • kits comprising an anti-TREM2 antibody are provided.
  • the anti-TREM2 antibody is an antibody as described in Section III above or an antigen-binding fragment thereof.
  • the kit further comprises one or more additional therapeutic agents.
  • the kit comprises an anti-TREM2 antibody as described herein and further comprises one or more additional therapeutic agents for use in the treatment of a neurodegenerative disease, e.g, Alzheimer’s disease.
  • the therapeutic agent is an agent for use in treating a cognitive or behavioral symptom of a neurodegenerative disease (e.g, an antidepressant, a dopamine agonist, or an anti-psychotic).
  • the therapeutic agent is a neuroprotective agent (e.g, carbidopa/levodopa, an anticholinergic agent, a dopaminergic agent, a monoamine oxidase B (MAO-B) inhibitor, a catechol-O-methyl transferase (COMT) inhibitor, a glutamatergic agent, a histone deacetylase (HD AC) inhibitor, a cannabinoid, a caspase inhibitor, melatonin, an anti-inflammatory agent, a hormone (e.g, estrogen or progesterone), or a vitamin).
  • a neuroprotective agent e.g, carbidopa/levodopa, an anticholinergic agent, a dopaminergic agent, a monoamine oxidase B (MAO-B) inhibitor, a catechol-O-methyl transferase (COMT) inhibitor, a glutamatergic agent, a histone deacetylase (HD AC) inhibitor, a cannabi
  • the kit comprises an anti-TREM2 antibody as described herein and further comprises one or more reagents for measuring sTREM2 levels. In some embodiments, the kit comprises an anti-TREM2 antibody as described herein and further comprises one or more reagents for measuring TREM2 activity (e.g, for measuring Syk phosphorylation).
  • the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g, instructions for using the kit for a therapeutic method as described in Section VI above).
  • instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g, magnetic discs, tapes, cartridges, chips), optical media (e.g, CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • the ecto domain (residues 19-172) of human TREM2 (UniProtKB ID - Q9NZC2) was subcloned into pRK vector with the secretion signal from mouse IgG kappa chain V-III, amino acids 1-20 (UniProtKB ID - P01661) at the N-terminal region, and a mouse Fc tag at the C-terminal region with a GGGGS (SEQ ID NO:34) between TREM2 ECD and Fc.
  • ExpiFectamineTM 293 Transfection Enhancer 1 and 2 were added to the cells 16 hours post transfection and the media supernatant was harvested 96 hours post transfection.
  • the clarified supernatant was supplemented with EDTA-free protease inhibitor (Roche) and was stored at -80°C.
  • the SEC mobile phase buffer was kept at 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 50 mM arginine, which was also the protein storage buffer. All chromatography steps were performed on AKTA pure or AKTA Avant systems (GE Healthcare Life Sciences).
  • the ecto domain (residues 19-172) of TREM2 (UniProtKB - Q9NZC2) was subcloned in the pRK vector with the secretion signal from mouse Ig kappa chain V-III, amino acids 1-20 (UniProtKB ID - P01661) at the N-terminal region, and a 6X-His tag (SEQ ID NO: 35) at the C-terminal region.
  • the insert was verified by sequencing and maxi prep plasmid purification was performed.
  • the bound His-tagged TREM2 eco domain was eluted with 20 mM Tris pH 8.0, 150 mM NaCl, and 200 mM imidazole. Eluted protein was concentrated using Amicon 10 kDa concentrators 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 lx PBS and eluted and fractionated using lx 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 the intact protein mass determination. Results from the PAGE and analytical characterization were used to pool the heavily glycosylated protein fractions and these were aliquoted and stored at -80°C.
  • Rodents (mice and rats) were immunized using standard protocols with rhTREM2- Fc immunogen or BWZ cells expressing full length Trem2 receptor. Titers were measured throughout immunization using sera collected at different time points. The detection of an antigen specific immune response was performed using flow cytometry with the rhTREM2- Fc immunogen and live BWZ cells expressing full-length TREM2. Selection criteria of candidate antibodies included rodent antibody production and specificity of binding to TREM2 as detected by flow cytometry. Antibody-secreting cells were isolated from animal immune tissues including spleen, lymph nodes and bone marrow.
  • Exemplary anti-TREM2 antibodies were sequence optimized and humanized, followed by characterization for binding kinetics and binding specificity.
  • Sequence optimization was conducted by searching within CDR sequences for residues that are susceptible to chemical modification (e.g asparagine deamidation motifs (NG), aspartic acid isomerization motifs (DS), and potential oxidation residues (tryptophan (W) and methionine (M)) and making amino acid substitutions with conservative and germline residues to remove such sequence liabilities.
  • NG asparagine deamidation motifs
  • DS aspartic acid isomerization motifs
  • W tryptophan
  • M methionine
  • the Fd (VH+CH1) region of a humanized, affinity matured anti-TREM2 antibody was cloned into expression vectors comprising a sequence encoding an Fc polypeptide engineered to bind to the human transferrin receptor (TfR) (CH3C.35.23.1.1, CH3C.35.23.3, CH3C.35.23.3 cisLALA, or CH3C.35.24) or a sequence encoding an Fc polypeptide that binds to the cynomolgus monkey transferrin receptor (CH3C.35.21).
  • TfR human transferrin receptor
  • the Fc polypeptide-encoding sequence also contained a “knob” (T366W) mutation to prevent homodimerization and promote heterodimerization with an Fc polypeptide comprising “hole” (T366S/L368A/Y407V) mutations.
  • the Fd region was also cloned into corresponding “hole” vectors comprising a sequence encoding an Fc polypeptide with hole mutations, but lacking the TfR binding mutations.
  • the coding sequences (both Fd- knob-Fc and Fd-hole-Fc constructs) also contained “LALA” (L234A; L235A) mutations in the hinge region to reduce effector function (Wines et al, ./. Immunol.
  • knob and hole vectors were co-transfected to ExpiCHO or Expi293 cells along with the corresponding light chain vector (SEQ ID NO:54) in the ratio knob:hole:light chain of 1:1:2.
  • 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-Fab was immobilized on a CM5 chip, and the anti-TREM2 protein was captured. Full-length human TfR or human TfR apical domain at serial dilution (e.g, concentrations of 1-1,000 nM) was flowed over the chip (180 second association time) and then allowed to dissociate. Fitting was performed using a 1 : 1 binding model.
  • the binding response was corrected by subtracting the RU value from a blank flow cell.
  • a 1:1 Languir model of simultaneous fitting of k on and k 0ff was used for kinetics analysis. KD binding values were calculated from k 0n and k 0ff .
  • a HEK 293 cell line stably expressing human TREM2/DAP12 was generated by transfecting the cells with a vector expressing wild type human TREM2 and DAP12, and DAP12 alone, respectively. Stable expressing clones were selected, and the cell surface TREM2 expression was evaluated by flow cytometry. APC-conjugated rat anti-human/mouse-TREM2 monoclonal antibody (R&D, Catalog No. MAB 17291) was used to detect surface TREM2 expression.
  • the clone showing the highest wild type TREM2 expression level was selected and named “HEK293-H6.”
  • the clones stably expressing DAP 12 were analyzed by Western blot, and the selected clone was named “HEK293- DAP12#1.”
  • HEK 293 overexpressing human TREM2 (HEK293-H6) and HEK 293 overexpressing GFP (B5) were harvested by 0.05% trypsin and incubated at 37°C for 2 hours. After incubation, the cells were centrifuged and washed in FACS buffer (PBS + 0.5% BSA) twice. Mixed cells were resuspended in FACS buffer with human Trustain FcX solution (Biolegend, Catalog No. 422302) at a density of 10 6 /mL per cell line. The mixed cell lines were seeded at 200,000 cells per well in a 96-well round-bottom plate and incubated for 20 minutes at room temperature.
  • the cells were centrifuged and incubated with a dose titration of anti-TREM2 antibodies for 45 minutes on ice. After incubation, the cells were centrifuged and washed with FACS buffer three times. The cells were then incubated with secondary antibody (Alexa Fluor 647 AffmiPure F(ab’)2 Fragment Goat Anti-human IgG(H+L), Jackson ImmunoResearch Laboratories, Catalog No. 109-606-088, 1:800 dilution) for 30 minutes on ice.
  • secondary antibody Alexa Fluor 647 AffmiPure F(ab’2 Fragment Goat Anti-human IgG(H+L), Jackson ImmunoResearch Laboratories, Catalog No. 109-606-088, 1:800 dilution
  • Activation of TREM2-dependent pSyk signaling was measured in human macrophage cells or in HEK293-H6 cells using a commercial AlphaLisa assay from Perkin- Elmer.
  • the lipid vesicles were prepared within two weeks of experiments as follows: 7 mg DOPC (l,2-dioleoyl-s «-glycero-3-phosphocholine) and 3 mg POPS (l-palmitoyl-2-oleoyl-5 «- glycero-3- phospho-L-serine) were combined in chloroform in a glass vial and dried under a stream of N2 gas for 1-2 hours, or until completely dry. The lipid mixture was re-suspended in 1 mL HBSS (for a final lipid concentration of about 10 mg/mL) and vortexed for 2-3 minutes. Subsequently, the lipid suspension was extruded using an Avanti mini-extruder constructed with one 100-nm pore size membrane to form small unilamellar vesicles at 10 mg/mL.
  • human macrophage cells or HEK293-H6 cells were plated at 100,000 cells/well or 40,000 cells/well, respectively, on a 96-well plate coated with poly-D- lysine.
  • Antibodies were diluted in a 10-point serial dilution with 3 -fold dilution between points into PBS.
  • lipid vesicles containing 70% DOPC and 30% POPS at 1 mg/mL final concentration were also included in the antibody/PBS mixture.
  • the cells were washed 3 times with HBSS using a Biotek 405/406 plate washer, after which 50 pL per well of the antibody/PBS (with or without vesicles) solution was added using a Hamilton Nimbus liquid handler. The cell plate was then transferred to a 37°C incubator for 5 minutes. The liposome/antibody solution was removed by flicking the plate, and 40 pL lysis buffer (Cell Signaling Technologies, CST) containing 1 pM PMSF was added using the liquid handler. The lysate was then either frozen at -80°C or immediately assayed in the AlphaLisa assay.
  • CST Cell Signaling Technologies
  • Human macrophage cells were prepared for assay as follows. Human monocytes were isolated following the RosetteSep human monocyte enrichment cocktail protocol (Stemcell Technologies, REF#15068) from fresh blood. Isolated monocytes were washed in wash buffer (PBS+2% FBS) and resuspended in 10 mL ACK lysis buffer (ThermoFisher Scientific, Catalog No. A10492) to lyse red blood cells.
  • wash buffer Twenty (20) mL of wash buffer was added to stop cell lysis, and the sample was centrifuged and washed once more with culture media (RPMI, 10% Hyclone FBS, 1% Sodium Pyruvate, 1% Glutamax, 1% non-essential amino acids, and 1% Penicillin-streptomycin). Human monocytes were then differentiated into macrophage cells in culture media in the presence of 50 ng/mL human recombinant M- CSF (Gibco, Catalog No. PHC9501) at 250-mL flask. Fresh human M-CSF was spiked on day 3 and human macrophages were subsequently harvested on day 5 and used for assay.
  • M- CSF Gibco, Catalog No. PHC9501
  • Cell lysates were assayed for pSyk using the standard protocol for the Perkin Elmer pSyk AlphaLisa kit.
  • 10 pL of lysate/well was transferred to a white opaque 384 well Optiplate (Perkin Elmer).
  • 5 pL of Acceptor Mix (containing the working solution of acceptor beads) was added per well, followed by sealing of plates with foil seals and incubation for 1 hour at room temperature.
  • 5 pL of Donor Mix (containing the working solution of donor beads) was added to each well under reduced light conditions. Plates were again sealed and incubated for 1 hour at room temperature.
  • the plates were read using AlphaLisa settings on a Perkin Elmer EnVision plate reader.
  • Human monocytes were isolated following the RosetteSep human monocyte enrichment cocktail protocol (Stemcell Technologies, Catalog No. 15068). Isolated monocytes were washed in wash buffer (PBS+2% FBS) and resuspended in 10 mL ACK lysis solution (ThermoFisher Scientific, Catalog No. A10492) to lyse red blood cells. Twenty (20) mL wash buffer was added to stop lysis. The cell suspension was centrifuged and washed once with culture media (RPMI 1640 + 10% FBS + penicillin/streptomycin). Cells were resuspended in culture media at a density of 10 6 cells pL/mL and used in the survival assay described below.
  • 96-well plates were pre-coated with anti-TREM2 antibody or isotype control in a dose titration (45 pL/well, total 12 points) and incubated overnight at 4°C. After overnight incubation, the pre-coated plate was washed twice with PBS and then loaded with human monocyte (10 5 cells/well) in the presence of low concentration human M- CSF (5 ng/mL, Gibco, Catalog No. PHC9501). After 5 days at 37°C, the media was aspirated, and 100 pL PBS + 100 pL Celltiter-glo media (Promega, Catalog No. G7571) was added to each well. After 10 minutes of incubation, the cell media was transferred to multiwell plates compatible for luminometer use, and luminescence for cell viability was recorded. Livid Storage Assay
  • iPSCs induced human pluripotent stem cells
  • HPCs hematopoietic progenitor cells
  • STEMdiff Hematopoietic Kit from StemCell Technologies
  • the concentration of anti-TREM2-antibody used was lOOnM.
  • media containing lOOnM anti-TREM2 antibody was serially diluted 3-fold for a total of 10 points.
  • RSV was used as a control. The cells were incubated for another 48 hours at 37°C before imaging cells using Bodipy stain, or extracting the cells for lipidomics, as described below.
  • Bodipy imaging the supernatant was removed, and cells were incubated at 37 °C for 30 minutes in live cell imaging buffer (Life Technologies, Catalog No. A14291DJ) containing 1:2500 of a lmg/mL Bodipy 493/503 solution in DMSO (Thermo-Fisher D3922) and 1 drop/mL of Nucblue (ThermoFisher, Catalog No. R37605). After the incubation period, the staining solution was removed, and the cells were either imaged live or fixed in 4% paraformaldehyde. The cells were imaged using the Alexa 488 channel for Bodipy, and DAPI illumination settings on an Opera Phoenix high content confocal imager. Lipid spots were analyzed using a spot-finding algorithm on the Harmony software supplied with the instrument.
  • Lipid levels were analyzed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled to electrospray mass spectrometry (QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 5 pL of sample was injected on a BEH C18 1.7 pm, 2.1x100 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.25 mL/min at 55°C.
  • mobile phase A consisted of 60:40 acetonitrile/water (v/v) with 10 mM ammonium formate + 0.1% formic acid
  • mobile phase B consisted of 90:10 isopropyl alcohol/acetonitrile (v/v) with 10 mM ammonium formate + 0.1% formic acid.
  • mobile phase A consisted of 60:40 acetonitrile/water (v/v) with 10 mM ammonium acetate
  • mobile phase B consisted of 90:10 isopropyl alcohol/acetonitrile (v/v) with 10 mM ammonium acetate.
  • Electrospray ionization was performed in either positive or negative ion mode applying the following settings: curtain gas at 30; collision gas set at medium; ion spray voltage at 5500 (positive mode) or 4500 (negative mode); temperature at 250°C (positive mode) or 600°C (negative mode); ion source Gas 1 at 50; ion source Gas 2 at 60.
  • Lipids were quantified using a mixture of non-endogenous internal standards. Lipids were identified based on their retention times and MRM properties of commercially available reference standards (Avanti Polar Lipids, Birmingham, AL, USA).
  • Wild-type iPSC-derived human microglia were cultured and treated with anti- TREM2 antibody (100 nM final concentration) and either DMSO or a commercial mTOR inhibitor (Selleckchem, Catalog No. AZD8055, 20 nM final concentration) for 96 hours.
  • the treated cells were subsequently lysed, and the cell lysates were prepared for Western blots to investigate phosphorylation of major signaling targets in the mTOR pathway.
  • Primary antibodies for Western blots were obtained from Cell Signaling Technologies: (1) phospho- mTOR (Ser2448), Product No. 5536T; (2) mTOR (7C10), Product No. 2983T; (3) phospho- AKT (Ser473), Product No.
  • results for an analysis of the binding characteristics of humanized and sequence- optimized variants of antibody CL0020188 are provided in Table 2 and FIGS. 1A-1H.
  • 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 provides KD values as measured by Biacore and ECso values as measured by dose-titrated binding assay in HEK293-H6 cells.
  • FIGS. 1A-1H include representative dose-response curves of binding to TREM2 expressed by HEK293-H6 cells for the humanized and sequence-optimized variants. Variants are represented by solid black circles ( ⁇ ), while isotype controls are represented by open white circles (°).
  • Results for 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 illustrated in FIG. 2.
  • FIG. 3 illustrates the results for an ATV:TREM2 variant (ATV:TREM2 #3) and a corresponding anti-TREM2 antibody.
  • the ATV:TREM2 was able to activate pSyk signaling in TREM2-expressing HEK293-H6 cells to a significantly greater extent than the corresponding TREM2 antibody, indicating that the addition of ATV to the molecule can increase its potency (FIG. 3).
  • the ATV:TREM2 induced macrophage survival with an ECso of 4.1 + 0.3 nM.
  • the anti-TREM2 antibodies demonstrated capability in reducing lipid accumulation in myelin-treated iMG (FIGS. 4 A and 4B) with an ICso for inhibition of lipid storage of 0.20 nM (97.7 + 0.3 % max. inhibition).
  • FIGS. 5A-5F and FIGS. 6A-6C show that a representative ATV:TREM2 variant (ATV:TREM2 #3) reduces lipid accumulation while enhancing fatty acid oxidation intermediates, suggesting a potential role of ATV:TREM2 in enhancing mitochondrial function.
  • Cells (iMG) treated with oleic acid lipid challenge (33 mM) followed by incubation with ATV:TREM2 were able to reduce lipid accumulation, as illustrated by Bodipy staining (FIGS. 5A and 5B).
  • FIG. 5C provides a heat map showing all TG, acyl carnitine, and TCA cycle intermediate species that illustrated a fold change of > 1.5 (p ⁇ 0.05), while FIGS. 5D-5F illustrate the changes of representative species in vehicle and myelin-challenged iMG that were incubated with ATV:TREM2 or isotype control following challenge.
  • FIGS. 5D-5F illustrate the changes of representative species in vehicle and myelin-challenged iMG that were incubated with ATV:TREM2 or isotype control following challenge.
  • FIGS. 6A-6C illustrate the changes of specific TG, acyl carnitine, and TCA cycle intermediate species in iMG incubated with ATV:TREM2 or isotype control following myelin challenge.
  • ATV:TREM2 reduces all species of TG and ceramides while increasing certain species of short chain acyl carnitines, indicating that ATV:TREM2 may enhance mitochondrial function.
  • FIG. 7A illustrates representative Western blot images of mTOR signal pathway targets. Quantification of Western blot data is provided in FIGS. 7B-7E (phosphorylation levels normalized to beta-actin loading control).
  • RPS6 is a signal target that is downstream of mTORCl complex
  • GSK3b is a signal target that is downstream of mTORC2 complex.
  • ISO isotype control
  • ATV:TREM2 The role of ATV:TREM2 in lysosomal function was investigated. To assess impact on lysosomal function, levels of progranulin (PGRN) and bis(monoacylglycero)phosphates (BMPs) were measured in iPSC-derived microglial cells (“iMG”) treated with an ATV:TREM2 variant.
  • PGRN progranulin
  • BMPs bis(monoacylglycero)phosphates
  • iMG were plated at a density of 30,000 cells per well in a 96-well plate, and incubated with ATV:TREM2 #3 (100 nM) for 72 hours, after which the cell supernatant and cell lysate were collected and analyzed for progranulin (PGRN) levels using a colorimetric sandwich ELISA.
  • iMG were challenged with myelin or vehicle for 24 hours, followed by incubation with ATV:TREM2 #3 (100 nM) for 48 hours.
  • Cellular lipids were extracted via addition of methanol containing an internal standard mixture and BMP abundance was measured by liquid chromatography-mass spectrometry (LC-MS/MS) on a Q- trap 6500 (SCIEX) similar to that described in International PCT Publication No. WO 2020/112889.
  • BMP species were quantified using BMP(14:0_14:0) as the internal standard and identified based on their retention times and MRM properties. Quantification was performed using Multi Quant 3.02 (Sciex) after correction for isotopic overlap.
  • BMP species were normalized to median lipid content of all species measured. Protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). A representative BMP result is illustrated in FIG. 9. As shown in FIG. 9, iMG challenged with myelin and subsequently treated with ATV:TREM2 reduces levels of BMP species, suggesting potential rescue of lysosomal challenge induced by myelin.
  • BCA bicinchoninic acid
  • iPSC-derived microglial cells (“iMG”) treated with an ATV:TREM2 variant or isotype control using a Seahorse XFe96 analyzer (Agilent) and using materials and protocol from the Seahorse XF Palmitate Oxidation Stress Kit (Agilent 103693).
  • iMG iPSC-derived microglial cells
  • Agilent Seahorse XFe96 analyzer
  • Agilent 103693 Materials and protocol from the Seahorse XF Palmitate Oxidation Stress Kit
  • FIG. 10A A representative kinetic graph of oxygen consumption is illustrated in FIG. 10A, and a bar graph illustrating the maximal respiratory capacity of the cells is provided in FIG. 10B.
  • ATV:TREM2 increases maximal respiration to an extent similar to that of fatty acid substrate palmitic acid (PAL). This effect is diminished in the presence of CPT1 inhibitor.
  • PAL fatty acid substrate palmitic acid
  • ATV:TREM2 #1 and ATV:TREM2 #3 were compared to those of reference antibodies that bind TREM2, which are described in WO 2019/028292.
  • the heavy chain and light chain sequences of reference antibody #1 (“Ref. Ab. #1”) are represented by SEQ ID NOs:74 and 75, respectively.
  • the heavy chain and light chain sequences of reference antibody #2 (“Ref. Ab. #2”) are represented by SEQ ID NOs:76 and 75, respectively.
  • Heavy and light chain sequences for the isotype controls of each anti-TREM2 antibody are provided in Table 5.
  • FIG. 11A illustrates cell viability dose-response curves with the anti-TREM2 antibodies, with “ISO” referring to the isotype control for ATV:TREM2 #3 (Table 5).
  • the results provided in FIGS. 11A-11C indicate that ATV:TREM2 #3 is more potent in promoting human macrophage survival in vitro than reference antibodies #1 and #2.
  • ATV:TREM2 #3, reference antibody #1, and reference antibody #2 are able to promote human macrophage survival and proliferation (FIG. 11 A).
  • ATV:TREM2 #3 shows stronger potency for cell survival with reduced overall cytokine signature relative to reference antibodies #1 and #2 (FIGS. 1 IB, 11C, and 12).
  • ATV:TREM2 is able to reduce trislvceride species levels after challenge
  • Anti-TREM2 antibodies were evaluated by lipid storage assay (using 10 mM oleic acid challenge) as described in Example 4. The results for ATV:TREM2 #3, reference antibody #1, and reference antibody #2 are provided in FIGS. 13A-13E.
  • FIGS. 13A-13C illustrate volcano plots with cut-offs of p ⁇ 0.05 and fold change > 1.5 for triglyceride species in iPSC-derived microglial cells (“iMG”) as quantified by LCMS. The data is normalized to the isotype control for each antibody. As shown in FIGS. 13A-13C, ATV:TREM2 #3 is able to modulate triglyceride (TG) species post-oleic acid dosing, while the reference antibodies did not significantly change levels of TG species after oleic acid challenge. FIGS. 13D and 13E illustrate bar graphs of representative TG species measurement, with data normalized to isotype control for ATV:TREM2 #3.
  • ATV:TREM2 is able modulate TREM2 levels in vitro
  • iMG iPSC-derived microglial cells
  • ATV:TREM2 #3 or reference antibody #1 for 72 hours at various concentrations, followed by measurement of TREM2 levels in iMG cell lysate and cell culture medium.
  • TREM2 was measured as follows. Briefly, MSD small spot streptavidin plates (Meso Scale Discovery) were coated with biotinylated goat anti-hTREM2 polyclonal antibody (R&D Systems, BAF1828) at room temperature for 1 hour. The plates were then blocked with MSD Block A buffer (Meso Scale Discovery) for 1 hour at room temperature.
  • Samples and standards were prepared/diluted with assay buffer (25% MSD Block A buffer in TBST), and 30 pL of samples and standards was loaded into the plate after blocking. After 1 hour of incubation at room temperature, the plates were washed with TBST and followed by binding the primary antibody (ATV:TREM2 #3) for 1- hour at room temperature. Afterwards, diluted sulfo-tagged goat anti-human IgG (Southern Biotech, 2049-01) was added to the plates, and incubated for one hour at room temperature. After washing with TBST, the MSD plates were developed using 2x MSD read buffer T, followed by detection using an MSD Sector plate reader. MSD values were converted to absolute quantities of TREM2 by fitting a standard curve using Meso Scale Discovery software (Discovery Workbench).
  • FIGS. 14A and 14B show plots of TREM2 levels as a function of antibody concentration in iMG cell lysates and cell culture media after incubation with anti-TREM2 antibodies. TREM2 levels for each antibody are normalized to their specific isotype control.
  • “ISO” represents isotype control for ATV:TREM2 #3.
  • the levels of total TREM2 increased with increasing amounts of antibody in the cell lysates of iMG treated with ATV:TREM2 #3 and reference antibody #1.
  • the levels of soluble TREM2 decreased with increasing amounts of antibody in the cell culture media of the antibody-treated cells.
  • ATV:TREM2 exhibits a superior pharmokine tic profile in non-human primates
  • PK pharmacokinetic
  • Table 6 shows the PK values of anti-TREM2 antibodies in the cerebrospinal fluid (CSF) of non-human primates.
  • CSF cerebrospinal fluid
  • ATV:TREM2 variants show that these antibodies have a higher exposure in CSF relative to TREM2 Ab (lacking binding capacity for transferrin receptor; heavy and light chain sequences represented by SEQ ID NOs: 83 and 54, respectively) and reference antibody #1.
  • FIG. 15 illustrates the PK profiles for anti-TREM2 antibodies in the CSF of dosed non-human primates. As illustrated in FIG. 15 and Table 6, ATV:TREM2 shows at least a 2- fold increase in CSF PK compared to reference antibody #1.
  • Human TREM2 copy number was determined using qPCR analysis of tail genomic DNA. Human TREM2 mRNA and protein levels were measured in brains, liver, lung, and spleen by qRT- PCR and anMSD assay. Human TREML1 and TREML2 mRNA were analyzed in brain- sorted microglia by qRT-PCR. Surface TREM2 expression was quantified by FACS in bone marrow derived macrophages (BMDM). Human TREM2 function was assessed by the in vitro BMDM survival assay.
  • BMDM bone marrow derived macrophages
  • Human TREML1 and TREML2 were undetectable in either TB36 or TB45, showing successful deletion of these genes.
  • qPCR analysis showed that there are two and one copies of human TREM2 transgenes in TB36 and TB45, respectively, with corresponding higher human TREM2 expression in TB36 relative to TB45 in brains and peripheral tissues.
  • the in vitro survival assay showed that human TREM2 agonist antibodies trigger stronger responses in TB36 line than in TB45 line.
  • TB36 for the following breeding and in vivo studies.
  • the hemizygous TB36 mice were further backcrossed to C57BL/6J for three rounds and then bred with hTfR KI mice (described in U.S. Patent No. 10,143,187) to generate human TREM2 BAC hemizygous; hTfR KI homozygous mice for in vivo studies.
  • ATV:TREM2 can trigger microglia response in vivo
  • a single dose of ATV:TREM2 #3 or a corresponding isotype control (ATV:RSV) (100 mg/kg) was intravenously administered to TB36/hTfR KI mice at day 0, and mice were sacrificed at day 1 or day 4 post-dose for ex vivo analysis.
  • animals were anesthetized by intraperitoneal injection of 2.5% Avertin. Terminal blood was collected through the cardiac puncture in an EDTA tube with slow inversion (10 times) and was then centrifuged at 15,350 g for 7 minutes at 4°C.
  • Plasma top layer was transferred to a 1.5-ml Eppendorf tube and stored at -80°C until measurement.
  • CSF samples were collected by pre-pulled glass capillary tubes from the cisterna magna and then transferred to 0.5 mL Protein LoBind Eppendorf tubes for centrifugation at 12,700 rpm for 7 minutes at 4°C.
  • the supernatants of CSF samples were snap frozen on dry ice and stored at -80°C until measurement. The animals were then perfused with cold PBS, and brains were dissected out, and the two hemispheres were separated.
  • Plasma and brain levels of human IgG were evaluated at 1 day and 4 days post-dose and are shown in Table 7 below.
  • ATV.TREM2 To determine the lowest effective dosage of ATV.TREM2 and to compare to non- ATV TREM2 antibodies, a single dose of ATV:TREM2 #3 (1, 3, 10, 30 mg/kg) or a corresponding TREM2 reference antibody (“TREM2 Ab;” 30 mg/kg), reference antibody #2 (30 mg/kg) or isotype control (ATV:RSV; 30 mg/kg) were intravenously administered to TB36/hTfR KI mice at day 0. To measure microglial proliferation, mice were IP administrated with EdU at day 0, day 1, day 2 and day 3 after antibody treatment. Mice were taken down at day 1 and day 4 for analysis.
  • EdU-Ibal double staining shows dosage- dependent increase of new bom microglia from 1 mg/kg to 10 mg/kg of the ATV:TREM2- dosed animals at day 4, with 10 mg/kg ATV:TREM2 showing the maximum effect on microglial proliferation (FIGS. 17A and 17B). Furthermore, 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 30-fold higher dosing.
  • cytokine levels were measured in terminal plasma and brain lysates (prepared by Cell Signaling lysis buffer #9803) of TB36/hTfR KI mice using the Mouse Cytokine Array/Chemokine Array 44-Plex (MD44).
  • ATV:TREM2 #3 did not change the cytokine levels measured in the plasma, but acutely increased some cytokine levels (e.g., IP-10 and MCP-5) measured in the brain at 24-hr post-dose, and these cytokine levels were restored to the baseline level at 96-hr post-dose.
  • ATV:TREM2 #3 at 3 mg/kg had maximum effect on brain cytokine levels (FIGS. 18A and 18B).
  • ATV:TREM2 increases brain CSF1R level in TR36/hTfR KI mice
  • the brain concentration of 10 mg/kg of ATV:TREM2 #3 was similar to TREM2 Ab at 30 mg/kg, and the brain concentration of l-3mg/kg of ATV:TREM2 #3 was similar to reference antibody #2 at 30 mg/kg. More 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.
  • the brain concentration of 10 mg/kg of ATV:TREM2 #3 was similar to TREM2 Ab and the reference antibody #2 at 30 mg/kg (FIG. 21). Overall, brain uptake of ATV:TREM2 #3 is more efficient than TREM2 Ab and reference antibody #2.

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