TREM2 agonists

Abstract

The present application discloses, inter alia, certain antibodies that specifically bind to human TREM2 (triggering receptor expressed on myeloid cells 2). In some embodiments, the antibodies act as TREM2 agonists. In some embodiments, the antibodies herein specifically bind to the stalk region of intact human TREM2 without binding to soluble TREM2, which is the product of TREM2 cleavage between residues H157 and S158. In some embodiments, the antibodies act as TREM2 agonists, specifically binding to the stalk region of TREM2 with a low dissociation constant ranging from 10 nM to 100-500 pM, 10-50 pM, or 1-10 pM, specifically binding to a TREM2 epitope spanning the H157-S158 cleavage site, and not binding to soluble TREM2. In some embodiments, the antibodies herein also inhibit soluble TREM2 shedding in human microglial cell models and in vivo in mouse models, reduce levels of soluble TREM2 in plasma, CSF and / or brain, enhance human microglial cell survival, and increase Aβ plaque formation and compaction in human microglial models (e.g., as measured by increased Aβ plaque intensity and / or increased X04 plaque intensity in human microglia), and thus may provide some neuroprotective activity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 188,800, filed May 14, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a computer-readable sequence listing entitled "01164-0014-00PCT_ST25", created on May 9, 2022, having a size of 115KB, which is incorporated herein by reference.

[0003] Field The present application discloses, inter alia, certain antibodies that specifically bind to TREM2. In some embodiments, the antibodies act as TREM2 agonists. [Background technology]

[0004] background TREM2 (triggering receptor expressed on myeloid cells 2) is an activating immune cell receptor expressed in the brain in microglia (Hickman et al., Nat. Neurosci. 16(12):1896-1905(2013); Zhang et al., Neuron 89:37-53(2016); Deczkowska et al., Cell 181:1207-1217(2020); Hansen et al., J. Cell Biology 217(2):459-472(2018); Yeh et al., Trends Mol. Med. 23:512-533(2017)). Certain TREM2 mutations have been identified as risk factors for Alzheimer's disease (AD) (Guerreiro et al., N. Engl. J. Med. 368:117-27 (2013); Jonsson et al., N. Engl. J. Med. 368:107-116 (2013)). Studies are also being conducted in mouse models to understand how TREM2 influences the development of AD pathologies such as β-amyloid (Aβ) plaques and phosphorylated aggregates of tau protein. The emerging consensus is that when TREM2 function is impaired, Aβ plaques have a less compacted morphology and are more damaging to surrounding neurites (Meilandt et al., J Neurosci. 40:1872-19(2020); Wang et al., J. Exp. Medicine 213:667-675(2016); Yuan et al., Neuron 90:724-739(2016)).

[0005] Aβ aggregates have been found to directly bind to recombinant TREM2 extracellular domain, while other studies suggest that certain phospholipids are relevant ligands for TREM2. TREM2 also binds APOE or APOJ when they are lipidated, promoting the uptake of lipophilic articles containing APOE / APOJ. (Yeh et al., Neuron 91:328-340(2016).) Furthermore, binding of Aβ oligomers to APOE / APOJ lipoparticles accelerates microglial uptake and degradation of Aβ, a process mediated in part by TREM2. After ligation, TREM2 triggers the DAP12 / Fyn / Syk signaling cascade that affects a host of microglial processes, including phagocytosis, endocytosis, chemotaxis, CSF-1-mediated survival, aggregate degradation, and metabolic changes. Thus, TREM2 appears to be central to microglial activity in response to amyloid and lipids. Summary of the Invention

[0006] overview The present disclosure relates to antibodies that specifically bind to TREM2, act as TREM2 agonists, and may have a particularly unique set of characteristics that make them useful for the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and multiple sclerosis (MS).

[0007] The TREM2 protein is a transmembrane protein that is cleaved at an extracellular region called the stalk domain, resulting in a soluble peptide called "soluble TREM2" or "sTREM2." TREM2 cleavage in vivo is believed to occur between residues H157 and S158, resulting in sTREM2. The present disclosure describes antibodies that have the unique property of specifically binding to the stalk domain in non-soluble intact TREM2 without binding to the cleavage product sTREM2. For example, certain antibodies herein not only act as TREM2 agonists, but also specifically bind to the stalk domain of TREM2 with a low dissociation constant of 10 nM to 100-500 pM, 10-50 pM, or 1-10 pM, and specifically bind to a TREM2 epitope spanning the H157-S158 cleavage site. These antibodies specifically bind to the TREM2 stalk domain, but do not bind to soluble TREM2.

[0008] The specificity of these antibodies for uncleaved TREM2 may have several potential advantages in vivo, including avoiding unwanted binding of the antibody to soluble TREM2 in the periphery and brain after in vivo administration and / or blocking the cleavage site and reducing the amount of sTREM2 released from the cell surface. The lack of soluble TREM2 binding may be beneficial in vivo, for example, by allowing more of the administered anti-TREM2 antibody to reach the desired target of uncleaved TREM2 on the surface of cells. By way of example and not limitation, this unique binding profile may prevent unwanted binding of the antibody to soluble TREM2 in vivo, ensuring that the antibody more specifically targets TREM2 at the surface of cells in the central nervous system rather than sTREM2 in the periphery.

[0009] Thus, the present disclosure encompasses certain antibodies that: (a) specifically bind to the TREM2 stalk domain, and more specifically to a TREM2 epitope spanning the H157-S158 cleavage site; (b) do not bind to soluble TREM2 and therefore may not present a risk of soluble TREM2 binding in vivo. In some embodiments, the antibodies of the present disclosure also (c) act as TREM2 agonists, for example by inducing NFAT regulated gene expression and / or Syk kinase phosphorylation in cultured cells; and (d) exhibit very high affinity for TREM2, for example, specifically binding to TREM2 with a dissociation constant of 100-500 pM, 10-50 pM, or 1-10 pM. In some embodiments, the antibodies herein also inhibit soluble TREM2 shedding in human microglial cell models and in vivo in mouse models, reduce levels of soluble TREM2 in plasma, CSF and / or brain, enhance human microglial cell survival, and increase Aβ plaque formation and compaction in human microglial models (e.g., as measured by increased Aβ plaque intensity and / or increased X04 plaque intensity in human microglia), and thus may provide some neuroprotective activity. The present disclosure relates, inter alia, to an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, 19, or 62, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, 20, 55, 63, 65, or 73, and a CDR-H3 comprising the amino acid sequence of: 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 are both either IL or L, and X 3 and a heavy chain variable region (VH) comprising:

[0010] The disclosure also provides, for example, an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, 19, or 62, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, 20, 55, 63, 65, or 73, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8, and a CDR-H4 comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8, 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 are both either IL or L, and X 3 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29, 39, 49, 59, or 69. The disclosure further encompasses an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), comprising a heavy chain variable region (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, or 20, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29, 39, 49, 59, or 69. The disclosure further encompasses an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), for example, an antibody comprising a heavy chain variable region (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, or 20, and a CDR-L3 comprising the amino acid sequence of: 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 are both either IL or L, and X 3 and a light chain variable region (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

[0011] In some embodiments herein, the antibody has one or more, two or more, three or more, four or more, five or more, six or more, or all of the following properties: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with higher affinity to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) than to a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibiting a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions, compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interference assays); (f) specifically binds to human and cynomolgus TREM2 with a KD of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM at 37° C. by surface plasmon resonance (SPR); and (g) a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM at 37° C. by surface plasmon resonance (SPR) D It specifically binds to human and cynomolgus monkey TREM2.

[0012] Further, in some embodiments, the antibody has a VH sequence derived from a rat IGHV6-8 germline segment and / or the antibody has a VL sequence derived from a rat IGKV2S11 germline segment. In some embodiments, the light chain variable region (VL) comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, 27, or 67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29, or 69. In some embodiments, the light chain variable region (VL) comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable region (VH) comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 10, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and the light chain variable region (VL) comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region (VH) comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and the light chain variable region (VL) comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 30, 40, 50, 60, 70, 76, 77, 78, 81, 82, 83, 133, 135, 137, 139, 146, 148, 150, or 152. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 133, 135, 137, 139, 146, 148, 150, or 152.In some embodiments, the antibody comprises a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8, 18, 31, 41, 51, 61, 71, 79, 80, 84, 85, 132, 134, 136, 138, 145, 147, 149, or 151. In some embodiments, the antibody comprises a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8, 18, 132, 134, 136, 138, 145, 147, 149, or 151. In some embodiments, the antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 7, 17, 133, 135, 137, 139, 146, 148, 150, or 152. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, 18, 132, 134, 136, 138, 145, 147, 149, or 151.

[0013] The present disclosure also provides, inter alia, an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody: (a) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (c) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (d) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (e) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 24, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29; (f) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (g) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 47, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 49; (h) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 57, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 58, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59, or (i) an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), comprising a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 69.

[0014] In some embodiments, the antibody comprises: or a VH comprising the CDRs of portion (a) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7; or a VH comprising the CDRs of portion (b) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7; or a VH comprising the CDRs of portion (c) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17; or a VH comprising the CDRs of portion (d) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17; or a VH comprising the CDRs of portion (e) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 30; or a VH comprising the CDRs of portion (f) above and having at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 40; or a VH comprising the CDRs of portion (g) above and having at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 50; or a VH comprising the CDRs of portion (h) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 60; or It further comprises a VH comprising the CDRs of portion (i) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:70.

[0015] In some embodiments, the antibody comprises: or a VL comprising the CDRs of portion (a) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:8; or a VL comprising the CDRs of portion (b) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:8; or a VL comprising the CDRs of portion (c) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18; or a VL comprising the CDRs of portion (d) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18; or a VL comprising the CDRs of portion (e) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31; or a VL comprising the CDRs of portion (f) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 41; or a VL comprising the CDRs of portion (g) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:51; or a VL comprising the CDRs of portion (h) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 61; or It further comprises a VL comprising the CDRs of portion (i) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:71.

[0016] In some examples, the antibody or a VH comprising the CDR of the above portion (a) and comprising the amino acid sequence of SEQ ID NO: 7; or a VH comprising the CDR of the portion (b) and the amino acid sequence of SEQ ID NO: 7; or a VH comprising the CDR of the above portion (c) and comprising the amino acid sequence of SEQ ID NO: 17; or a VH comprising the CDR of the above portion (d) and comprising the amino acid sequence of SEQ ID NO: 17; or a VH comprising the CDR of the above portion (e) and the amino acid sequence of SEQ ID NO: 30; or a VH comprising the CDR of the above portion (f) and the amino acid sequence of SEQ ID NO: 40; or a VH comprising the CDR of the above portion (g) and the amino acid sequence of SEQ ID NO:50; or a VH comprising the CDR of portion (h) above and comprising the amino acid sequence of SEQ ID NO: 60; or It further comprises a VH comprising the CDR of the above portion (i) and the amino acid sequence of SEQ ID NO:70.

[0017] In some examples, the antibody or a VL comprising the CDR of the above portion (a) and the amino acid sequence of SEQ ID NO:8; or a VL comprising the CDR of portion (b) above and the amino acid sequence of SEQ ID NO:8; or a VL comprising the CDR of portion (c) above and the amino acid sequence of SEQ ID NO: 18; or or a VL comprising the CDR of portion (e) above and the amino acid sequence of SEQ ID NO: 31; or a VL comprising the CDR of portion (f) above and comprising the amino acid sequence of SEQ ID NO: 41; or a VL comprising the CDR of portion (g) above and comprising the amino acid sequence of SEQ ID NO: 51; or a VL comprising the CDRs of portion (h) above and comprising the amino acid sequence of SEQ ID NO: 61; or It further comprises a VL comprising the CDRs of the above portion (i) and comprising the amino acid sequence of SEQ ID NO:71.

[0018] In some examples, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some examples, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18. In some examples, the antibody comprises a VL comprising 1-5 amino acid substitutions in the framework regions compared to human IGKV2-28*01 germline, optionally, the amino acid substitutions include Q100P and / or V104L. In some examples, the antibody comprises or a VL comprising the CDR of portion (a) above and comprising the amino acid sequence of SEQ ID NO: 145; or a VL comprising the CDR of portion (b) above and comprising the amino acid sequence of SEQ ID NO: 145; or a VL comprising the CDR of portion (a) above and comprising the amino acid sequence of SEQ ID NO: 147; or a VL comprising the CDRs of portion (b) above and the amino acid sequence of SEQ ID NO: 147; or a VL comprising the CDR of portion (a) above and comprising the amino acid sequence of SEQ ID NO: 149; or a VL comprising the CDR of portion (b) above and comprising the amino acid sequence of SEQ ID NO: 149; or a VL comprising the CDR of portion (a) above and comprising the amino acid sequence of SEQ ID NO: 151; or a VL comprising the CDR of portion (b) above and the amino acid sequence of SEQ ID NO: 151; or a VL comprising the CDR of portion (c) above and comprising the amino acid sequence of SEQ ID NO: 132; or or a VL comprising the CDR of portion (c) above and comprising the amino acid sequence of SEQ ID NO: 134; or or a VL comprising the CDR of portion (c) above and comprising the amino acid sequence of SEQ ID NO: 136; or or a VL comprising the CDRs of portion (c) above and comprising the amino acid sequence of SEQ ID NO: 138; or It further comprises a VL comprising the CDRs described in part (d) above and comprising the amino acid sequence of SEQ ID NO:138.

[0019] In some embodiments, the antibodies described herein have one or more, two or more, three or more, four or more, five or more, six or more, or all of the following properties: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with higher affinity to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) than to a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibiting a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions, compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interference assays); (f) a K of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM at 37° C. by surface plasmon resonance (SPR), or a K of between 100 and 500 pM or between 100 and 200 pM D specifically binds to human and cynomolgus TREM2; and (g) a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or a K of 10 to 50 pM, or 10 to 25 pM at 37°C by surface plasmon resonance (SPR) D It specifically binds to human and cynomolgus monkey TREM2.

[0020] The present application also provides, for example, an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody: (a) a VH comprising the amino acid sequence of SEQ ID NO: 146, and a VL comprising the amino acid sequence of SEQ ID NO: 145; (b) a VH comprising the amino acid sequence of SEQ ID NO: 148, and a VL comprising the amino acid sequence of SEQ ID NO: 147; (c) a VH comprising the amino acid sequence of SEQ ID NO: 150, and a VL comprising the amino acid sequence of SEQ ID NO: 149; or (d) an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), comprising a VH comprising the amino acid sequence of SEQ ID NO: 152, and a V comprising the amino acid sequence of SEQ ID NO: 151. In some embodiments, the antibody has one or more, two or more, three or more, four or more, five or more, or all of the following properties: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with higher affinity to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) than to a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibits a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry); and (f) a K of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM at 37° C. by surface plasmon resonance (SPR), or a K of between 100 and 500 pM or between 100 and 200 pM D It specifically binds to human and cynomolgus monkey TREM2.

[0021] The present application further provides, for example, an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody: (a) a VH comprising the amino acid sequence of SEQ ID NO: 133, and a VL comprising the amino acid sequence of SEQ ID NO: 132; (b) a VH comprising the amino acid sequence of SEQ ID NO: 135, and a VL comprising the amino acid sequence of SEQ ID NO: 134; (c) a VH comprising the amino acid sequence of SEQ ID NO: 137, and a VL comprising the amino acid sequence of SEQ ID NO: 136; or (d) an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), comprising a VH comprising the amino acid sequence of SEQ ID NO: 139, and a VL comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody has one or more, two or more, three or more, four or more, five or more, or all of the following properties: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with higher affinity to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) than to a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibits a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry); and (f) a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or a K of 10 to 50 pM, or 10 to 25 pM at 37°C by surface plasmon resonance (SPR) D It specifically binds to human and cynomolgus monkey TREM2.

[0022] In some examples, the antibodies herein have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following characteristics: (a) Induce luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (b) reducing the levels of sTREM2 in plasma in vivo; (c) inhibiting sTREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1; (g) inhibiting sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and (i) increase total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia (e.g., as described in the assay of Example 17 herein). In some embodiments, the antibodies herein also have a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (e.g., as described in Example 10 herein).

[0023] In some embodiments, the antibody is an Fv, a single chain Fv (scFv), a Fab, a Fab', or a (Fab') 2In some embodiments, the antibody is an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some such examples, the antibody comprises a wild-type human IgG1 or IgG4 Fc region. In some such examples, the antibody comprises a human IgG1 Fc region comprising (a) an N297G substitution, (b) an L234A, L235A, and P329G substitution (LALAPG substitution), or (c) an N297G, M428L, and N434S substitution. In some embodiments herein, the antibody has reduced effector function or is effectorless, or does not bind to FcγR. In some embodiments, the antibody comprises a human IgG1 Fc region comprising an N297G substitution. In some embodiments herein, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 144 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, but lacking the C-terminal lysine of SEQ ID NO: 144, or lacking the C-terminal glycine and lysine of SEQ ID NO: 144, and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 144 and / or a light chain consisting of the amino acid sequence of SEQ ID NO: 176. In some embodiments herein, the antibody comprises a human IgG4 Fc region comprising an S228P substitution, or comprising S228P, M252Y, S254T and T256E substitutions. In some embodiments herein, the antibody is a full-length antibody. In some embodiments herein, the antibody is an IgG antibody lacking the C-terminal lysine in the heavy chain constant region.

[0024] The disclosure herein also encompasses, for example, an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), where the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 17, and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 18. The disclosure herein further encompasses an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), where the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176. The disclosure herein also encompasses an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, but lacking the C-terminal lysine of SEQ ID NO: 144, or lacking the C-terminal glycine and lysine of SEQ ID NO: 144, and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176. The disclosure also encompasses an isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 144, and / or a light chain consisting of the amino acid sequence of SEQ ID NO: 176.In some embodiments, the antibody specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), the antibody specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site, and / or specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with greater affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94). In some embodiments, the antibody specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), and the antibody is a TREM2 agonist. In some embodiments herein, the antibody has one or more, two or more, three or more, four or more, five or more, six or more, or all of the following properties: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with higher affinity to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) than to a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibiting a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions, compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interference assays); (f) specifically binds to human and cynomolgus TREM2 with a KD of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM at 37° C. by surface plasmon resonance (SPR); and

[0025] (g) a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM at 37° C. by surface plasmon resonance (SPR) D In some embodiments, the antibodies specifically bind to human and cynomolgus TREM2 at 37° C. by surface plasmon resonance (SPR) of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM or between 10 and 25 pM. D Additionally, in some embodiments, the antibody has one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following characteristics: (a) Induce luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (b) reducing the levels of sTREM2 in plasma in vivo; (c) inhibiting sTREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1; (g) inhibiting sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and

[0026] (i) increases total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia (e.g., as described in the assay of Example 17 herein). In some embodiments, the antibody also has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (e.g., as described in Example 10 herein).

[0027] The disclosure herein also provides an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and further wherein the antibody specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), and wherein the antibody , an isolated antibody that specifically binds to the triggering receptor expressed on myeloid cells-2 (TREM2), which is a TREM2 agonist, wherein the antibody specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site and / or specifically binds to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) with greater affinity than a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94). In some embodiments, the antibodies exhibit reduced binding affinity to TREM2 stalk domain polypeptides comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain (e.g., as measured by bilayer interferometry) and / or have a K for human and cynomolgus TREM2 by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM or between 10 and 25 pM. DAdditionally, in some embodiments, the antibody has one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following characteristics: (a) Induce luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (b) reducing the levels of sTREM2 in plasma in vivo; (c) inhibiting sTREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1; (g) inhibiting sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and

[0028] (i) increase total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia (e.g., as described in the assay of Example 17 herein). In some embodiments, the antibody also has a low off-target binding score (e.g., a score less than 1) in an off-target binding assay (e.g., as described in Example 10 herein). Further, in some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, 133, 135, 137, or 139, or the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144. In some examples, the antibody comprises a human IgG1 Fc region comprising (a) an N297G substitution, (b) an L234A, L235A, and P329G substitution (LALAPG substitution), or (c) an N297G, M428L, and N434S substitution. In some examples, the antibody has reduced effector function or is effectorless, or does not bind to FcγR.

[0029] In some embodiments herein, the antibody is a bispecific or multispecific antibody or is covalently or non-covalently conjugated to at least one other molecule, in some examples, the antibody is covalently or non-covalently conjugated to at least one other molecule, where the at least one other molecule comprises a detection label and / or a drug.

[0030] The present disclosure also includes pharmaceutical compositions comprising the antibodies described herein and a pharma- ceutically acceptable carrier. The present disclosure also includes an isolated nucleic acid or set of two or more nucleic acids encoding the antibodies disclosed herein. The present disclosure also includes an isolated vector comprising one or more nucleic acids encoding the heavy and light chains of the antibodies herein. The present disclosure further includes an isolated host cell comprising a nucleic acid or vector. The present disclosure also includes a method of producing an antibody that specifically binds to TREM2, comprising culturing a host cell under conditions suitable for expression of the antibody. In some examples, the method includes recovering the antibody from the host cell. The present disclosure also includes the antibody produced by the method.

[0031] The disclosure further includes a method of treating a condition associated with loss of TREM2 function in a subject in need thereof, comprising administering to the subject an antibody herein or a pharmaceutical composition herein. In some embodiments, the disclosure includes a method of reducing levels of sTREM2 in a subject in need thereof, comprising administering to the subject an antibody herein or a pharmaceutical composition herein. In some examples, the condition is a neuroinflammatory or neurodegenerative disease, or the subject is afflicted with a neuroinflammatory or neurodegenerative disease. In some examples, the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some examples, the disease is Alzheimer's disease. In some examples, the disease is MS.

[0032] The disclosure also encompasses an antibody or pharmaceutical composition herein for use in treating a condition associated with loss of TREM2 function in a subject in need of such treatment. In some embodiments, the disclosure herein encompasses an antibody or pharmaceutical composition herein for use in reducing levels of sTREM2 in a subject in need of such treatment. In some examples, the condition is a neuroinflammatory or neurodegenerative disease, or the subject is afflicted with a neuroinflammatory or neurodegenerative disease. In some examples, the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some examples, the disease is Alzheimer's disease. In some examples, the disease is MS.

[0033] The disclosure further includes the use of an antibody herein or a pharmaceutical composition herein in the preparation of a medicament for treating a condition associated with loss of TREM2 function in a subject in need of such treatment. The disclosure herein also includes the use of an antibody or a pharmaceutical composition herein in the preparation of a medicament for reducing levels of sTRE2 in a subject in need of such treatment. In some examples, the condition is a neuroinflammatory or neurodegenerative disease, or the subject is afflicted with a neuroinflammatory or neurodegenerative disease. In some examples, the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some examples, the disease is Alzheimer's disease. In some examples, the disease is MS.

[0034] Additional objects and advantages will be set forth in part in the following description and in part be understood from the description or may be learned by practice. The objects and advantages will be realized and attained by the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the claims. For example, in addition to the various embodiments shown and claimed herein, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, certain features presented herein, particularly as aspects or embodiments, may be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The descriptions of certain embodiments of the disclosed subject matter are presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the disclosed subject matter to the disclosed embodiments. [Brief description of the drawings]

[0035] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows an overview of anti-TREM2 clones obtained by hybridomas with different immunization strategies.

[0036] [Figure 2A-2B] Figures 2A-B provide peptide sequences of various TREM2 stalks and the binding strength to each peptide by three different antibodies. Figure 2A shows the sequence of a peptide fragment within the TREM2 stalk domain. The TREM2 residues adjacent to the TREM2 cleavage site between residues 157 and 158 are shown in bold. Figure 2B shows the binding data (nM) of the three antibodies to the peptides in Figure 2A. Binding is shown in shades of grey (darker shading indicates stronger binding) along with the A620 absorbance values.

[0037] [Figure 3A-3B]Figures 3A-B show the light and heavy chain variable region amino acid sequences of anti-TREM2 antibodies. Amino acid differences compared to the 3.10C2 variable region sequence are shown in black. CDR regions according to Chothia or Kabat are shown above the sequences, and Kabat CDR definitions are also underlined. Figure 3A shows the light chain variable region sequences for five clones. Figure 3B shows the heavy chain variable region sequences for six clones.

[0038] [Figure 4] Figure 4 shows ELISA screening of antibodies with IGHV6-8 clones from the rat repertoire deep sequencing dataset in combination with different light chains. Stalk, 149-158, 159-175 and inhibin in the table refer to the stalk domain of TREM2, stalk fragments 149-158 and 159-175, and a 30-mer inhibin control peptide, respectively. ELISA signals are shown in grey shading. The CDR H3 sequences of the clones in the IMGT definition (length 5) are shown in the left column. Clone 3.10C2 was paired only with its own cognate light chain. KV2S11 refers to a light chain with the IGK2S11 germline segment in germline sequence configuration and a VJ junction sequence similar to the 3.10C2 antibody group.

[0039] [Diagram 5] Figure 5 shows an overview of alanine / glycine mutations that affect binding of anti-TREM2 antibodies. Grey boxes indicate mutations that affect either off-rate binding or total binding of the different antibodies. Amino acid residues and positions in TREM2 are shown at the top of the table. Two residues in TREM2 adjacent to the cleavage site of the protein are highlighted in grey on the same line. Antibody AL2p-31 is a humanized high affinity variant of the murine antibody 9F5. Antibody Para.09 has the antibody 3.2H7 light chain.

[0040] [Figure 6A-6B]Figures 6A-B show humanized 3.10C2 CDR grafted variant sequences. The variable region sequences are aligned to the human framework sequences used for humanization. Differences from the framework sequence (top sequence) are highlighted in black. CDR regions in the Kabat definition are underlined. "3.10C2" in each figure refers to the rat 3.10C2 variable region sequence. Figure 6A shows the light chain variable region sequences of three variants. Figure 6B shows the heavy chain variable region sequences of four variants.

[0041] [Figure 7A-7B] Figures 7A-7B show the expression yields of 3.10C2 CDR-grafted clones. Figure 7A shows the expression yields as measured by titer (mg / L) of humanized IgG variants with different heavy and light chain humanized variants. Clones L9 and L10 are identical to clones L1 and L5, respectively (see Figure 6A), but with a Q100P mutation in framework 4. Figure 7B shows the expression yields (mg) of Fab fragments with humanized 3.10C2-L1 variant or humanized 3.10C2-H1 heavy chain variant in combination with rat 3.10C2 light chain.

[0042] [Figure 8A-8B] Figures 8A-B show humanized 3.27H7 light chain and Para.09 heavy chain CDR grafted variant sequences. The variable region sequences are aligned to the human framework sequences used for humanization. Differences from the framework sequence (top sequence) are highlighted in black. CDR regions in the Kabat definition are underlined. "3.27H7" and "Para.09" are rat variable region sequences. Figure 8A shows the light chain variable region sequences of three variants. Figure 8B shows the heavy chain variable region sequences of four variants.

[0043] [Figure 9A-9B] Figures 9A-9B show the variable region and CDR sequences of antibody h3.10C2.v1. Kabat CDR regions are underlined. Figure 9A shows the light chain variable region sequence. Figure 9B shows the heavy chain variable region sequence.

[0044] [Figure 10] Figure 10 shows the scanning mutagenesis of antibody 3.10C2. Figure 10 shows the distribution of dissociation rates of antibody 3.10C2 heavy chain CDR single point mutants at 37°C. The dissociation rates of antibody 3.10C2 determined in the same set of experiments are shown as dotted lines. Two 3.10C2 point mutants with lower dissociation rates compared to wild type 3.10C2 are shown, with the respective improvement rates shown in brackets. Data for 440 mutants is shown.

[0045] [Figure 11] 11 shows the expression yield (mg / L) after Protein A chromatography of humanized variants of Para.09 produced in rat and CHO cells. The isotype of each antibody is indicated.

[0046] [Figure 12A-12B] Figures 12A-B show the amino acid sequences of the variable regions of antibody hPara.09.v2. Kabat CDR regions are underlined. Figure 12A shows the light chain variable region sequence. Figure 12B shows the heavy chain variable region sequence.

[0047] [Figure 13A-13B] Figures 13A and 13B show the light chain (Figure 13A) and heavy chain (Figure 13B) sequences and CDR regions of hPara.09.v2 and hPara.09.v2 mutants. The Q100P, I58V and V104L mutations, as well as other mutations in the light chain compared to hPara.09.v2, are highlighted. The Kabat CDR regions are underlined.

[0048] [Figure 14A-14B] Figures 14A-B show the variable region sequences and CDR regions of the light chain (Figure 14A) and heavy chain (Figure 14B) antibody h3.10C2.v1 and the h3.10C2.v1 mutant. The Q100P, I58V and L104V mutations in the light chain are highlighted. The Kabat CDR regions are underlined.

[0049] [Figure 15] FIG. 15 shows normalized baculovirus (BV) ELISA reactivity of four anti-TREM2 antibodies (parental and humanized): a chimeric clone with a rat variable region and a human IgG1 constant region, and humanized variants h3.10C2.v1 and hPara.09.v2 with a human IgG1 constant region with the N297G mutation to eliminate Fc receptor binding.

[0050] [Figure 16] Figure 16 shows antibodies that do and do not bind to soluble TREM2 (sTREM2) in vitro. Figure 16 shows normalized values ​​for sTREM2 binding for the antibodies tested: control antibody 1.16B8 (CTL), rat Para.09-LC 3.27H7 mIgG2a LALAPG (Para09), rat 3.10C2 mIgG2a LALAPG (3.10C2), rat 3.18E5 mIgG2a LALAPG (3.18E5), rat 3.50G1 mIgG2a LALAPG (3.50G1), rat 3.27H7 mIgG2a LALAPG (3.27H7), rat 3.36F5 mIgG2a LALAPG (3.36F5), A.9F5 (also referred to herein as 9F5), AL2p-12, AL2p-31, AL2p-58, BM.3D3, BM.42E8, BM.RS9, BM.14D3, and BM.14D8.

[0051] [Figure 17] Figure 17 shows Jurkat reporter activity of recombinant antibodies in mIgG2a format. Jurkat cells were engineered to express luciferase under the control of NFAT (nuclear factor of activated T cells) and express human TREM2. Figure 17 shows eight antibodies that induced moderate to strong luciferase expression in the cells: 3.10C2, 3.18E5, 3.27H7, 3.50G1, 3.17G12, 3.27H5, 3.41B10, and 3.47B1.

[0052] [Figure 18A-18B]Figures 18A-B show the effect of expressing increasing concentrations of rat anti-human and humanized anti-TREM2 antibodies on luciferase in Jurkat-NFAT luciferase reporter cells expressing human TREM2. Luciferase signal in the presence of various concentrations of rat anti-human antibodies in the mIgG2 LALAPG format (Figure 18A) and humanized hPara.09.v2 and h3.10C2.v1 antibodies (Figure 18B) are shown after 24 hours.

[0053] [Figures 19A-19C] Figures 19A-C show induction of luciferase expression by rat anti-human 3.10C2, 3.18E5, 3.27H7 and 3.50G1 mIgG2a LALAPG antibodies in Jurkat-NFAT luciferase reporter cell lines expressing hTREM2 mutants R47H (Figure 19A), R62H (Figure 19B) or H157Y (Figure 19C).

[0054] [Figure 20A-20B] Figures 20A-B and 20D show the effect of rat anti-human and humanized antibodies on sTREM2 shedding in a Jurkat-NFAT luciferase reporter cell line expressing hTREM2 after 24 hours as measured by ELISA. Figure 20A shows the effect of rat 1.20A2 mIgG2a LALAPG, rat Para.09 mIgG2a LALAPG and rat 3.10C2 mIgG2a LALAPG antibodies on increasing concentrations of sTREM2 levels. Figure 20B shows the effect of humanized h3.10C2.v1 and hPara.09.v2 in human IgG1 N297G format. Figure 20 shows the effect of humanized antibodies hPara.09 v2, hPara.09.v2 Q100P / V104L, and hPara.09.v2 Q100P. FIG. 20C shows luciferase expression induced by hPara.09 v2, hPara.09.v2 Q100P / V104L and hPara.09.v2 Q100P antibodies.

[0055] [Figure 21]FIG. 21 shows the effect of antibodies 3.10C2, 3.18E5, 3.27H7, 3.50G1, and 9F5, including mouse IgG2 LALAPG, on TREM2 shedding in human induced pluripotent stem cell (iPSC)-derived microglia.

[0056] [Figures 22A-22C] Figures 22A-C show pan phospho-tyrosine (pY) levels (Figure 22A) and phospho-SYK (pSYK) levels (Figure 22B-C) in primary human monocyte-derived macrophage (hMDM) cells after incubation with humanized anti-TREM2 antibodies for a defined 10-20 min time period (Figure 22A-B) or dose range (Figure 22C). In Figure 22B, phosphorylation was also compared to hMDM cells engineered to reduce TREM2 expression (gTREM2). Figure 22C shows the effect of SYK phosphorylation with increasing doses of humanized antibodies hPara.09.v2 N297G or hPara.09.v2 Q100P / V104L N297G, or a control antibody. pSYK levels were measured and normalized to total SYK. n=6 from three independent biological specimens.

[0057] [Figures 23A-23E]Figures 23A-E show that rat anti-human and humanized anti-TREM2 antibodies promote survival of human induced pluripotent stem cell (iPSC) microglia. Figure 23A shows survival of wild type (WT, thin line) or TREM2 KO (KO, dotted line) iPSC microglia induced by treatment with increasing doses of hPara.09 v2,h3.10C2.v1, measured as fold of response induced by control antibody (gD hIgG1 N297G) treatment. Error bars + / - SEM. Figure 23B shows survival of iPSC-MG cells in response to treatment with antibodies at increasing doses (0, 1, 10, 100 ng). Antibodies are grouped according to TREM2 binding (span of residues shown at the top of the figure). Error bars + / - SEM. FIG. 23C shows pSYK induction by humanized antibodies (hPara09.v2 N297G, hPara09.v2.Q100P / V104L N297G) in iPSC-MG at 1, 4 and 24 hours. Error bars + / - SEM. *P<0.05, **P<0.01, ****<0.0001. FIG. 23D shows the effect of increasing concentrations of humanized Para.09.v2 antibodies or anti-gD control antibodies on microglial survival in the presence and absence of a SYK inhibitor (SYKi). Error bars + / - SEM. FIG. 23E shows microglial survival and EC50 (nM shown in table below graph) of the listed anti-TREM2 antibodies compared to the control anti-gD antibody. Error bars + / - SEM.

[0058] [Figures 24A-24C]Figures 24A-C show that humanized anti-TREM2 antibodies increase iPSC-MG amyloid beta plaque formation and compaction. Figure 24A shows immunofluorescence images of Aβ plaque-like structures and Aβ aggregate formation around iPSC-microglia treated with hPara.09.Q100P hIgG1 N297G or control gD.hIgG1.N297G. From left to right, the panels show: staining for Aβ amyloid plaques (Methoxy X04 staining), microglial staining (IBA1 staining), and a merged image from all three stainings showing the co-location of staining for IBA1 positive microglia, Aβ and amyloid plaques. Figure 24B shows total Aβ plaque intensity in the presence of increasing concentrations of several humanized anti-TREM2 antibodies or control anti-gD antibodies, all in human IgG1 N297G format. EC50 values ​​in nM for each antibody are shown in the table below the graph. Error bars + / - SEM. Figure 24C shows the average X04 iPSC-MG plaque intensity at increasing antibody concentrations for several humanized anti-TREM2 antibodies or a control anti-gD antibody, both in the human IgG1 N297G format. EC50 values ​​are shown in nM for each antibody in the table below the curves.

[0059] [Figures 25A-25C] Figures 25A-C show sTREM2 levels in mice treated with anti-TREM2 or control antibodies. Figures 25A-B show the percent of sTREM2 relative to control antibody (gp120) in plasma (Figure 25A) and brain homogenate (Figure 25B) samples from mice treated with 3.10C2 and Para.09 in mouse IgG2 LALAPG format. Figure 25C shows sTREM2 levels (pg / ml) in plasma of mice treated with increasing doses of Para.09 and AL2p58 antibodies in mouse IgG2a LALAPG format compared to isotype control.

[0060] [Fig. 26A-26F]Figures 26A-F show sTREM2 levels over time in CSF, plasma and brain lysate samples from cynomolgus monkeys dosed with h3.10C2.v1.hIgG1.N297G, hPara.09.v2.hIgG1.N297G, or anti-gD.hIgG1.N297G control antibody. Figures 26A-C show sTREM2 levels measured in CSF (Figure 26A), plasma (Figure 26B) and brain (Figure 26C) over a 2 day period. Figures 26D-F show sTREM2 levels measured in CSF (Figure 26D), plasma (Figure 26E) and brain (Figure 26F) over a 28 day period.

[0061] [Figure 27] Figure 27 shows brain sections from mice treated with 3.10C2, Para.09, or control antibody stained for the proliferation marker Ki67. Cell proliferation is expressed as the average number of Ki67+ cells per number of 4',6-diamidino-2-phenylindole (DAPI) positive cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Description of exemplary embodiments definition As used herein, the term about refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly stated. The term about generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values ​​or ranges, those terms modify all of the values ​​or ranges provided in the list. In some instances, the term about may include numerical values ​​that are rounded to the nearest significant figure.

[0063] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0064] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of multiple dependent claims, the use of "or" refers only in the alternative to more than one preceding independent or dependent claim. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and element, and components that contain more than one subunit, unless specifically stated otherwise.

[0065] Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions and purification methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2002), among others. nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0066] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0067] As used herein, "TREM2" or "triggering receptor expressed on myeloid cells-2" refers to the human TREM2 protein ("hTREM2") unless otherwise specified (i.e., mouse TREM2 or cynomolgus TREM2, etc.). An exemplary hTREM2 amino acid sequence including the signal sequence at amino acids 1-18 is set forth in SEQ ID NO:21, and an exemplary sequence without the signal sequence is set forth in SEQ ID NO:22. TREM2 including the signal sequence is sometimes referred to as the "precursor" or "preprotein" form of the protein, and TREM2 without the signal sequence is sometimes referred to as the "mature" form of the protein. The membrane-bound form of the protein includes a V-type immunoglobulin (Ig) domain (at amino acids 19-128), followed by the TREM2 "stalk" domain (at amino acids 129-174; SEQ ID NO:90), which collectively form the "extracellular domain" of the protein, followed by the transmembrane domain (residues 175-197), and the cytosolic domain (residues 198-230). A soluble form of the protein, herein "soluble TREM2" or "sTREM2", can be generated in vivo by either cleavage or alternative splicing at the stalk domain. For example, a protease may cleave the protein between H157 and S158, cleaving soluble TREM2 including the N-terminal portion of the protein up to H157, e.g., residues 19-157. An exemplary sTREM2 amino acid sequence is shown in SEQ ID NO: 23. The portion of the protein that can be cleaved to form sTREM2 is also known as the "ectodomain" and includes residues 19-157. In general, as used herein, the term "TREM2" refers to the membrane-bound form of the protein, unless the context explicitly makes clear that both forms of the protein are being referenced. Additionally, hTREM2 includes several isoforms or alleles, the native sequence or splicing of which may differ from that shown in SEQ ID NOs: 21 and 22 and described in the Examples section herein. The term TREM2 includes all of these naturally occurring forms of TREM2, unless a specific isoform or sequence is referred to.

[0068] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by methods common in the art, including those described herein. Specific illustrative exemplary methods for measuring binding affinity are described below.

[0069] The term "antibody" as used herein refers to a molecule comprising at least the complementarity determining region (CDR)1, CDR2 and CDR3 of a heavy chain and at least the CDR1, CDR2 and CDR3 of a light chain, which molecule is capable of binding to an antigen. The term is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), full-length antibodies, single-chain antibodies, antibody conjugates and antibody fragments, so long as they exhibit the desired TREM2-specific binding activity.

[0070] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0071] "Antigen" refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds. The term "epitope" refers to a site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes on proteins can be both formed from a contiguous stretch of amino acids (linear epitopes) or can include non-contiguous amino acids that are in spatial proximity, for example, due to antigen folding (i.e., by tertiary folding of a proteinaceous antigen) (conformational epitopes). Linear epitopes are typically still bound by antibodies after exposure of a proteinaceous antigen to a denaturing agent, whereas conformational epitopes are typically destroyed by treatment with a denaturing agent. In some instances, a TREM2 "epitope" herein may "span" a site, such as a naturally occurring proteinase cleavage site or cleavage point within a molecule, meaning that there is close contact between one or more antibody residues and the antigen on either side of the cleavage site, or that the antibody binds to a TREM2 peptide that spans the site. In such cases, the TREM2 epitope includes the cleavage site or residues on either side of the breakpoint.

[0072] In this disclosure, "specifically binds" or "specific binding" and similar terms mean that the binding affinity is strong enough that the interaction between the members of a binding pair cannot be attributed to random molecular association (i.e., "non-specific binding"). Specific binding typically has a dissociation constant (K D Specific binding often requires, for example, a K for TREM2 of 10 nM or less. D may include.

[0073] "Anti-TREM2 antibody" or "TREM2 antibody" or "antibody that specifically binds to TREM2" or "antibody that binds to TREM2" and similar phrases refer to an antibody that specifically binds to TREM2 as defined herein.

[0074] Certain antibodies herein "do not bind soluble TREM2". As used herein, this means that the antibody may not exhibit more than 10% binding to sTREM2, and in some instances, not more than 5% binding to sTREM2, when assayed in an ELISA assay, with the % binding normalized to the binding of the positive control antibody 1.16B8, whose binding to sTREM2 is set to 100%. The light chain sequence of the control antibody 1.16B8 is shown in SEQ ID NO: 170, and its heavy chain sequence is shown in SEQ ID NO: 171. In some embodiments, an ELISA assay may be performed as shown in Example 11 and FIG. 16 below. In some embodiments, the antibody 3.17A9 is used as a detection agent in the assay. The light chain sequence of the detection antibody 3.17A9 is shown in SEQ ID NO: 168, and its heavy chain sequence is shown in SEQ ID NO: 169. Certain antibodies herein "do not bind FcγR" or have a heavy chain constant region or Fc region that "does not bind FcγR", meaning that binding is not detectable above trace levels in an appropriate in vitro binding assay.

[0075] The term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain includes at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0076] The term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain also comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0077] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable region that is hypervariable in sequence and determines antigen binding specificity, e.g., "complementarity determining regions" (CDRs). Generally, antibodies contain six CDRs; three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Unless otherwise indicated, the CDRs are determined according to the sequence listing herein, and the amino acid positions of the heavy and light chain regions and domains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda MD (1991). In some examples, both Kabat and Chothia heavy chain CDR1 and CDR2 are provided in the sequence listing. In the case of SEQ ID NOs: 1-6 and 9-10, and 11-16 and 19-20, the Kabat and Chothia CDRs differ in CDR-H1 and CDR-H2, but are the same in the remaining four CDRs. Those skilled in the art will understand that the designation of the CDRs may also be determined according to McCallum or any other scientifically accepted naming system. See, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Kabat et al., Sequences of Proteins of Immunological Interest, 5 th See Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); MacCallum et al. J. Mol. Biol. 262:732-745 (1996). The antigenic contacts making amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) constitute the MacCallum CDRs.

[0078] "Framework" or "FR" refers to the residues of the variable region that are not part of the complementarity determining regions (CDRs). The FR of a variable region generally consists of four FRs: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally occur in the following order in a VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0079] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, and each domain contains four conserved framework regions (FR) and three complementarity determining regions (CDR). See, for example, Kindt et al. Kuby Immunology, 6 th ed., W. H. Freeman and Co., page 91 (2007). The variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3, with or without all or a portion of FR1 and / or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3, with or without all or a portion of FR1 and / or FR4. That is, the variable domain may lack a portion of FR1 and / or FR4, so long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, a library of complementary VL or VH domains, respectively, may be screened to isolate antibodies that bind to a particular antigen using the VH or VL domain of the antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0080] The "constant region" of the light and heavy chains of an antibody refers to the FR and CDR and additional sequence portions outside the variable region. Certain antibody fragments may lack all or part of the constant region. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light chain (CL) domain.

[0081] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, an antibody produced by a host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may comprise a full-length heavy chain or may comprise a cleaved variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Thus, for example, "full-length IgG1" includes IgG1 with Gly446 and Lys447, or without Lys447, or without both Gly446 and Lys447. The amino acid sequence of the heavy chain comprising the Fc region is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one aspect, the heavy chain comprising the Fc region as specified herein, contained in the antibody according to the invention, may comprise Gly446 and Lys447 (numbering according to the EU index). In one aspect, the heavy chain comprising the Fc region as specified herein, contained in the antibody according to the invention, may comprise Gly446 (numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th It follows the EU numbering system (also called the EU Index), as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0082] "Effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. For example, an antibody with "intact effector function" includes a heavy chain constant region or Fc region with a native intact effector function of that particular isotype, such as a wild-type heavy chain constant region or Fc region, or one with a modification that has not been shown to affect effector function. In contrast, an antibody heavy chain constant region or Fc region can be modified in various ways, such as amino acid substitutions, insertions or deletions, or glycosylation modifications, to reduce or enhance effector function, depending on the use of the antibody. As used herein, some antibodies may have "intact effector function", while others may be "effectorless", meaning that they do not exhibit detectable CDC or ADCC activity, or that they comprise a heavy chain constant region or Fc region that has previously been shown to have no detectable CDC or ADCC activity. In other cases, an antibody may have a variant Fc region or heavy chain constant region that has "reduced effector function" compared to a corresponding wild-type Fc or heavy chain constant region (e.g., one that has previously been shown to have reduced effector function, or has reduced effector function, in the context of the antibodies herein). Such antibodies with reduced effector function may, for example, exhibit a lower degree of CDC and / or ADCC activity and / or FcγR binding activity compared to a corresponding wild-type Fc region, and / or may retain some effector function, such as binding to a particular Fc receptor, such as FcRn, but have, for example, low or no detectable CDC or ADCC or FcγR binding activity.

[0083] The "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are subdivided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 In certain embodiments, the antibody is an IgG 1 In a particular embodiment, the antibody is an IgG1 antibody with P329G, L234A, and L235A mutations to reduce Fc region effector function. 1 In other embodiments, the antibody is of the IgG 2 In certain embodiments, the antibody is of the IgG 4 IgG with S228P mutation in the hinge region to improve antibody stability 4 isotype. In some embodiments, the antibody may have a non-human IgG constant region, e.g., a murine IgG2a antibody, such as the murine IgG2a LALAPG antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0084] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen (i.e., TREM2) to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2Examples of antibody fragments include, but are not limited to, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv, and scFab), single domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0085] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or, in the case of IgG antibodies, an antibody having a heavy chain including an Fc region as defined herein.

[0086] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portions of the heavy and / or light chain are derived from a different source or species.

[0087] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises all or nearly all of the CDRs corresponding to the variable domains of a non-human antibody, and nearly all of at least one, usually two, variable domains in all or nearly all of the FRs corresponding to the variable domains of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0088] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (which may, for example, include naturally occurring mutations or arise during the manufacture of the monoclonal antibody preparation, and such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which usually include different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0089] A "multispecific" antibody is an antibody that specifically binds to two or more target antigens, and a "bispecific" antibody is an antibody that specifically binds to two antigens. An "antibody conjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a therapeutic agent or a label.

[0090] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity for the alignment. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, such as publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code is on file in the user documentation of the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0091] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, followed by the BLOSUM50 comparison matrix. The FASTA program package is certified by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison", Meth. Enzymol. 266:227-258; and Pearson et. Al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.Ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring a global rather than local alignment. Percent amino acid identity is given in the output alignment header.

[0092] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including derivatized sugar or phosphate backbone linkages or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo. (See, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0093] An "isolated" nucleic acid is a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0094] "Isolated nucleic acid encoding an anti-TREM2 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the term TREM2 antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) are present in one or more locations within a host cell.

[0095] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0096] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.

[0097] The term "signal sequence" or "leader sequence" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of the polypeptide from a mammalian cell. The leader sequence may be cleaved upon export of the polypeptide from a mammalian cell to form the mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are bound. Non-limiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, the antibody lacks a leader sequence. In some embodiments, the antibody comprises at least one leader sequence, which may be selected from a native antibody leader sequence and a heterologous leader sequence.

[0098] As used herein, the term "shedding" refers to the process of generating soluble TREM2 from membrane-bound TREM2 by proteolytic cleavage of the stalk domain of the protein. In vivo, "shedding" can occur at the cell surface, for example, by cleavage of TREM2 on the cell membrane by metalloproteinases or other enzymes. In some instances, cleavage occurs between H157 and S158 of the protein, forming sTREM2 from ectodomain residues 19-157.

[0099] As used herein, the term "agonist" refers to a substance, such as an antibody, that causes an increase in at least one activity or function of a molecule to which it binds, or that otherwise activates or helps activate the molecule. As used herein, the term "antagonist" refers to a substance, such as an antibody, that causes a decrease in at least one activity or function of a molecule to which it binds, or that otherwise blocks or inhibits at least one activity or function of the molecule.

[0100] An "agonist anti-TREM2 antibody" or similar phrase herein refers to an antibody that, for example, induces luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2, and / or induces SYK phosphorylation (p-SYK) in Jurkat-NFAT reporter cells expressing human TREM2, human monocyte-derived macrophage (MDM) cells, and / or human induced pluripotent stem cell (iPSC)-derived microglial cells. Agonist anti-TREM2 antibodies may also have additional activities, such as, for example, enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1, and activating TREM2 signaling in human macrophages and microglia, among other activities, as described herein.

[0101] The terms "subject" and "patient" are used interchangeably herein to refer to humans. In some embodiments, methods of treating other mammals are also provided, including, but not limited to, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets.

[0102] As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a human or other mammal, including inhibiting or slowing the progression of a disease, inhibiting or slowing the progression of a disease, arresting or delaying the onset of at least one symptom of a disease, delaying the time to onset of a disease, preventing the onset of at least one disease symptom, delaying the time to onset of at least one disease symptom, partially or completely alleviating a disease, or curing a disease, for example by causing regression or by restoring or repairing a lost, missing, or defective function, or stimulating an inefficient process. The term "inhibition" or "inhibiting" refers to the reduction or cessation of any symptom or phenotypic characteristic, or the reduction or cessation of the incidence, extent, or likelihood of that symptom or characteristic.

[0103] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with a therapeutic agent that together comprises a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is administered orally, the carrier may be a gel capsule. If the therapeutic agent is administered subcutaneously, the carrier is ideally not irritating to the skin and does not cause injection site reactions.

[0104] As used herein, the term "effective amount" refers to an amount sufficient to bring about a desired result, such as treating, inhibiting, or reducing as described above.

[0105] Development of specific anti-TREM2 antibodies The disclosure herein encompasses anti-TREM2 antibodies. In some embodiments herein, the antibodies have a unique set of properties. For example, certain antibodies herein (a) specifically bind to the TREM2 stalk domain at an epitope spanning the cleavage site between residues 157 and 158, and also specifically bind to a polypeptide consisting of residues 151-165 of TREM2, (b) do not bind to soluble TREM2 and therefore may not present a significant risk of soluble TREM2 binding in vivo, (c) act as a TREM2 agonist, and (d) specifically bind to TREM2 with a dissociation constant of, for example, 100-500 pM, 10-50 pM, or 1-10 pM.

[0106] As described in the Examples section below, certain exemplary antibodies herein were obtained in part following an initial screening of rat anti-human TREM2 antibodies. A particular set of rat anti-human TREM2 antibodies that specifically bind to a binding epitope in the region of amino acids 151-165 of TREM2 was found to have both relatively high affinity and a lack of binding to soluble TREM2, unlike other antibodies found in the screening, and unlike other previously described anti-TREM2 antibodies. This set of antibodies includes rat anti-human antibodies 3.10C2, 3.50G1, 3.18E5, 3.27H7 (also referred to as "3.10C2 group antibodies") and 3.36F5. (See also Figures 3A-3B, and Figures 2A-2B and 16).

[0107] Further TREM2-immunized rat immune repertoire deep sequencing experiments were performed on the heavy chain variable region to obtain antibodies with even higher affinity for TREM2. This experiment identified the Para.09 heavy chain variable region, which is clonally unrelated to the 3.10C2 group antibodies and has a different heavy chain CDR3 compared to previously identified antibodies in the 3.10C2 group. (See Figures 4 and 8B). The Para.09 heavy chain variable region was combined with a light chain variable region from a previously identified 3.10C2 group antibody. The resulting Fab fragments had affinities for human and cynomolgus TREM2 of the order of 1-5 pM (i.e., 1-5E×10 -12 M). (See Table 1 and Example 5).

[0108] The rat anti-human antibodies were then humanized. Antibodies h3.10C2.v1 and hPara.09.v2 are examples of humanized antibodies herein. (See Figures 9A-9B and 12A-12B). Further modifications of the light chain framework regions of the humanized antibodies were then made to improve properties such as, for example, expression yield. For example, specific modifications at positions 58, 100 and / or 104 of the light chain framework regions (e.g., I58V, Q100P and / or V104L, (as used herein the nomenclature convention is "amino acid position-new amino acid", i.e., I58V indicates that the amino acid I at position 58 has been changed to amino acid V)) resulted in humanized antibodies that not only retained characteristics of the starting rat anti-human antibody such as high affinity for the TREM2 stalk domain, but also expressed with relatively high yields. Such exemplary antibodies are shown in Figures 13A-13B and 14A-14B. As further described below in Examples 11 and 13-17, for example, the antibodies herein are TREM2 agonists and also exhibit several other beneficial biological properties in vitro and in vivo.

[0109] Exemplary Anti-TREM2 Antibody Sequences In some embodiments, the disclosure encompasses an isolated antibody that specifically binds to TREM2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, 19, or 62, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, 20, 55, 63, 65, or 73. In some embodiments, the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, 27, 37, 47, 57, or 67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, or SEQ ID NO: 69. In some embodiments, the VH comprises a CDR-H3 comprising the amino acid sequence of: X 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 are both either IL or L, and X 3 is either D or E). In some embodiments, the antibody comprises a VH derived from a rat IGHV6-8 germline segment. In some embodiments, the antibody comprises a VL derived from a rat IGKV2S11 germline segment. In some embodiments, the antibody comprises a VH derived from a rat IGHV6-8 and a VL derived from a rat IGKV2S11.

[0110] In some embodiments, the disclosure provides an isolated antibody that specifically binds to TREM2, comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, 19, or 62, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, 20, 55, 63, 65, or 73, and a CDR-H3 comprising the amino acid sequence of: 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 are both either IL or L, and X 3and a light chain variable region (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, 27, 37, 47, 57 or 67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29, 39, 49, 59 or 69. For example, such antibodies are shown in Figures 3A-3B. Examples include antibodies comprising heavy chain Kabat CDRs of 3.10C2, 3.50G1, 3.18E5, 3.36F5 and 3.27H7, as well as Para.09. In some examples, the antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11 or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12 or 20, and a CDR-L3 comprising the amino acid sequence of the following: X, 29, 39, 49, 59 or 69. 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 are both either IL or L, and X 3 and a light chain variable region (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0111] In some examples, the light chain variable region (VL) comprises the light chain CDRs of either 3.10C2 or 3.27H7. In some embodiments, the antibody light chain variable region may comprise CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, 27 or 67, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29 or 69. In some examples, the antibody comprises the light chain CDRs of 3.27H7, i.e., CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0112] In some examples, the heavy chain variable region (VH) comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 10, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the light chain variable region (VL) comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In other cases, the heavy chain variable region (VH) comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and the light chain variable region (VL) comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0113] In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence depicted in FIG. 3B. In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence depicted in FIG. 6B. In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence depicted in FIG. 8B. In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence depicted in FIG. 9B. In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence depicted in FIG. 12B. In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence shown in Figure 13B. In some embodiments, the antibodies herein may comprise a heavy chain variable region amino acid sequence that is at least 90%, 95%, 97% or 99% identical to that of the sequence shown in Figure 14B. In some embodiments, the antibody framework region may be chimeric or humanized.

[0114] In some examples, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7 or 17. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 30, 40, 50, 60, 70, 76, 77, 78, 81, 82, 83, 133, 135, 137, 139, 146, 148, 150, or 152. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 133, 135, 137, 139, 146, 148, 150, or 152. In other cases, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, 17, 30, 40, 50, 60, 70, 76, 77, 78, 81, 82, 83, 133, 135, 137, 139, 146, 148, 150, or 152. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, 17, 133, 135, 137, 139, 146, 148, 150, or 152.

[0115] In some embodiments, the antibody light chain variable region comprises an amino acid sequence at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in FIG. 3A. In some embodiments, the antibody light chain variable region comprises an amino acid sequence at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in FIG. 6A. In some embodiments, the antibody light chain variable region comprises an amino acid sequence at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in FIG. 8A. In some embodiments, the antibody light chain variable region comprises an amino acid sequence at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in FIG. 9A. In some embodiments, the antibody light chain variable region comprises an amino acid sequence at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in FIG. 12A. In some embodiments, the antibody light chain variable region comprises an amino acid sequence that is at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in Figure 13 A. In some embodiments, the antibody light chain variable region comprises an amino acid sequence that is at least 90%, 95%, 97% or 99% identical to the amino acid sequence of the sequence depicted in Figure 14 A. In some embodiments, the light chain variable region framework is humanized or chimeric.

[0116] In some embodiments, the antibody comprises a VL that is at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO: 8 or 18. In some embodiments, the antibody comprises a VL that is at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO: 8, 18, 31, 41, 51, 61, 71, 79, 80, 84, 85, 132, 134, 136, 138, 145, 147, 149 or 151. In some embodiments, the antibody comprises a VL that is at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO: 8, 18, 132, 134, 136, 138, 145, 147, 149 or 151. In some embodiments, the antibody comprises a VL that comprises the amino acid sequence of SEQ ID NO: 8 or 18. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, 18, 31, 41, 51, 61, 71, 79, 80, 84, 85, 132, 134, 136, 138, 145, 147, 149, or 151. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, 18, 132, 134, 136, 138, 145, 147, 149, or 151.

[0117] In some embodiments, the antibody comprises the heavy chain CDRs of antibody 3.10C2, 3.50G1, 3.18E5, 3.36F5, 3.27H7 or Para.09 and the light chain CDRs of antibody 3.27H7. In some embodiments, the antibody comprises the heavy chain CDRs of antibody 3.10C2 or Para.09 and the light chain CDRs of antibody 3.27H7. (See Figures 3A-B). In some embodiments, the antibody is humanized or chimeric.

[0118] In some embodiments, the antibody comprises one of the following sets of CDRs: (a) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (c) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (d) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (e) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 24, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29; (f) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (g) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 47, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 49; (h) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 57, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 58, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59, or (i) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 69. CDRs (a) to (i) are also referred to in the following four paragraphs.

[0119] In some embodiments, the antibody comprises: (a) comprising the CDRs of (a) or (b) in the above paragraph and further comprising a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7; or (c) a VH comprising the CDRs of (c) or (d) in the above paragraph and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17; or (e) a VH comprising the CDRs of (e) in the above paragraph and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 30; or (f) a VH comprising the CDRs of (f) in the above paragraph and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 40; or (g) a VH comprising the CDRs of (g) in the above paragraph and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 50; or (h) a VH comprising the CDRs of (h) in the above paragraph and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 60; or (i) comprising the CDRs of (i) in the above paragraph and further comprising a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:70.

[0120] In some embodiments, the antibody comprises: (a) a VL comprising the CDRs of portion (a) or (b) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:8; or (c) further comprising a VL comprising the CDR of portion (c) or (d) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18; or (e) a VL comprising the CDR of (e) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31; or (f) a VL comprising the CDR of (f) above and having at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 41; or (g) a VL comprising the CDR of (g) above and having at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 51; or (h) a VL comprising the CDRs of (h) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 61; or (i) The antibody further comprises a VL comprising the CDR of (i) above and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:71.

[0121] In some examples, the antibody (a) comprising the CDRs according to (a) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 7; or (a) or (b) further comprising a VH comprising the CDRs of SEQ ID NO: 7; or (c) comprising the CDRs according to (c) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 17; or (d) comprising the CDRs according to (d) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 17; or (e) comprising the CDRs according to (e) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 30; or (f) comprising the CDRs according to (f) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 40; or (g) comprising the CDRs according to (g) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 50; or (h) comprising the CDRs according to (h) and further comprising a VH comprising the amino acid sequence of SEQ ID NO: 60; or (i) (i) further comprising a VH comprising the CDRs described in (i) and the amino acid sequence of SEQ ID NO: 70.

[0122] In some examples, the antibody (a) comprising the CDRs according to (a) and further comprising a VL comprising the amino acid sequence of SEQ ID NO:8; or (a) or (b) comprising the CDRs described therein and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 8; (c) comprising the CDRs according to (c) and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 18; or (d) comprising the CDRs according to (d) and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 18; or (e) comprising the CDRs according to (e) and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 31; or (f) comprising the CDRs according to (f) and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 41; or (g) comprising the CDRs according to (g) and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 51; or (h) comprising the CDRs according to (h) and further comprising a VL comprising the amino acid sequence of SEQ ID NO: 61; or (i) (i) further comprises a VL comprising the CDRs described in (i) and the amino acid sequence of SEQ ID NO: 71.

[0123] As noted above, in some embodiments, the VH and / or VL are humanized. In some embodiments, the antibody comprises a light chain and a heavy chain as shown in Figures 6A-6B, e.g., 3.10C2L1 combined with 3.10C2H1, H3 or H5, or 3.10C2L5 combined with 3.10C2H1, H3 or H5. In some embodiments, the antibody comprises a light chain and a heavy chain as shown in Figures 8A-8B, e.g., 3.27H7L1 combined with Para.09H1, H5 or H7, or 3.27H7L6 combined with Para.09H1, H5 or H7. In some embodiments, the antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or 11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 12, and a CDR-H3 comprising the amino acid sequence of the following amino acid sequence: 1 -X 2 -X 3 -Y(wherein, X 1 and X 2 together are either IL or L, and X 3is either D or E), and further comprising a VH framework region at least 90%, at least 95%, at least 97%, or at least 99% identical to the VH framework region of human IGHV-73*01 (SEQ ID NO: 74). (See Figures 6B and 8B). In some embodiments, the antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, 14, or 27, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, 15, or 28, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 16, or 29, and further comprising a VL framework region at least 90%, at least 95%, at least 97%, or at least 99% identical to the VL framework region of human IGKV-28*01 (SEQ ID NO: 75). (See Figures 6A and 8A). In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76, 77, or 78. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 79 or 80. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76, 77, or 78, and further comprises a VL comprising the amino acid sequence of SEQ ID NO: 79 or 80. (See Figures 6A-6B). In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 81, 82, or 83. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 84 or 85. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 81, 82, or 83, and further comprises a VL comprising the amino acid sequence of SEQ ID NO: 84 or 85. (See Figures 8A-8B).

[0124] In some of the above embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8. (See Figures 9A-9B; i.e., h3.10C2.v1). In some of the above embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:17 and a VL comprising the amino acid sequence of SEQ ID NO:18. (See Figures 10A-10B; i.e., hPara.09.v2.) In any of the above embodiments, the antibody can be humanized or chimeric (i.e., having humanized or chimeric framework regions).

[0125] In some embodiments, the framework regions of the antibody are further modified after humanization, for example to improve properties such as expression yield. In some embodiments, the antibody comprises a light chain according to SEQ ID NO: 18, but with 1-5 residues substituted in the framework region. In some embodiments, the antibody comprises a light chain according to SEQ ID NO: 18, but with substitutions at one or more of I58, Q100 and V104, e.g., I58V, Q100P and / or V104L, in the framework region. In some embodiments, the antibody comprises a light chain according to SEQ ID NO: 18, but with a Q100P substitution, both an I58V and a Q100P substitution, or both a Q100P and a V104L substitution. (See, e.g., Figures 13A and 14A). In some such embodiments, the antibody comprises one of the following sets of VH and VL: (a) SEQ ID NOs: 133 and 132 (antibody hPara.09.v2 Q100P); (b) SEQ ID NOs: 135 and 134 (antibody hPara.09.v2 I58V / Q100P); (c) SEQ ID NOs: 137 and 136 (antibody hPara.09.v2 Q100P / V104L); (d) SEQ ID NOs: 146 and 145 (antibody h3.10C2.v1 Q100P); (e) SEQ ID NOs: 148 and 147 (antibody h3.10C2.v1 I58V / Q100P); or (f) SEQ ID NOs: 150 and 149 (antibody h3.10C2.v1 Q100P / V104L). In other embodiments, the antibody comprises the VH and VL of SEQ ID NOs: 139 and 138 (h3.10C2.H1-3.10C2.L10). In yet other embodiments, the antibody comprises the VH and VL of SEQ ID NOs: 152 and 151 (hPara.09.H5-3.10C2.L10).

[0126] In some embodiments, the antibody comprises a VL that comprises between 1 and 10 amino acid substitutions in the framework regions compared to human IGKV2-28*01 germline (see FIG. 8A showing the Kabat / Chothia framework and CDR regions of IGKV2-28*01). In some examples, the antibody comprises a VL that comprises between 1 and 5 amino acid substitutions in the framework regions compared to human IGKV2-28*01. In some embodiments, these amino acid substitutions include Q100P or V104L, or both Q100P and V104L. In some such examples, the VL comprises a Val at position 58, and in other cases, the VL comprises an He at position 58 (e.g., Q100P and 58V or Q100P and V58I compared to IGKV2-28*01).

[0127] In any of the embodiments herein, the antibody may be an Fv, a single chain Fv (scFv), a Fab, a Fab', or a (Fab') 2 In other embodiments, the antibody may be an antibody fragment such as. In other embodiments, the antibody may be a whole antibody (i.e., including a heavy chain constant region and a light chain constant region). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgG1 Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P / M252Y / S254T / T256E Fc region, a human IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. In the case of a murine IgG antibody, the antibody may be an mIgG1 or mIgG2 or mIgG2 LALAPG antibody. The antibody may, in some instances, include a full-length heavy chain and / or a full-length light chain. In some instances, the antibody may lack the C-terminal Lys or C-terminal Lys and Gly residues of the heavy chain constant region, while in other cases, the antibody contains one or both of these C-terminal residues.

[0128] In some embodiments, the antibody comprises a heavy chain that comprises or consists of the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises a light chain that comprises or consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain that comprises or consists of the amino acid sequence of SEQ ID NO: 144 and a light chain that comprises or consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain that comprises or consists of the amino acid sequence of SEQ ID NO: 144 but lacking the C-terminal lysine of SEQ ID NO: 144 or lacking the C-terminal glycine and lysine of SEQ ID NO: 144 and a light chain that comprises or consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain that comprises an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises a light chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 176.In some embodiments, the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, optionally without a C-terminal lysine or glycine-lysine, and a light chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, optionally excluding the C-terminal lysine or glycine-lysine, and a light chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176.In any of the above cases allowing for sequence variation in SEQ ID NO: 144 and / or SEQ ID NO: 176, in some embodiments such sequence variation is limited to the antibody framework and / or constant regions, such that the antibody also comprises the heavy chain CDRs of SEQ ID NOs: 11, 12 and 13, or alternatively 19, 20 and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15 and 16. In other embodiments, such sequence variation in SEQ ID NO: 144 and / or SEQ ID NO: 176 is limited to the antibody constant regions, such that the antibody also comprises SEQ ID NOs: 17 and / or 18.

[0129] In some instances, the antibody may be bispecific or multispecific. In some instances, the antibody may be conjugated to another molecule, such as a label or a drug, directly or through a linker.

[0130] In some instances, antibodies have intact effector functions. In some instances, antibodies have Fc regions with reduced effector functions. In other cases, antibodies have effector-less Fc regions. For example, the Fc region of an antibody therapeutic can bind complement component C1q and Fc gamma receptors (FcγRs) to trigger cellular effector responses such as phagocytosis, cytokine release, and production of reactive oxygen species. (See, e.g., X. Wang, et al., Protein & Cell 9:63-73 (2018); SBMkaddem et al., Frontiers in Immunology, available at https: / / doi.org / 10.3389 / fimmu.2019.00811 (2019).) Overactivation of these pathways can be detrimental in the CNS, especially in pathological conditions. (DJDiSabato et al., J.Neurochemistry 139(S2):136-153(2016).) Clinically, antibody therapeutics against amyloid beta that have intact effector function and bind to amyloid beta aggregates significantly increase the incidence of ARIA (amyloid-related imaging abnormality), a potentially harmful side effect, but ARIA has not been observed from antibodies that only bind to monomeric forms of amyloid beta or antibodies with reduced effector function. (M.Filippi et al., JAMA Neurol.79(3):291-304(2022).) In some embodiments, for example as described in the Examples and Figures herein, antibodies herein with reduced effector function (comprising a human IgG1 heavy chain constant region with either the LALAPG mutation or the N297G mutation, as described above) were sufficient to induce a microglial response in the CNS via TREM2 stimulation.

[0131] Exemplary Antibody Variants, Fragments, and Constant Regions In further aspects, the antibodies that specifically bind to TREM2 herein may incorporate any of the features, either alone or in combination, as described in the following sections.

[0132] antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,87,458. Fab and F(ab') fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives 2 For a discussion of fragments, see US Pat. No. 5,869,046.

[0133] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0134] Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0135] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of an intact antibody and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0136] Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0137] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody, and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0138] Humanized antibodies and methods for producing the same are described, for example, in Almagro and Fransson, Front. Biosci., vol. 13: pp. 1619-1633 (2008), and further described, for example, in Riechmann et al., Nature, vol. 332: pp. 323-329 (1988); Queen et al., Proc. Nat'l, vol. 10: 1111-1115 (1997); Acad. Sci. USA, vol. 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods, vol. 36:25-34 (2005) (describing grafting of specificity determining regions (SDRs)); Padlan, Mol. Immunol., vol. 28:489-498 (1991) (describing grafting of specificity determining regions (SDRs)); in "FR shuffling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005), and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0139] Framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0140] In some embodiments, a humanized antibody may comprise a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0141] Bispecific or multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for TREM2 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of TREM2. Bispecific antibodies can also be used to localize drugs, such as cytotoxic agents, or to localize detection labels to cells expressing TREM2. In some embodiments, multispecific antibodies (e.g., bispecific antibodies) comprise a first variable domain that comprises the CDRs or variable regions described herein. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0142] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knobs-into-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by a number of techniques, including the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); the use of leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 18(5):1547-1553 (1992)); the use of "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999, 93:111-112 (1993)). al., J. Immunol., 152:5368 (1994); and, for example, by preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0143] Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0144] Further antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding. Specific examples of humanized variants that have been generated are described, for example, in Figures 6A-6B, 8A-8B, 9A-9B, and 12A-12B herein.

[0145] Substitution, insertion, and deletion mutants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table A under the heading of "preferred substitutions." More substantial changes are shown in Table A under the heading of "exemplary substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or increased or decreased ADCC or CDC activity. Table A [Table 1]

[0146] Amino acids may be classified according to common side chain properties as follows: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0147] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have a modification (e.g., improvement) in a particular biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have a particular biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0148] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting a secondary library is described, for example, in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0149] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such changes may, for example, be outside of antigen contact residues within the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0150] A useful method for identifying antibody residues or regions that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. Mutants may be screened to determine whether they have the desired properties.

[0151] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of an antibody or a polypeptide to an enzyme which extends the serum half-life of the antibody (e.g., for ADEPT).

[0152] Glycosylation variants Alterations in the glycosylation of the Fc region, as well as certain Fc region mutations, can affect the effector function of the antibody by either enhancing or reducing effector function, or in some instances may render the antibody effectorless.

[0153] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0154] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain a branched, biantennary oligosaccharide, usually attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may contain various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNac), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the bisecting oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the invention may be performed to generate antibody variants with specific improved properties.

[0155] In one aspect, antibodies may be modified to reduce or eliminate glycosylation at Asn297, for example, by mutating that residue to glycine or another amino acid (N297G). In other cases, other residues in the Fc region may be modified to reduce ADCC and / or CDC activity, or to reduce or modify Fc-gamma receptor binding.

[0156] In another embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 of the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300 (EU numbering), due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; 2004 / 01 32140; 2004 / 0110704; 2004 / 0110282; 2004 / 0109865; WO 2003 / 085119; 2003 / 084570; 2005 / 035586; 2005 / 035778; 2005 / 053742; 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. 2003 / 0157108, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107). In some embodiments, the antibody may have a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region that includes a mutation at Asn297 (EU numbering), e.g., to reduce fucosylation or to eliminate glycosylation. In some embodiments, the antibody may have an Asn297Ala or Asn297Gly mutation.

[0157] Further provided are antibody variants having bisected oligosaccharides, for example, where the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0158] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0159] In certain embodiments, the invention contemplates antibody variants that have some, but not all, effector functions (i.e., generating antibodies with reduced effector functions or generating effectorless antibodies), which may make them desirable candidates for applications where the half-life of the antibody in vivo is important, but where certain effector functions (e.g., complement and ADCC, etc.) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express Fc(RI, FcγRII and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (Bruggemann, M. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0160] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Pat. No. 6,737,056; EU numbering of residues). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581; EU numbering). In some embodiments, the antibody comprises an engineered alanine at amino acid position 265 according to the EU numbering rules. In some embodiments, the antibody comprises an engineered alanine at amino acid position 297 according to the EU numbering rules.

[0161] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0162] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0163] In some embodiments, modifications are made within the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0164] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (EU numbering), e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260; 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0165] In some embodiments, the antibody may have a wild-type human IgG1 Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P / M252Y / S254T / T256E Fc region, a human IgG1 N297G Fc region, a human IgG1 LALAPG(L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. (All positions are EU numbering.)

[0166] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain embodiments, the replaced residues are located at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which may be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0167] Antibody Derivatives and Conjugates In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when more than one polymer is attached, the polymers may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0168] In another embodiment, a conjugate of an antibody and a non-protective moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protective moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that will not harm normal cells but will heat the non-protein moiety to a temperature that will kill cells proximal to the antibody-non-protein moiety.

[0169] In some embodiments, the anti-TREM2 antibody is conjugated to a detection label and / or a drug. As used herein, a detection label is a moiety that facilitates detection of the antibody and / or the molecule to which the antibody binds. Non-limiting exemplary detection labels include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal binding tags, and the like.

[0170] Exemplary Properties of Certain Anti-TREM2 Antibodies As described in the Examples herein, a set of rat anti-human anti-TREM2 antibodies were identified from the screening herein followed by deep sequencing analysis that have a unique set of properties including, for example, (a) specific binding to the TREM2 stalk domain at an epitope spanning the cleavage site between residues 157 and 158, and also specific binding to a polypeptide consisting of residues 151-165, and more specifically within residues 151-161 of hTREM2, (b) not binding to soluble TREM2, (c) acting as TREM2 agonists, and (d) specific binding to TREM2 with high affinity. These rat anti-human antibodies, 3.10C2, 3.27H7, 3.50G1, 3.18E5 ("3.10C2 group"), 3.36F5, and Para.09, and their humanized variants, may have these and additional properties, which are described in more detail below.

[0171] binding affinity In some embodiments, the antibodies herein specifically bind to both human TREM2 and cynomolgus TREM2 with high affinity. For example, in some embodiments, the anti-TREM2 antibodies herein have a K of less than 1 nM, less than 0.5 nM, less than 100 pM, less than 50 pM, less than 25 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., by surface plasmon resonance (SPR) at 37° C. D In some embodiments, the anti-TREM2 antibodies herein can bind to human TREM2 with a K of less than 1 nM, less than 0.5 nM, less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., by SPR at 37°C. D and can bind to cynomolgus monkey TREM2.

[0172] In some embodiments, the anti-TREM2 antibodies herein have a K of less than 500 pM (i.e., less than 0.5 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to hTREM2 with a K of less than 200 pM (i.e., less than 0.2 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to hTREM2 with a K of less than 100 pM (i.e., less than 0.1 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to hTREM2 with a K of less than 50 pM (i.e., less than 5E-11 M or less than 0.05 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to hTREM2 with a K of 10-100 pM, 10-50 pM, 10-25 pM, or 1-10 pM. D In some embodiments, the anti-TREM2 antibodies herein bind to hTREM2 with a K of less than 500 pM (i.e., less than 5E-10 M or less than 0.5 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to cynomolgus TREM2 with a K of less than 200 pM (i.e., less than 2E-10 M or less than 0.2 nM). DIn some embodiments, the anti-TREM2 antibodies herein bind to cynomolgus TREM2 with a K of less than 100 pM (i.e., less than 1E-10 M or less than 0.1 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to cynomolgus TREM2 with a K of less than 50 pM (i.e., less than 5E-11 M or less than 0.05 nM). D In some embodiments, the anti-TREM2 antibodies herein bind to cynomolgus TREM2 with a K of 10-100 pM, 10-50 pM, or 10-25 pM. D In some embodiments, the anti-TREM2 antibodies herein bind to cynomolgus TREM2 with a K of 100-500 pM, 10-100 pM, 10-50 pM, 10-25 pM, or 1-10 pM. D It binds to both human and cynomolgus TREM2.

[0173] For example, both the Fab and IgG versions of the original rat anti-human 3.10C2 antibody have a K D For example, h3.10C2.v1 bound to both human and cynomolgus TREM2 and had a K D For example, rat anti-human antibodies with Para.09 heavy chains but different light chains based on the 3.10C2 antibody group had K values ​​of 1-5 pM. D Humanized hPara.09.v2 bound to human and cynomolgus TREM2 with K values ​​of 10–25 pM. D Further humanized versions of 3.10C2 bound to TREM2 in humans and cynomolgus monkeys with K values ​​of 100–200 pM. D 09 binds to human and cynomolgus TREM2 with a K of 10–50 pM, and a further humanized version of Para.09 D It bound to human and cynomolgus monkey TREM2 at 100 ng / mL.

[0174] In this specification, K Dcan be measured by SPR, for example, using a BIACORE® SPR assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 or BIACORE®-T200 (BIAcore, Inc., Piscataway, NJ) can be performed with immobilized antibody chips. Association rates (kon) and dissociation rates (koff) can be calculated using a simple one-to-one Langmuir binding model (e.g., BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K D ) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0175] In the following examples, affinity was measured by SPR in one of two formats. In one format, TREM2-Fc was immobilized on a Protein A chip and a soluble anti-TREM2 Fab fragment was used as the ligand. In the second format, anti-TREM2 IgG was immobilized on an anti-CH1 Biacore™ chip and soluble monomeric TREM2 was used as the ligand. For example, the K D Measurements were determined by SPR at 37°C.

[0176] Lack of epitope and soluble TREM2 binding and inhibition of TREM2 shedding As shown in the Examples section, the present disclosure provides antibodies that specifically bind within the stalk domain of TREM2 spanning residues 129-174 of hTREM2 (see SEQ ID NO: 90) but not soluble TREM2 (sTREM2; residues 19-157 of hTREM2), in contrast to other anti-TREM2 antibodies previously described in the literature as stalk binders. sTREM2 is formed by cleavage of TREM2 between residues H157 and S158, and generally comprises residues 19-157 of TREM2. In some embodiments, binding to the stalk domain of TREM2 can be demonstrated by performing a binding assay to a polypeptide comprising residues 129-174 (SEQ ID NO: 90) or residues 129-175 of TREM2 (SEQ ID NO: 177; FIG. 2A), which includes the stalk domain and the first transmembrane domain residues. The results provided herein show that the 3.10C2, Para.09, 3.50G1, 3.18E5, 3.36F5 or 3.27H7 antibodies of the present disclosure, as well as humanized versions of those antibodies described herein, such as Para.09.v2, 3.10C2.v1, bind to an epitope spanning the H157-S158 cleavage site that is destroyed upon cleavage of TREM2 at the H157-S158 cleavage site and is likely absent in sTREM2 (e.g., Figures 2A-B and 5). In contrast, several anti-TREM2 antibodies described in the literature show binding to soluble TREM2 and likely recognize a different epitope that is not destroyed by cleavage of intact TREM2, even though in some instances they may contact residues surrounding the cleavage site (see, e.g., Figure 5). Thus, the present disclosure provides antibodies with properties not found in other anti-TREM2 antibodies, namely, the ability to bind to intact TREM2 but not sTREM2, despite appearing to interact with a similar region of the protein as previous antibodies that bind sTREM2.

[0177] As mentioned above, certain antibodies herein do not bind to soluble TREM2. As used herein, this means that the antibody may not exhibit more than 10% binding to sTREM2, and in some instances, not more than 5% binding to sTREM2, when assayed in an ELISA assay, with the % binding normalized to the binding of the positive control antibody 1.16B8, whose binding to sTREM2 is set to 100%. The light chain sequence of the control antibody 1.16B8 is shown in SEQ ID NO: 170, and its heavy chain sequence is shown in SEQ ID NO: 171. In some embodiments, antibody 3.17A9 is used as a detection agent in the ELISA assay. The light chain sequence of the detection antibody 3.17A9 is shown in SEQ ID NO: 168, and its heavy chain sequence is shown in SEQ ID NO: 169. In some embodiments, binding may be assayed using a polypeptide comprising residues 19-157 of hTREM2 (e.g., residues 19-157 of SEQ ID NO: 21). In some embodiments, the antibody does not bind to soluble TREM2 in an ELISA assay in which binding is normalized to binding of control antibody 1.16B8, detection antibody 3.17A9 is used as the detection reagent, and sTREM2 is a polypeptide comprising residues 19-157 of SEQ ID NO: 21. In some embodiments, an ELISA assay may be performed as shown in Example 11 and FIG.

[0178] For example, as shown in Example 11 herein, a human TREM2 ELISA assay measured binding of antibodies to sTREM2 by coating the antibody being tested onto plate wells as a capture reagent and using biotinylated IgV-reactive monoclonal antibody 3.17A9 as a detection reagent. Control antibody 1.16B8 was used as a positive control antibody to establish a standard for sTREM2 binding. For example, in this assay, Para.09-LC 3.27H7 mIgG2a LALAPG (Para09), rat 3.10C2 mIgG2a LALAPG (3.10C2), rat 3.18E5 mIgG2a LALAPG (3.18E5), rat 3.50G1 mIgG2a LALAPG (3.50G1), rat 3.27H7 mIgG2a LALAPG (3.27H7), rat 3.36F5 mIgG2a LALAPG (3.36F5) all showed no binding to sTREM2 (see FIG. 16 , which shows near zero normalized sTREM2 binding for these antibodies in contrast to the 1.116B8 control). The humanized antibody Para.09.v2 antibody with hIgG1 N297G constant region was also tested for sTREM2 binding and showed no binding to sTREM2, as did the rat Para.09 with mIgG2 LALAPG. Thus, in some embodiments, the antibodies herein, such as those shown in Figure 16, and humanized variants of such antibodies, such as Para.09.v2, 3.10C2.v1, do not bind soluble TREM2. In some embodiments, the antibodies bind the TREM2 stalk domain but do not bind soluble TREM2.

[0179] Lack of binding to sTREM2 may be advantageous in vivo, as it may significantly reduce or eliminate the possibility of undesired binding between the antibody and sTREM2 in vivo. For example, without being limited by mechanism, sTREM2 may otherwise act as a "decoy" for anti-TREM2 antibodies, such that in vivo antibody molecules are not available to bind to TREM2 at the cell surface.

[0180] As shown in the Examples and accompanying Figures herein, the antibodies herein may also bind to residues within the stalk domain of TREM2, in the region spanning the H157-S158 cleavage site, specifically at residues 149-168, 146-169, 146-161, and 151-165 of TREM2. This unique cleavage site-spanning epitope may explain why the antibodies herein bind to the TREM2 stalk and not to soluble TREM2. (See, e.g., FIG. 5). In some embodiments, the antibodies herein specifically bind to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) and / or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94). In some examples, the antibodies herein specifically bind to a TREM2 polypeptide consisting of amino acids 149 to 168 (SEQ ID NO: 93), 146 to 169 (SEQ ID NO: 95), 146 to 161 (SEQ ID NO: 96), and / or 151 to 165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94). In some embodiments, the antibodies herein specifically bind to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) and / or 151 to 165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and a TREM2 polypeptide consisting of amino acids 159 to 175 (SEQ ID NO: 94). In some examples, the antibodies herein specifically bind to a TREM2 polypeptide consisting of amino acids 149 to 168 (SEQ ID NO: 93), 146 to 169 (SEQ ID NO: 95), 146 to 161 (SEQ ID NO: 96) and / or 151 to 165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and a TREM2 polypeptide consisting of amino acids 159 to 175 (SEQ ID NO: 94).

[0181] As shown in the Examples provided herein, data suggest that the antibodies of the present disclosure specifically bind to an epitope within residues 151-161 of TREM2. (See FIG. 2A and FIG. 2B). In some embodiments, the antibodies herein bind to an epitope that includes one or more of Asp-152, His-154, Val-155, Glu-156, His-157, Ser-158, Ile-159, and Ser-160 that span the TREM2 cleavage site at H157-S158. (See FIG. 5). In some embodiments, the antibodies herein bind to an epitope that includes residues 152, 154, 157, 158, and 159. In some embodiments, the antibodies herein specifically bind to an epitope that includes Asp-152, His-157, and Ile-159. In certain embodiments, the antibodies herein bind to an epitope comprising one or more of residues Asp-152, His-154, Val-155, Glu-156, His-157, and Ile-159. In some embodiments, the antibodies herein bind to an epitope comprising Glu-156 and Ser-160. Alanine scanning analysis shows that TREM2 binding of antibody Para.09, comprising the 09 heavy chain variable region paired with the light chain variable region of antibody 3.27H7, is affected by alanine mutations at, for example, Asp-152, His-157, and Ile-159 (Figure 5). Para.09 and 3.10C2 have similar patterns in the alanine scanning assay, except that the binding of 3.10C2 is further affected by alanine mutations at H154 and S158 (Figure 5).

[0182] In some embodiments, antibodies herein, such as Para.09 or 3.10C2, including humanized variants of such antibodies, such as Para.09v2 and 3.10C2.v1, also reduce the levels of sTREM2 in vivo, such as in mouse plasma, CSF or brain tissue, as compared to a control antibody that does not specifically bind to TREM2 (e.g., an isotype control), as measured by ELISA. For example, in some embodiments, an ELISA assay may be performed by determining the change in the level of soluble TREM2 from plasma, CSF or brain tissue samples incubated in the presence of an anti-TREM2 antibody, as compared to an isotype control antibody. Reduced levels of sTREM2 in the presence of an anti-TREM2 antibody indicate inhibition of sTREM2 shedding.

[0183] As shown in Examples 18A-B below, the lack of binding to sTREM2 also correlates with this depletion of sTREM2 in vivo (in mouse plasma, CSF and brain tissue). In contrast to the comparative antibody, treatment with the exemplary antibody Para.09 resulted in substantially lower levels of plasma sTREM2 below baseline control levels, whereas treatment with the comparative anti-TREM2 antibody increased the levels of sTREM2 in plasma several-fold (Example 18B and Figure 25C). For example, the Para.09 antibody in a mouse mIgG2 LALAPG background caused a 70-85% reduction in soluble TREM2 levels in plasma for at least 7 days after administration, whereas the comparative antibody increased sTREM2 levels by more than 2-fold over the same period (Figure 25C). Thus, in some embodiments, the antibodies herein, such as Para.09 and 3.10C2, and their humanized variants, such as Para.09.v2 and 3.10C2.v1, cause a reduction in soluble TREM2 levels in mouse plasma for at least 7 days after administration to the mice. Thus, the antibodies of the present disclosure, with their increased specificity for intact TREM2 and lack of binding to sTREM2 in vivo, may be particularly useful in situations where elevated sTREM2 levels are undesirable.

[0184] Thus, the antibodies herein, such as Para.09 and 3.10C2, and humanized variants thereof, may have any one or more of the following properties: (a) specific binding to the stalk domain of TREM2; (b) not binding to soluble TREM2 (sTREM2); (c) specific binding to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (d) specific binding to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94); (e) a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry); (f) a K by surface plasmon resonance (SPR) at 37°C of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM or between 10 and 25 pM. D Specific binding to human and cynomolgus TREM2 at 200 ng / mL. In some examples, the antibodies may have two or more of the above properties, three or more of the above properties, four or more of the above properties, five or more of the above properties, or all of the above properties. For example, in some examples, the antibodies herein do not bind soluble TREM2, and exhibit specific binding to a TREM2 epitope spanning the H157-S158 cleavage site, and / or exhibit specific binding to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or amino acids 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or amino acids 159-175 (SEQ ID NO: 94). In some such examples, the antibody also has a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM, or between 10 and 25 pM. DSpecifically binds to human and cynomolgus TREM2 at 37° C. For example, in some examples, the antibodies herein do not bind to soluble TREM2 but specifically bind to the stalk domain of TREM2. In some examples, the antibodies herein do not bind to soluble TREM2 but have a reduced binding affinity to TREM2 stalk domain polypeptides comprising D152A, H157A and I159A substitutions compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). In some such examples, the antibodies also have a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM or less than 2 pM at 37° C. by surface plasmon resonance (SPR), or a K of 10-50 pM or 10-25 pM. D The antibodies specifically bind to human and cynomolgus TREM2 at 100 ng / mL. Additionally, in some instances, the antibodies also have intact effector function. In some instances, the antibodies also have an Fc region with reduced effector function. In other cases, the antibodies also have an effector-less Fc region. For example, in some instances, the antibodies comprise a hIgG1 Fc region with an N297G substitution.

[0185] In certain exemplary embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, further wherein the antibody does not bind soluble TREM2 (sTREM2), and the antibody is a TREM2 agonist, as further described in the following section. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, 133, 135, 137, or 139, or comprises an amino acid sequence 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% identical to the amino acid sequence of SEQ ID NO: 17, 133, 135, 137, or 139. or the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144 with or without the C-terminal lysine or C-terminal glycine-lysine, or comprises an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144. In some examples, the antibody also specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site and / or specifically binds with greater affinity to a TREM2 polypeptide consisting of amino acids 146 to 161 (SEQ ID NO: 96) or 151 to 165 (SEQ ID NO: 97) than to a TREM2 polypeptide consisting of amino acids 139 to 158 (SEQ ID NO: 92) and / or 159 to 175 (SEQ ID NO: 94). In some examples, the antibody does not bind to soluble TREM2 and has reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry).In some examples, the antibody also has a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM, or between 10 and 25 pM. D The antibody specifically binds to human and cynomolgus TREM2 at 3.10C2, 3.50G1, 3.18E5, 3.36F5, or 3.27H7. Additionally, in some examples, the antibody also has an intact effector function. In some examples, the antibody also has an Fc region with reduced effector function. In other cases, the antibody also has an effector-less Fc region. For example, in some examples, the antibody comprises a hIgG1 Fc region with an N297G substitution. In any of these cases, in some embodiments, the antibody can have light chain CDRs 1-3 of 3.10C2, 3.50G1, 3.18E5, 3.36F5, or 3.27H7.

[0186] The antibodies described herein may also have any combination of the above properties described in this section, as well as one or more of the additional biological activities described in the next section.

[0187] Further biological activity In some embodiments, the antibodies herein, such as Para.09 and 3.10C2 and their humanized variants, may have one or more, two or more, three or more, four or more, five or more, or six or more, seven or more, eight or more, or all of the following additional characteristics: (a) induce luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (b) induce in plasma luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (c) reducing the levels of sTREM2 in vivo; (d) inhibiting sTREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) inducing tyrosine phosphorylation in human MDM cells; (e) inducing SYK phosphorylation in human MDM cells; (f) enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1; (g) inhibiting sTREM2 shedding in human iPSC-derived microglia; (h) inducing SYK phosphorylation in human iPSC-derived microglia; (i) increasing total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia (e.g., as described in the assay of Example 17 herein).

[0188] Thus, in some embodiments, the antibodies herein, such as Para.09 and 3.10C2 and their humanized variants, also reduce sTREM2 levels (i.e., inhibit sTREM2 shedding) in cultured cells, including Jurkat cells engineered to express human TREM2, or in human monocyte-derived macrophages (MDMs) or human induced pluripotent stem cell (iPSC)-derived microglia. In some examples, sTREM2 levels in cell culture assays may be measured by an ELISA assay.

[0189] In some embodiments, the antibodies herein are TREM2 agonists. For example, in some embodiments, the antibodies herein induce reporter gene expression in Jurkat-NFAT reporter cells expressing human TREM2. For example, TREM2 activity can result in cell signaling events that result in enhanced gene expression under the control of NFAT (nuclear factor of activated T cells). NFAT activity can be assessed using Jurkat cells engineered to express a reporter gene, such as firefly luciferase, in response to activation of gene expression by NFAT transcription factors. To test the effect of anti-TREM2 antibodies on NFAT-controlled gene expression, Jurkat-NFAT reporter cells expressing human TREM2 can be constructed. In some embodiments, the antibodies herein enhance reporter gene expression in Jurkat-NFAT reporter cells expressing human TREM2. (See Figures 18A-B). In some embodiments, the antibodies herein also enhance reporter gene expression in Jurkat-NFAT reporter cells expressing human TREM2 mutants such as hTREM2 R47H, hTREM2 R62H and / or H157Y (see Figures 19A-C).

[0190] TREM2 activity, and thus agonism by anti-TREM2 antibodies, can also be assessed by phosphorylation levels of tyrosine residues in general, or by phosphorylation levels of Syk kinase, a kinase that is phosphorylated upon activation of TREM2-associated cell signaling. Thus, in some embodiments, the antibodies herein increase TREM2 activity as measured by an increase in one or both of pan phospho-tyrosine (pY) and phosphorylated Syk kinase (p-SYK) levels in one or more of Jurkat-NFAT reporter cells expressing human TREM2, human iPSC-derived microglia, or human MDM cells. In some embodiments, the antibodies herein increase SYK phosphorylation in human iPSC-derived microglia and human MDM cells.

[0191] In some embodiments, the antibodies herein also enhance survival of human iPSC-derived microglia when those cells are cultured in the absence of IL-34 and CSF-1, which are normally required to promote cell survival and growth. For example, humanized antibodies hPara.09.v2, h3.10C2.v1, hPara.09Q100PV104L, and hPara.09Q100P each enhance survival of iPSC-derived microglia and EC 50 (See Example 17 and FIG. 23E). Thus, in some embodiments, the antibodies herein inhibit the survival of iPSC-derived microglia in the absence of IL-34 and CSF-1 with an EC in the range of 60 pM to 700 pM, e.g., 60 pM to 400 pM, or 60 pM to 200 pM. 50 Additionally, in some embodiments, the antibodies may enhance microglial survival in the absence of IL-34 and CSF-1 by at least 3-fold, or 3-8-fold, e.g., 3-6-fold, 3-5-fold, 5-8-fold, or 3-4-fold over that of an isotype control antibody (see Figures 23D-E).

[0192] In some embodiments, the antibodies herein increase Aβ plaque intensity in the presence of Aβ oligomers and / or increase X04 plaque intensity in human iPSC-derived microglia. (See Example 17 and Figures 24A-C). Plaque formation and compaction can be measured as an increase in intensity, for example, when staining iPSC-derived microglia with a specific marker dye, such as one that recognizes Aβ (i.e., measuring "Aβ intensity" or "Aβ plaque intensity") or one that recognizes Aβ plaques (X04 marker, measuring "X04 intensity" or "X04 plaque intensity"). In some examples, "total" Aβ plaque intensity may be determined. In some examples, "average" X04 plaque intensity may be measured. Examples of assays for measuring plaque formation and compaction, total Aβ plaque intensity, and average X04 plaque intensity are shown in Example 17 and Figures 24A-C.

[0193] In some embodiments, the antibodies herein increase Aβ plaque formation and compaction in iPSC-derived microglia as indicated by an increase in one or both of total Aβ plaque intensity and mean X04 intensity compared to an isotype control antibody. For example, in some embodiments, the presence of the antibody increases total Aβ plaque intensity by 4-6 fold compared to an isotype control antibody. In some embodiments, the antibodies herein have an EC for increasing total Aβ plaque intensity between 100 nM and 800 nM. 50 For example, as shown in FIG. 24B, antibodies hPara.09.v2, h3.10C2.v1, hPara.09Q100PV104L, and Para.09 Q100P increased total Aβ plaque intensity in a human iPSC-derived microglia model plaque assay and EC 50 were 330 nM, 130 nM, 770 nM and 120 nM, respectively. Further, in some embodiments, the presence of the antibody increases the mean X04 plaque intensity in the assay by 2-3 fold compared to an isotype control antibody. In some embodiments, the antibodies herein have an EC 50 For example, as shown in FIG. 24C, antibodies hPara.09.v2, h3.10C2.v1, hPara.09Q100PV104L, and hPara.09Q100P increased mean X04 plaque intensity in a human iPSC-derived microglia model plaque assay and EC 50 were 320 nM, 11 nM, 40 nM and 500 nM, respectively.

[0194] Data from Jurkat-NFAT reporter cells expressing human TREM2, as well as data from MDM and iPSC-derived microglia models, indicate that the antibodies herein may have, for example, neuroprotective activity. For example, the antibodies herein may have a combination of biological activities that enhance human iPSC-derived microglia survival, increase p-SYK phosphorylation and / or NFAT-regulated gene expression, and promote Aβ plaque formation and compaction in the presence of Aβ oligomers in human iPSC-derived microglia. In some embodiments, the antibodies herein also show little or no significant off-target binding in BV ELISA assays. (Hotzel et al., Landes Bioscience dx.doi.org / 10.4161 / mabs.22189(2012)). An exemplary BV ELISA assay is as described in Example 10 below. In such an assay, for example, an antibody may have high (score >5), moderate (score 1-5) and low (undetectable, score <1) off-target binding. In some embodiments, when the antibodies herein were tested in parallel with antibodies previously determined to have high moderate and low off-target binding scores, the antibodies herein were found to have low off-target binding scores, i.e., less than 1. (See FIG. 15). In particular, 3.10C2.v1 and Para.09.v2, prepared in a human IgG1 N297G constant region background, showed low off-target binding in this assay (i.e., scores less than 1), despite also having affinities for TREM2 in the 100-500 pM and 10-50 pM ranges, respectively. A low score in this assay may indicate, for example, that the antibody is less likely to bind to the wrong target in vivo, which correlates with favorable pharmacokinetic properties in humans and cynomolgus monkeys and may be beneficial in reducing toxicity and side effects due to off-target binding in vivo.

[0195] Certain Exemplary Humanized Antibody Sequences and Properties Antibody h3.10C2.v1. In some aspects, the disclosure relates to an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence comprising the heavy chain CDRs of the 3.10C2 antibody, comprising SEQ ID NOs: 1, 2, and 3 (Kabat), or comprising SEQ ID NOs: 9, 10, and 3 (Chothia). (See FIG. 9B). In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising an amino acid sequence comprising the light chain CDRs of the 3.10C2.v1 antibody, based on the light chain CDRs of the 3.27H7 Fab, comprising SEQ ID NOs: 4, 5, and 6. (See FIG. 9A). In some embodiments, the antibody comprises the heavy and light chain CDRs of 3.10C2.v1, comprising SEQ ID NOs: 1-6. In some embodiments, the antibody comprises the heavy and light chain CDRs of 3.10C2.v1, comprising SEQ ID NOs: 9, 10, 3, 4, 5, and 6.

[0196] In some embodiments, the antibody further comprises a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NOs: 1, 2, and 3, or 9, 10, and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5, and 6, and comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody further comprises a light chain variable region comprising the heavy chain CDRs of SEQ ID NOs: 1, 2, and 3, or 9, 10, and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5, and 6, and comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 1, 2 and 3, or 9, 10 and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5 and 6, and further comprises a heavy chain variable region comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 1, 2 and 3, or 9, 10 and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5 and 6, and further comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 1, 2 and 3, or 9, 10 and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5 and 6, and further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 1, 2 and 3, or the light chain CDRs of SEQ ID NOs: 9, 10 and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5 and 6, and further comprises a heavy chain variable region in a heavy chain framework region of SEQ ID NO: 7 comprising the amino acid sequence of SEQ ID NO: 7, but with up to five, e.g., 1, 2, 3, 4 or 5 amino acid substitutions, insertions or deletions. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 1, 2 and 3, or the light chain CDRs of SEQ ID NOs: 9, 10 and 3, and / or the light chain CDRs of SEQ ID NOs: 4, 5 and 6, and further comprises a light chain variable region in a light chain framework region of SEQ ID NO: 8 comprising the amino acid sequence of SEQ ID NO: 8, but with up to five, e.g., 1, 2, 3, 4 or 5 amino acid substitutions, insertions or deletions. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8, but with up to five amino acid substitutions, insertions or deletions, such as 1, 2, 3, 4 or 5 amino acid substitutions, insertions or deletions, in the framework regions of SEQ ID NO:7 and / or SEQ ID NO:8.

[0197] In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0198] In any of the embodiments herein, the antibody may be an Fv, a single chain Fv (scFv), a Fab, a Fab', or a (Fab') 2In other embodiments, the antibody may be an antibody fragment such as. In other embodiments, the antibody may be a whole antibody (i.e., including a heavy chain constant region and a light chain constant region). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgG1 Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P / M252Y / S254T / T256E Fc region, a human IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. In the case of a murine IgG antibody, the antibody may be an mIgG1 or mIgG2 or mIgG2 LALAPG antibody. The antibody may, in some instances, include a full-length heavy chain and / or a full-length light chain. In some instances, the antibody may lack the C-terminal Lys or C-terminal Lys and Gly residues of the heavy chain constant region. In other cases, the antibody contains one or both of these C-terminal residues. In some instances, the antibody may be bispecific or multispecific. In some instances, the antibody may be conjugated directly or via a linker to another molecule, such as a label or a drug. In some instances, the antibody has intact effector function. In some instances, the antibody has an Fc region with reduced effector function. In other cases, the antibody has an effector-less Fc region. For example, in some instances, the antibody comprises a hIgG1 Fc region with an N297G substitution.

[0199] In some embodiments, the antibody may have certain properties, which are described in more detail below. For example, in some aspects, the antibody has one or more, two or more, three or more, four or more, five or more, or all of the following characteristics: (a) specifically binds to the stalk domain of TREM2 and specifically binds to a polypeptide consisting of residues 151-165 of hTREM2 (SEQ ID NO:97); (b) does not bind to soluble TREM2 (sTREM2); and (c) has a K by surface plasmon resonance (SPR) of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or between 100 and 500 pM, or between 100 and 200 pM at 37° C. DIn further embodiments, the antibody may also (d) specifically bind to an epitope spanning the H157-S158 cleavage site of TREM2; (e) specifically bind to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94); and (f) exhibit reduced binding affinity to TREM2 stalk domain polypeptides individually comprising the D152A, H157A, and I159A substitutions as compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). Thus, in some examples, the antibody (a) does not bind to soluble TREM 2 and (b) specifically binds to the stalk domain of TREM 2. In some examples, the antibody (a) does not bind soluble TREM2, (b) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site of TREM2, and / or (c) specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with greater affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94). In some examples, (a) the antibody does not bind soluble TREM2, and (b) exhibits reduced binding affinity to TREM2 stalk domain polypeptides individually comprising the D152A, H157A and I159A substitutions compared to wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry). In further cases, the antibody (c) has a K by surface plasmon resonance (SPR) at 37°C of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or a K by surface plasmon resonance (SPR) at 37°C of between 100 and 500 pM or between 100 and 200 pM. D It specifically binds to human and cynomolgus monkey TREM2.

[0200] In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2 and has a K of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or between 100 and 500 pM, or between 100 and 200 pM, by surface plasmon resonance (SPR) at 37°C for human and cynomolgus TREM2. D In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In any of these cases, the antibody may further have one or more of the additional properties (a)-(i) listed below.

[0201] In some examples, the antibody also has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay, such as a BV ELISA assay (one example is an assay performed as described in Example 10 herein). In some examples, such antibodies may act as TREM 2 agonists and may have one or more of the properties (a)-(i) listed below.

[0202] Specifically, in some embodiments, the antibody may also have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following characteristics: (a) induce luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2; (b) induce luciferase reporter activity in plasma. (c) reduce the levels of sTREM2 in vivo; (d) inhibit sTREM2 shedding and / or induce internalization of human TREM2 in Jurkat-NFAT luciferase reporter cells; (d) induce tyrosine phosphorylation in human MDM cells expressing human TREM2; (e) induce SYK phosphorylation (p-SYK) in human MDM cells expressing human TREM2; (f) enhance survival of human iPSC-derived microglia; (g) inhibit sTREM2 shedding in human iPSC-derived microglia; (h) activate TREM2 signaling in human iPSC-derived microglia; and (i) increase total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia.

[0203] h3.10C2.v1 mutant antibody In some embodiments, an antibody herein may comprise the heavy and light chain CDRs of h3.10C2.v1, and the heavy chain framework region of the h3.10C2.v1 antibody, as described above and shown in Figures 9A-9B, but with additional modifications in the antibody light chain, e.g., as shown in Figure 14 A. For example, in some embodiments, certain light chain modifications may be made to the framework regions to improve expression yields, etc., as described in the Examples below.

[0204] In some embodiments, the antibody comprises one of the following sets of VH and VL: (a) SEQ ID NOs: 146 and 145 (antibody h3.10C2.v1 Q100P); (b) SEQ ID NOs: 148 and 147 (antibody h3.10C2.v1I58V / Q100P); or (c) SEQ ID NOs: 150 and 149 (antibody h3.10C2.v1 Q100P / V104L). In some embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0205] In any of the embodiments herein, the antibody may be an Fv, a single chain Fv (scFv), a Fab, a Fab', or a (Fab') 2 In other embodiments, the antibody may be an antibody fragment such as. In other embodiments, the antibody may be a whole antibody (i.e., including a heavy chain constant region and a light chain constant region). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgG1 Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P / M252Y / S254T / T256E Fc region, a human IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. In the case of a murine IgG antibody, the antibody may be an mIgG1 or mIgG2 or mIgG2 LALAPG antibody. The antibody may, in some instances, include a full-length heavy chain and / or a full-length light chain. In some instances, the antibody may lack the C-terminal Lys or C-terminal Lys and Gly residues of the heavy chain constant region. In other cases, the antibody contains one or both of these C-terminal residues. In some instances, the antibody may be bispecific or multispecific. In some instances, the antibody may be conjugated directly or via a linker to another molecule, such as a label or a drug. In some instances, the antibody has intact effector function. In some instances, the antibody has an Fc region with reduced effector function. In other cases, the antibody has an effector-less Fc region. For example, in some instances, the antibody comprises a hIgG1 Fc region with an N297G substitution.

[0206] In some embodiments, the antibody may have certain properties, which are described in more detail below. For example, in some aspects, the antibody has one or more, two or more, three or more, four or more, five or more, or all of the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); and (c) has a K by surface plasmon resonance (SPR) of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM at 37° C., or between 100 and 500 pM, or between 100 and 200 pM. DIn further embodiments, the antibody may also (d) specifically bind to an epitope spanning the H157-S158 cleavage site of TREM2; (e) specifically bind to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94); and (f) exhibit reduced binding affinity to TREM2 stalk domain polypeptides individually comprising the D152A, H157A, and I159A substitutions as compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). Thus, in some examples, the antibody (a) does not bind to soluble TREM 2 and (b) specifically binds to the stalk domain of TREM 2. In some examples, the antibody (a) does not bind soluble TREM2, (b) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site of TREM2, and / or (c) specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with greater affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94). In some examples, (a) the antibody does not bind soluble TREM2, and (b) exhibits reduced binding affinity to TREM2 stalk domain polypeptides individually comprising the D152A, H157A and I159A substitutions compared to wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry). In some of these examples, the antibody also has (c) a K by surface plasmon resonance (SPR) at 37° C. of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or of between 100 and 500 pM, or between 100 and 200 pM. D It specifically binds to human and cynomolgus monkey TREM2.

[0207] In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2 and has a K of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or between 100 and 500 pM, or between 100 and 200 pM, for human and cynomolgus TREM2 by surface plasmon resonance (SPR) at 37°C. D In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2.

[0208] In some examples, the antibody also has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (performed as described in Example 10 herein). In some examples, such antibodies may act as TREM 2 agonists and may have one or more of the properties (a)-(i) listed below.

[0209] Specifically, in some embodiments, the antibody may also have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following additional characteristics: (a) induce luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2; (b) induce luciferase reporter activity in plasma. (c) reduces sTREM2 levels in vivo; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhances survival of human iPSC-derived microglia in the absence of IL34 and CSF1; (g) inhibits sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and (i) increases total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia.

[0210] In some embodiments, the antibody comprises one of the following sets of VH and VL: (a) SEQ ID NOs: 146 and 145 (antibody h3.10C2.v1 Q100P); (b) SEQ ID NOs: 148 and 147 (antibody h3.10C2.v1I58V / Q100P); and (c) SEQ ID NOs: 150 and 149 (antibody h3.10C2.v1 Q100P / V104L).

[0211] Antibody hPara.09.v2 In some embodiments, the antibody comprises the heavy and light chain CDRs of antibody Para.09.v2, where the heavy chain CDRs are based on the Para.09 heavy chain and the light chain CDRs are based on 3.27H7 Fab. In some embodiments, the antibody herein comprises the heavy chain CDRs of SEQ ID NOs: 11, 12 and 13 (Kabat) or alternatively 19, 20 and 13 (Chothia), and / or the light chain CDRs of SEQ ID NOs: 14, 15 and 16. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13 (Kabat), or 19, 20, and 13 (Chothia), and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a heavy chain variable region comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13, or 19, 20, and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a light chain variable region comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13, or 19, 20, and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a heavy chain variable region comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 17, and a light chain variable region comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 18.In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13, alternatively 19, 20, and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13, alternatively 19, 20, and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13, alternatively 19, 20, and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, but with up to five, e.g., 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions in the heavy chain framework regions of SEQ ID NO: 17. In some embodiments, the antibody comprises the heavy chain CDRs of SEQ ID NOs: 11, 12, and 13, or the light chain CDRs of SEQ ID NOs: 19, 20, and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15, and 16, and further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18 but with up to five amino acid substitutions, insertions or deletions, for example 1, 2, 3, 4 or 5 amino acid substitutions, insertions or deletions, in the light chain framework regions of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18 but with up to five amino acid substitutions, insertions or deletions, for example 1, 2, 3, 4 or 5 amino acid substitutions, insertions or deletions, in the framework regions of SEQ ID NO: 17 and / or SEQ ID NO: 18.

[0212] In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0213] In any of the embodiments herein, the antibody may be an Fv, a single chain Fv (scFv), a Fab, a Fab', or a (Fab') 2In other embodiments, the antibody may be an antibody fragment such as. In other embodiments, the antibody may be a whole antibody (i.e., including a heavy chain constant region and a light chain constant region). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgG1 Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P / M252Y / S254T / T256E Fc region, a human IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. In the case of a murine IgG antibody, the antibody may be an mIgG1 or mIgG2 or mIgG2 LALAPG antibody. The antibody may, in some instances, include a full-length heavy chain and / or a full-length light chain. In some instances, the antibody may lack the C-terminal Lys or C-terminal Lys and Gly residues of the heavy chain constant region. In other cases, the antibody contains one or both of these C-terminal residues. In some instances, the antibody may be bispecific or multispecific. In some instances, the antibody may be conjugated directly or via a linker to another molecule, such as a label or a drug. In some instances, the antibody has intact effector function. In some instances, the antibody has an Fc region with reduced effector function. In other cases, the antibody has an Fc region that is effectorless. For example, in some instances, the antibody comprises a hIgG1 Fc region with an N297G substitution.

[0214] In some embodiments, the antibody comprises a heavy chain that comprises or consists of the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises a light chain that comprises or consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain that comprises or consists of the amino acid sequence of SEQ ID NO: 144 and a light chain that comprises or consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain that comprises or consists of the amino acid sequence of SEQ ID NO: 144 but lacking the C-terminal lysine of SEQ ID NO: 144 or lacking the C-terminal glycine and lysine of SEQ ID NO: 144 and a light chain that comprises or consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain that comprises an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises a light chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 176.In some embodiments, the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, optionally without a C-terminal lysine or glycine-lysine, and a light chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, optionally excluding the C-terminal lysine or glycine-lysine, and a light chain comprising an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 144, and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 176.In any of the above cases allowing for sequence variation in SEQ ID NO: 144 and / or SEQ ID NO: 176, in some embodiments such sequence variation is limited to the antibody framework and / or constant regions, such that the antibody also comprises the heavy chain CDRs of SEQ ID NOs: 11, 12 and 13, or alternatively 19, 20 and 13, and / or the light chain CDRs of SEQ ID NOs: 14, 15 and 16. In other embodiments, such sequence variation in SEQ ID NO: 144 and / or SEQ ID NO: 176 is limited to the antibody constant regions, such that the antibody also comprises SEQ ID NOs: 17 and / or 18.

[0215] In some embodiments, the antibodies may have particular properties, which are described in more detail below.

[0216] For example, in some embodiments, the antibody has one or more, two or more, three or more, four or more, five or more, or all of the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) has a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. DIn further embodiments, the antibody may also (d) specifically bind to an epitope spanning the H157-S158 cleavage site of TREM2; (e) specifically bind to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94); and (f) exhibit reduced binding affinity to TREM2 stalk domain polypeptides comprising the D152A, H157A, and I159A substitutions, individually, as compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). Thus, by way of example, in some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind to soluble TREM2. In some such examples, the antibody also has a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. D In some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and / or specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94), and does not bind to soluble TREM2. In some such examples, the antibody also has a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., 10-50 pM or 10-25 pM. DIn some embodiments, the antibody specifically binds to the stalk domain of TREM2 and does not bind to soluble TREM2. In some such examples, the antibody also has a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. D In some embodiments, the antibody specifically binds to human and cynomolgus TREM2 at 37° C. and has a K by surface plasmon resonance (SPR) of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., 10-50 pM or 10-25 pM. D In some examples, the antibodies specifically bind to human and cynomolgus monkey TREM2 at 10 pM, 50 pM, 10 pM, 7 pM, 5 pM, 4 pM, 3 pM, or 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. In some examples, the antibodies do not bind to soluble TREM2 and exhibit reduced binding affinity to TREM2 stalk domain polypeptides that individually contain the D152A, H157A, and I159A substitutions, as compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). In some such examples, the antibodies also have a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. D The antibody specifically binds to human and cynomolgus TREM2 at 100 ng / mL. In some instances, the antibody has intact effector function. In some instances, the antibody has an Fc region with reduced effector function. In other cases, the antibody has an Fc region that is effector-less. For example, in some instances, the antibody comprises a hIgG1 Fc region with an N297G substitution.

[0217] In some examples, the antibody also has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (performed as described in Example 10 herein). In some examples, the antibody has a lower K for human TREM2 than an antibody comprising the heavy and light chain CDRs of 3.10C2. D may have:

[0218] In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2; does not bind soluble TREM2; and has a K by surface plasmon resonance (SPR) of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., 10-50 pM or 10-25 pM at 37°C. D The antibodies are TREM2 agonists that specifically bind to human and cynomolgus TREM2 at 100 ng / mL. In some instances, the antibodies have intact effector function. In some instances, the antibodies have an Fc region with reduced effector function. In other cases, the antibodies have an Fc region that is effector-less. For example, in some instances, the antibodies comprise a hIgG1 Fc region with an N297G substitution.

[0219] In any of these cases, the antibody may further have one or more of the additional properties (a)-(i) listed below.

[0220] Specifically, in some embodiments, the antibody may also have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following characteristics: (a) induce luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2; (b) induce luciferase reporter activity in plasma. (c) reduce levels of sTREM2 in vivo; (d) induce tyrosine phosphorylation in human MDM cells; (e) induce SYK phosphorylation in human MDM cells; (f) enhance survival of human iPSC-derived microglia in the absence of IL34 and CSF1; (g) inhibit sTREM2 shedding in human iPSC-derived microglia; (h) induce SYK phosphorylation in human iPSC-derived microglia; and (i) increase total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia. In some embodiments, the antibody may have higher luciferase reporter activity (e.g., lower EC50 in a Jurkat-NFAT reporter assay as described in the Examples herein) than an antibody comprising the heavy and light chain CDRs of 3.10C2. In some embodiments, the antibodies may exhibit greater inhibition of sTREM2 shedding in Jurkat-NFAT reporter cells (eg, in the assay described in the Examples) compared to an antibody comprising the heavy and light chain CDRs of 3.10C2.

[0221] hPara.09.v2 mutant antibody In some embodiments, the antibodies herein may comprise the hPara.09.v2 heavy and light chain CDRs and heavy chain framework regions of the hPara.09.v2 antibody as described above and shown in Figures 12A-B, but with additional modifications in the antibody light chain, e.g., as shown in Figure 13 A. For example, in some embodiments, certain light chain modifications may be made to the framework regions to improve expression yields, as described in the Examples below.

[0222] In some such embodiments, the antibody comprises one of the following sets of VH and VL: (a) SEQ ID NOs: 133 and 132 (antibody hPara09.v2 Q100P); (b) SEQ ID NOs: 135 and 134 (antibody hPara09.v2I58V / Q100P); (c) SEQ ID NOs: 137 and 136 (antibody hPara09.v2 Q100P / V104L). In some embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region. In some embodiments, the antibody light chain comprises or consists of the amino acid sequence of SEQ ID NO: 176.

[0223] In any of the embodiments herein, the antibody may be an Fv, a single chain Fv (scFv), a Fab, a Fab', or a (Fab') 2In other embodiments, the antibody may be an antibody fragment such as. In other embodiments, the antibody may be a whole antibody (i.e., including a heavy chain constant region and a light chain constant region). In other embodiments, the antibody may be an IgG, IgA, or IgM antibody. In some embodiments, the antibody may have a wild-type human IgG1 Fc region or a wild-type human IgG4 Fc region, a human IgG4 S228P Fc region, a human IgG4 S228P / M252Y / S254T / T256E Fc region, a human IgG1 N297G Fc region, a human IgG1 LALAPG (L234A / L235A / P329G) Fc region, or a human IgG1 N297G / M428L / N434S Fc region. In the case of a murine IgG antibody, the antibody may be an mIgG1 or mIgG2 or mIgG2 LALAPG antibody. The antibody may, in some instances, include a full-length heavy chain and / or a full-length light chain. In some instances, the antibody may lack the C-terminal Lys or C-terminal Lys and Gly residues of the heavy chain constant region. In other cases, the antibody contains one or both of these C-terminal residues. In some instances, the antibody may be bispecific or multispecific. In some instances, the antibody may be conjugated directly or via a linker to another molecule, such as a label or a drug. In some instances, the antibody has intact effector function. In some instances, the antibody has an Fc region with reduced effector function. In other cases, the antibody has an Fc region that is effectorless. For example, in some instances, the antibody comprises a hIgG1 Fc region with an N297G substitution.

[0224] In some embodiments, the antibody may have certain properties, which are described in more detail below. For example, in some aspects, the antibody has one or more, two or more, three or more, four or more, five or more, or all of the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) has a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM or between 10 and 25 pM. DIn further embodiments, the antibody may also (d) specifically bind to an epitope spanning the H157-S158 cleavage site of TREM2; (e) specifically bind to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94); and (f) exhibit reduced binding affinity to TREM2 stalk domain polypeptides comprising the D152A, H157A, and I159A substitutions, individually, as compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). Thus, by way of example, in some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind to soluble TREM2. In some such examples, the antibody also has a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. D In some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and / or specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO: 96) or 151-165 (SEQ ID NO: 97) with higher affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO: 92) and / or 159-175 (SEQ ID NO: 94), and does not bind to soluble TREM2. In some such examples, the antibody also has a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., 10-50 pM or 10-25 pM. DIn some embodiments, the antibody specifically binds to the stalk domain of TREM2 and does not bind to soluble TREM2. In some such examples, the antibody also has a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. D In some embodiments, the antibody specifically binds to human and cynomolgus TREM2 at 37° C. and has a K by surface plasmon resonance (SPR) of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., 10-50 pM or 10-25 pM. D In some examples, the antibodies specifically bind to human and cynomolgus monkey TREM2 at 10 pM, 50 pM, 10 pM, 7 pM, 5 pM, 4 pM, 3 pM, or 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. In some examples, the antibodies do not bind to soluble TREM2 and exhibit reduced binding affinity to TREM2 stalk domain polypeptides that individually contain the D152A, H157A, and I159A substitutions, as compared to wild-type TREM2 stalk domain polypeptides (e.g., as measured by bilayer interferometry). In some such examples, the antibodies also have a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., between 10 and 50 pM or between 10 and 25 pM. D The antibody specifically binds to human and cynomolgus TREM2 at 100 ng / mL. In some instances, the antibody has intact effector function. In some instances, the antibody has an Fc region with reduced effector function. In other cases, the antibody has an Fc region that is effector-less. For example, in some instances, the antibody comprises a hIgG1 Fc region with an N297G substitution.

[0225] In some examples, the antibody also has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (performed as described in Example 10 herein). In some examples, the antibody has a lower K for human TREM2 than an antibody comprising the heavy and light chain CDRs of 3.10C2. DIn some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In some embodiments, the antibody is a TREM2 agonist that specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2 and does not bind soluble TREM2. In some embodiments, the antibody specifically binds to an epitope spanning the H157-S158 cleavage site of TREM2; does not bind soluble TREM2; and has a K by surface plasmon resonance (SPR) of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, e.g., 10-50 pM or 10-25 pM at 37°C. D The antibodies are TREM2 agonists that specifically bind to human and cynomolgus TREM2 at 100 ng / mL. In some instances, the antibodies have intact effector function. In some instances, the antibodies have an Fc region with reduced effector function. In other cases, the antibodies have an Fc region that is effector-less. For example, in some instances, the antibodies comprise a hIgG1 Fc region with an N297G substitution.

[0226] In any of these cases, the antibody may further have one or more of the additional properties (a)-(i) listed below.

[0227] In some embodiments, the antibody may also have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following characteristics: (a) induces luciferase reporter activity in Jurkat-NFAT reporter cells expressing human TREM2; (b) induces luciferase reporter activity in plasma. (c) reduces sTREM2 levels in vivo; (d) inhibits sTREM2 human TREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhances survival of human iPSC-derived microglia in the absence of IL34 and CSF1; (g) inhibits sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and (i) increases total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia. In some embodiments, the antibody may have higher luciferase reporter activity (e.g., lower EC50 in a Jurkat-NFAT reporter assay as described in the Examples herein) than an antibody that comprises the heavy and light chain CDRs of 3.10C2. In some embodiments, the antibody may show greater inhibition of sTREM2 shedding in Jurkat-NFAT reporter cells (e.g., in the assay described in the Examples) compared to an antibody that comprises the heavy and light chain CDRs of 3.10C2.

[0228] In some embodiments, the antibody comprises one of the following sets of VH and VL: (a) SEQ ID NOs: 133 and 132 (antibody hPara09.v2 Q100P); (b) SEQ ID NOs: 135 and 134 (antibody hPara09.v2I58V / Q100P); (c) SEQ ID NOs: 137 and 136 (antibody hPara09.v2 Q100P / V104L).

[0229] Nucleic acid encoding the antibody Nucleic acid molecules comprising polynucleotides encoding one or more chains of an anti-TREM2 antibody are also provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an anti-TREM2 antibody. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an anti-TREM2 antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0230] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides, either from one nucleic acid molecule, or from two separate nucleic acid molecules, in some embodiments, for example when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0231] In some embodiments, a polynucleotide encoding a heavy or light chain of an anti-TREM 2 antibody comprises a nucleotide sequence that, when translated, encodes a leader sequence located at the N-terminus of the heavy or light chain. As noted above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0232] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0233] Vectors, host cells and methods of production Vectors are provided that include a polynucleotide encoding an anti-TREM2 heavy chain and / or an anti-TREM2 light chain. Vectors are also provided that include a polynucleotide encoding an anti-TREM2 heavy chain and / or an anti-TREM2 light chain. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector includes a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, such as when the antibody is an scFv.

[0234] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into the host cell in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a molar or mass ratio of 5:1 to 1:5 of the first vector and the second vector is transfected into the host cell. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0235] In some embodiments, a vector is selected that is optimized for expression of a polypeptide in CHO or CHO-derived cells or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0236] In some embodiments, vectors are selected for in vivo expression of anti-TREM2 heavy chains and / or anti-TREM2 light chains in animals, including humans. In some such embodiments, expression of the polypeptide is under the control of a promoter that functions in a tissue-specific manner. For example, liver-specific promoters are described, for example, in PCT Publication No. WO 2006 / 076288.

[0237] For recombinant production of an anti-TREM2 antibody, nucleic acid encoding the antibody, such as those described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0238] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies of the invention can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0239] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are suitable as cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0240] Also suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0241] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標) Please refer to the following reference document (which describes the technique).

[0242] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT060562); see, e.g., Mather et al., Annals Other useful mammalian host cell lines include DHFR cells, TRI cells, described in NYAcad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. - Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0243] Anti-TREM2 antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to TREM 2 ECD and antibody constant regions. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to bind the constant region and purify anti-TREM2 antibodies. Hydrophobic interaction chromatography, such as butyl or phenyl columns, can also be suitable for purifying some polypeptides. Many methods of purifying polypeptides are known in the art. In some embodiments, anti-TREM2 antibodies are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0244] Methods of Pharmaceutical Use The present disclosure also includes methods of using the anti-TREM2 antibodies herein, for example, in pharmaceutical treatment. For example, the present disclosure includes methods of treating conditions associated with TREM2 loss of function in a subject. The present disclosure also includes methods of reducing the level of sTREM2 in a subject.

[0245] In some instances, the condition is a neuroinflammatory or neurodegenerative disease. Examples include, for example, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behçet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some instances, the condition is Alzheimer's disease. In some instances, the condition is MS.

[0246] For example, pathological hallmarks of Alzheimer's disease (AD) include extracellular deposits of beta-amyloid peptides that form amyloid plaques, and intracellular deposits of aggregated hyperphosphorylated tau called neurofibrillary tangles. These pathologies are followed by increased brain inflammation involving activated astrocytes and microglia, and neurodegeneration. Familial forms of AD can be caused by mutations in the presenilin 1 / 2 and amyloid precursor protein genes. Transgenic mice expressing these human mutations as well as mutations in the tau protein show similar age-dependent increases in Abeta pathology, hyperphosphorylated tau, and neurodegeneration. Mutations that cause loss of TREM2 function result in irregular microglial compaction of plaques, elevated neuritic dystrophy surrounding the plaques, elevated Abeta-induced tau pathology, and neurodegeneration. Without wishing to be bound by theory, enhancing TREM 2 activity may enhance microglial activity and facilitate the removal of toxic amyloid-beta peptides by phagocytosis or compaction into less toxic amyloid plaques, thus treating AD.

[0247] Multiple sclerosis (MS) is a disease characterized by autoimmune-related demyelination in the CNS. In patients, the disease presents with symptomatic episodes such as ataxia, limb weakness, and optic neuritis, among other neurological effects. Current treatments include immunosuppressants, which may have limited efficacy, but no treatments effectively prevent or reverse the disease. Remyelination therapies have been proposed as an approach to treat and, in some cases, reverse MS. Compounds and therapies that promote oligodendrocyte differentiation from progenitor cells (OPCs) to mature myelinating oligodendrocytes are considered possible routes to remyelination therapy, as well as therapies that modify glial cells. Specifically, microglia are believed to have a role in removing myelin debris and promoting the formation of new myelin. Loss of function of TREM2 results in hypomyelinating leukodystrophy, and TREM2 knockout mice have significant impairments in remyelination and recovery in animal models of MS. Without wishing to be bound by theory, activation of the TREM2 pathway may accelerate remyelination and therefore treat MS.

[0248] In various embodiments, the anti-TREM2 antibody may be administered in vivo by various routes, including but not limited to oral, intravenous, subcutaneous, parenteral, intranasal, intramuscular, intradermal, topical, transdermal, and intrathecal, or by other methods of implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gas form, including but not limited to tablets, capsules, powders, granules, ointments, liquids, suppositories, enemas, injections, inhalants, and aerosols. The nucleic acid molecule encoding the anti-TREM2 antibody may be administered directly or in a vector, such as a viral vector. The appropriate formulation and administration route may be selected according to the intended use.

[0249] In various embodiments, compositions comprising anti-TREM2 antibodies can be administered in a wide variety of pharma- ceutically acceptable carriers (e.g., as described in Gennaro, Remington: The Science and Practice of Pharmacology, 1999). ywith Facts and Comparisons:Drugfacts Plus,20th ed.(2003);Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems,7 th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000)). A variety of pharma- ceutically acceptable carriers are available, including vehicles, adjuvants, and diluents. In addition, a variety of pharma-ceutically acceptable auxiliary substances are available, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0250] In various embodiments, compositions comprising anti-TREM2 antibodies may be formulated for injection or infusion by dissolving, suspending, or emulsifying in an aqueous or non-aqueous solvent, such as vegetable or other oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, optionally with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives. In various embodiments, the compositions may be formulated for inhalation, using pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. The compositions may also be formulated in various embodiments, for example, with biodegradable or non-biodegradable polymers into sustained release microcapsules. Non-limiting exemplary biodegradable formulations include polylactic-glycolic acid polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. Certain methods for producing such formulations are described, for example, in EP 1 125 584 A1.

[0251] Pharmaceutical packs and kits are also provided that include one or more containers, each containing one or more doses of an anti-TREM2 antibody. In some embodiments, unit doses are provided that include a predetermined amount of a composition that includes an anti-TREM2 antibody, with or without one or more additional agents. In some embodiments, such unit doses are provided in a single-use pre-filled syringe for injection. In various embodiments, the composition included in the unit dose may include a buffer, such as saline, sucrose, etc.; phosphate, and / or is formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of an appropriate liquid, such as sterile water. In some embodiments, the composition includes one or more substances that inhibit protein aggregation, such as, but not limited to, sucrose and arginine. In some embodiments, the composition of the present invention includes heparin and / or proteoglycan. EXAMPLES

[0252] Example 1: Preparation of rat anti-TREM2 antibody A. Materials and Methods To generate anti-human TREM2 antibodies, Sprague Dawley rats (Charles River, Hollister, CA) or TREM2 knockout mice (Genentech, South San Francisco) were immunized with different antigen formats: human TREM2 extracellular domain (ECD) protein emulsified in complete Freund's or Ribi adjuvant; extracellular vesicles (EVs) expressing human full-length TREM2; or pDNA encoding full-length human TREM2 delivered via gene gun or hydrodynamic tail vein (HTV) injection (Figure 1).

[0253] Lymph nodes and spleens from immunized animals were harvested and B cells were isolated as follows: for rats, class-switched B cells were enriched using a cocktail of biotinylated antibodies (CD4, CD8a, CD11b / c, CD161, HIS48, IgM) from BD Biosciences, followed by magnetic separation using streptavidin beads (Miltenyi Biotec, San Diego, CA). For mice, B cells were enriched using a pan B cell isolation kit (Miltenyi Biotec, San Diego, CA), followed by magnetic depletion of IgM-positive B cells (biotinylated anti-IgM II / 41, BD Biosciences). IgM-depleted B cells were fused with Sp2ab myeloma cells (Abeome, Athens, GA) by electrofusion (Harvard Apparatus, Holliston, MA). After overnight recovery in Clonacell-HY medium C (StemCell Technologies, Canada), cells were selected in HAT (hypoxanthine-aminopterin-thymidine) (Sigma-Aldrich). Fused hybridomas were then harvested and stained with anti-rat IgG-Alexa 488 (Jackson ImmunoResearch) for rat hybridomas or anti-mouse IgG FITC (Bethyl Labs) for mouse hybridomas, and fluorescently conjugated human TREM 2-Alexa Fluor 643 (Novus Biological). Single IgG+ / human TREM2+ hybridoma cells were sorted into 96-well plates using a FACSAriaIII sorter (BD, Franklin Lakes, NJ) and cultured for 7 days. Supernatants were screened by ELISA against human TREM2 antigen, and ELISA-positive clones were scaled up. Purified IgG (Gamma Bind Plus, GE Healthcare, Pittsburgh, PA) from hybridoma supernatants was screened by FACS for cell surface binding using DOX-inducible human TREM2-expressing 293 cells and functional activity.

[0254] B. Results A total of 1,650 hybridoma clones bound specifically to immobilized human TREM2 in an ELISA format (Figure 1). Of these, 947 bound by FACS to 293 cells expressing human TREM2 (Figure 1). The variable regions of the immunoglobulin genes of FACS-positive hybridoma clones were then sequenced.

[0255] A total of 229 unique clones were identified, the majority of which were derived from SD rats immunized with human TREM2 ECD (Figure 1). Variable region sequences of unique FACS-positive anti-TREM2 clones were used to clone as mouse IgG2a by gene synthesis, effectorless mouse IgG2a with LALAPG (L234A / L235A / P329G, Eu numbering system), chimeric human IgG1, chimeric effectorless human IgG1 LALAPG (L234A / L235A / P329G) and chimeric effectorless human IgG1 N297G (GeneWiz). Recombinant IgG was transiently expressed in CHO cells and purified by protein A and size exclusion chromatography. Selected clones were also cloned as chimeric human Fab fragments in CHO cells and purified by anti-CH1 affinity chromatography followed by size exclusion chromatography.

[0256] Example 2: Epitope specificity of anti-TREM2 clones A. Materials and Methods Antibodies were screened for binding to peptides derived from the human TREM2 stalk (TREM2 residues 129-174). Peptides with overlapping sequences ranging from TREM2 residues 129-175 were used to provide coverage of the entire stalk (Figure 2A). Antibody epitopes were mapped to the stalk domain of human TREM2 using enzyme-linked immunosorbent assay (ELISA). Overlapping biotinylated peptides were captured on streptavidin-coated ELISA plates. Binding of antibodies to the peptides was detected using anti-mouse IgG conjugated to horseradish peroxidase.

[0257] B. Results Several antibody groups were found that bind to the stalk peptide fragment of human TREM2, with three rat anti-human antibodies generally binding to residues 151-161 of TREM2: antibodies 3.10C2, 3.18E5, and 3.50G1 in FIG. 2B (binding affinity in nM). In particular, these three antibodies bind peptides of residues 146-169, 146-161, 149-168, or 151-165 of the hTREM2 stalk, and bind at least as well, if not better, than a peptide consisting of residues 129-175. Each of these four peptides encompasses the region of the hTREM2 stalk from residue 151 to residue 161, including the His157-Ser158 cleavage site on the hTREM2 stalk, also shown in FIG. 2A. The affinity of these antibodies for all other peptides tested was significantly reduced compared to their affinity for the 129-175 peptide.

[0258] Example 3: Affinity of rat anti-TREM2 antibodies A. Materials and Methods 46 rat anti-human antibodies were tested for binding affinity to the stalks of hTREM2 and cynomolgus TREM2. Antibody binding affinity to the hTREM2 stalk and cynomolgus TREM2 stalk was measured by surface plasmon resonance (SPR) using a Biacore™ T200 instrument. TREM2 stalk antigen (human or cynomolgus) was immobilized on an anti-human IgG1 Fc Biacore™ chip and anti-TREM2 antibody clones were allowed to bind to immobilized TREM2 at 37° C. Binding affinity K D (nM) was measured and recorded.

[0259] B. Results The binding affinity of 46 tested antibodies to the hTREM2 stalk was measured, and the measured K DValues ​​ranged from 1.13 μM to 0.01 nM. Several antibodies were selected for binding measurements to the cynomolgus monkey TREM2 stalk. From the first group of antibodies tested, five antibodies (3.10C2, 3.50G1, 3.18E5, 3.36F5, and 3.27H7) showed particularly low K D The binding affinities of antibodies 3.10C2, 3.50G1, 3.18E5, 3.36F5 and 3.27H7 to the hTREM2 stalk were 0.24 nM, 0.61 nM, 0.26 nM, 10 nM and 0.22 nM, respectively. The binding affinities of antibodies 3.10C2, 3.50G1, 3.18E5, 3.36F5 and 3.27H7 to the cynomolgus monkey TREM2 stalk were 0.25 nM, 0.57 nM, 0.30 nM, 12 nM and 0.26 nM, respectively. These clones are independent clones in the same B cell clonal lineage, and their sequences of the CDRs and variable regions of 3.10C2, 3.50G1, 3.18E5, 3.36F5 and 3.27H7 are shown in Figures 3A-B. Of those first five antibodies, four had K values ​​below 1 nM D values, specifically 3.10C2, 3.50G1, 3.18E5 and 3.27H7. These four antibodies are hereinafter collectively referred to as the "3.10C2 group" of antibodies.

[0260] Example 4: Deep sequencing of rat antibody clones and identification of clone Para.09 Further studies were conducted with the aim of identifying clones from the deep sequencing dataset of the immunized rat antibody repertoire that have similar binding and activity characteristics to the 3.10C2, 3.50G1, 3.18E5, and 3.27H7 groups of antibodies (collectively "3.10C2 group") and the 3.36F5 antibody clone, but with even higher affinity for TREM2.

[0261] A. Rationale and Introduction In the rodent antibody repertoire, the same V H and V LIt has previously been shown that antibodies specific for antigens that share germline segments tend to share epitope specificity at a high frequency despite CDR H3 diversity (Hsiao et al., mAbs 12:1, 1722541, doi:10.1080 / 19420862.2020.1722541 (2020)). Thus, additional antibodies from immunized rats bearing IGHV6-8 and IGKV2S11 germline segments but different CDR H3 sequences could potentially recognize the same epitope as the 3.10C2 group of antibodies if they were TREM2 specific. Mining for clonally independent antibodies that bind to antigen in a similar manner has been described for anti-HIV-1 gp120 antibodies from infected donors (Zhu et al., Proc. Natl. Acad. Sci. USA 110:E4088-E4097, doi:10.1073 / pnas.1306262110(2013).). However, for the 3.10C2 group of antibodies, phylogenetic techniques similar to those used for HIV-1 antibodies were not available. There were at least three reasons for this: (1) the mutation load of the 3.10C2 group of antibodies was relatively low; (2) the key features of the 3.10C2 group of antibodies for antigen binding were unknown; and (3) clonally independent antibodies of the same class as the 3.10C2 group were not known. Mining cross-lineage cross-donor antibodies by deep sequencing has only been described in the context of highly similar long CDR H3 sequences that provide substantial sequence information for mining specific clonotypes from the repertoire (Zhu et al., Proc. Natl. Acad. Sci. USA 110:E4088-E4097, doi:10.1073 / pnas.1306262110 (2013)). In contrast, in this case, the information available to search the repertoire of clonally independent antibodies in the 3.10C2 group is relatively limited, including very short CDR H3 sequences that provide little specific sequence information for the search. Thus, in the 3.10C2 group, V H and V LOnly germline segment pairing information from 1441 was available for mining, but this information lacked specificity for mining because many clones of different specificities in the repertoire, including those specific for antigens other than TREM2, share the same germline segment pairings.

[0262] Furthermore, clone 3.36F5 has a highly similar V-protein complex to clone 3.10C2, which contains the rat IGHV6-8 germline segment and the Kabat CDR H3 sequence LDY (or TGLDY in the IMGT system). H 3.10C2 clonal group, but has a different light chain with germline segment IGKV2U18 (germline segment described in Goldstein et al., Commun. Biol. 2:304, doi:10.1038 / s42003-019-0551-y (2019)). (See FIG. 3B). Thus, light chains with similar IGKV segments can also replace the 3.10C2 clonal group to mine deep sequencing datasets. Finally, because these clones do not descend from the same B cells, clone-independent VH sequences that share the same specificity as the clones in the 3.10C2 group can be derived from any TREM2-immunized rat expressing the heavy chain IGHV6-8 germline segment and the light chain IGKV2S11, IGKV2U18 or similar germline segment.

[0263] A mining strategy for clones outside of clonal lineages in deep sequencing datasets has been recently described (Richardson et al., mAbs doi:10.1080 / 19420862.2020.1869409(2021)). However, that method, unlike the one described herein, relies on amino acid identity including the CDR H3 and is limited to sequences with the same CDR H3 length (Richardson et al., 2021). The search strategy used here completely ignores the CDR H3 sequence, even allowing for different CDR H3 lengths, contrary to the currently held view that the CDR H3 is the primary specificity determinant and is implicit in the search strategy of Richardson et al., which includes the CDR H3 sequence in the identity calculation for clone selection. Furthermore, the method described herein does not explicitly require a given level of amino acid identity across any set of residues between clones, but rather V H It uses only germline segments and actually seeks to identify clones with widely divergent CDR H3 sequences. Therefore, it is important to consider the V H Mining by germline segments would, in principle, allow a broader search for independent clones with the same epitope specificity as the Richardson et al. method, while avoiding the need for informative sequence characterization as performed by Zhu et al.

[0264] B. Materials and Methods V of three immunized rats H The repertoire was sequenced by paired-end Illumina sequencing using rat-specific primers for cDNA synthesis and PCR amplification. Briefly, total RNA was extracted using bone marrow and spleen tissue from the same pooled set of three rats from which the 3.10C2 group clones were derived by hybridoma using lymph node tissue, and used in an RT-PCR step to identify rat V specific for the sequence encoding the first residue of the framework 1 region. HEntire repertoire V was cloned using germline segment primers and constant region primers specific for rat IgG and IgA isotypes. H Segments were amplified. Amplicons were subjected to paired-end sequencing on an Illumina HiSeq™ instrument. Sequence reads were assembled into full-length VH sequences and analyzed for germline segments and CDR boundaries using Absolve™ as previously described (Goldstein et al., Commun. Biol. 2:304, doi:10.1038 / s42003-019-0551-y (2019)).

[0265] Twenty-nine clones from independent clonal types with IGHV6-8 germline segments and diverse CDR H3 sequences were selected and paired with different light chains of the 3.10C2 clone group for expression of IgG fragments. DNA clones encoding the full-length heavy and light chains of the selected variants were transfected into Expi293 cells at a 1 ml scale, and IgG was purified by protein A chromatography as previously described (Bos et al., Biotechnol. Bioeng. 112:1832-4, doi:10.1002 / bit.25601 (2015); Luan et al., Mabs 10:624-35, doi:10.1080 / 19420862.2018.1445450 (2018)).

[0266] Clones were tested for binding to peptides from the TREM2 stalk in an ELISA format, and the heavy chain CDR3 of each clone is shown in Figure 4 (left column).

[0267] C. Results Of the 29 selected clones, one clone, designated Para.09, bound robustly to a peptide of the human TREM2 stalk (TREM2 residues 129-175) and to a TREM2 peptide fragment of residues 149 to 158 in ELISA when paired with the light chain of the 3.10C2 group, similar to the 3.10C2 control (Figure 4). The CDR H3 sequence of clone Para.09 is one residue longer than the CDR H3 of the 3.10C2 group and has a different IGHJ germline segment (IGHJ3 instead of IGHJ2), indicating that Para.09 is not a clonal variant of the 3.10C2 group, but rather a clonal lineage generated independently in the rat repertoire. Para.09 also has two amino acid differences in CDR H3 compared to the 3.10C2 group (Figure 3B), ignoring the insertions (TG-LDY vs. TDILEY for 3.10C2 and Para.09, respectively, insertions are underlined and differences are highlighted in bold and italics). That is, only half of the residues in CDR H3 of Para.09 (3 of 6 in the IMGT system and 2 of 4 in the Kabat system) are the same in similar positions in clone 3.10C2. Highlighting the independent clonal origin of Para.09 compared to the 3.10C2 class of clones, Para.09 has rat IGHJ3 germline segments in the heavy chain framework 4 region, whereas the 3.10C2 group of clones has rat IGHJ2 germline segments.

[0268] Furthermore, CDR H3 sequences alone do not predict TREM2 binders. This is exemplified by clone Para.10, which does not bind to the TREM2 stalk or peptide fragment 149-158, an epitope bound by antibodies of the 3.10C2 group when paired to either the 3.10C2 group or germline light chain (Figures 3A-B). This clone has a CDR H3 that is one residue longer than the CDR H3 of the 3.10C2 group (and the same length as the Para.09 CDR H3), but does not differ from them at other positions (TGL-DY vs. TGLGDY for 3.10C2 and Para.10, respectively, insertions are underlined). That is, there are no obvious sequence features in the Para.09 CDR H3 region that make this clone an obvious anti-TREM2 candidate. Finally, the frequency of 3.10C2 group and Para.09 lineage sequences in the deep sequencing dataset explains the unlikely discovery of Para.09 by hybridomas, but not the 147 unique 3.10C2 group Vs in the deep sequencing dataset. H There were 8,283 read counts for the sequence, with 4 unique Para.09 related Vs. H There were only 47 sequence reads for the clone. Given that the count frequency in the 3.10C2 group was such that only 4 of 174 hybridomas were in this group, it is estimated that the likelihood of identifying the low frequency Para.09 clone by hybridoma or similar techniques is very low.

[0269] Example 5: Binding affinity of Para.09 Fab fragment A. Materials and Methods Antibody fragments containing the heavy chain of clone Para.09 paired with light chain antibodies 3.10C2, 3.18E5, 3.27H7 and 3.50G1 ("3.10C2 group") were tested for affinity to human and cynomolgus TREM2 by SPR on a Biacore™ T200 instrument. Briefly, clone variants were expressed as Fab fragments and purified by anti-CH1 affinity chromatography followed by size exclusion chromatography. TREM2-Fc antigen was immobilized on an anti-human IgG1 Fc Biacore™ chip and soluble Para.09 variants and 3.10C2 clone Fab fragments were bound to immobilized TREM2 at 37°C.

[0270] B. Results All Para.09 clone variants showed significantly higher affinity than Fabs containing the heavy and light chain variable regions of antibody 3.10C2, regardless of the light chain used and despite the high similarity of CDRs H1 and H2 of Para.09 compared to 3.10C2 (Table 1). This indicates that CDR H3 is important for higher binding affinity. Higher affinity is associated with a higher association rate (k a ) and a lower dissociation rate (k d The results are shown in Table 1 below. [Table 2]

[0271] Example 6: Mapping residues in the TREM2 stalk that interact with antibodies A. Materials and Methods On the other hand, based on how the antibody was generated, the epitope of Para.09 was predicted to be in the same region as the antibodies of the 3.10C2 family, and more detailed studies were performed to evaluate the importance of individual residues for binding. Epitope mapping of clone Para.09, antibody 3.10C2, and other antibodies of the 3.10C2 family, such as 3.50G1, was performed by testing binding to synthetic TREM2 peptides with a single alanine point mutation at residues 149 to 161 or an alanine to glycine mutation at position 153 (Figure 5). Binding to the synthetic peptides was tested by Biolayer Interferometry (BLI) on an Octet Red instrument, immobilization of the synthetic peptides on a streptavidin-coated sensor chip, and binding of the peptides to free soluble Fab fragments to avoid avidity effects that could obscure effects on binding. This was necessary because the effect of single mutations on antibody binding was not evident due to their high affinity, especially Para.09, which allows robust binding to mutant peptides even with reduced affinity for the mutant. Therefore, reduced binding or faster dissociation of the Fab fragment was used as a readout of the effect of the mutations on antibody binding.

[0272] B. Results Overlapping peptide mapping provided the approximate span of the epitopes of 3.10C2, 3.18E5, and 3.50G1, spanning the interval from residue Glu-151 to residues Ile-159 to Arg-161 (Figures 2A-B). Further mapping of 3.10C2, 3.50G1, and Para.09 by mutational scanning as described in this Example indicated that residues with side chains important for binding were located within the range Asp-152 to Ile-159, consistent with binding to the overlapping peptides. As shown in Figure 5, antibodies 3.10C2 and 3.50G1, both in the 3.10C2 group, had similar binding profiles, with the synthetic peptides containing alanine mutations at positions 152, 154, 157, 158, and 159 spanning the TREM2 cleavage site, each of which affected binding of the Fab fragment to the peptide. Specifically, critical TREM2 residue side chains for binding include at least Asp-152, His-157, and Ile-159 for the 3.10C2, 3.50G1, and Para.09 antibodies, respectively. Examination of the crystal structures of huPara.09.v2 Q100P and hu3.10C2.v1 Q100P in complex with a peptide containing residues 148-165 of TREM2 (resolution = 2 Å) further demonstrated that the primary contacts between TREM2 and the two antibodies are mediated by residues Asp-152, His-154, Val-155, Glu-156, His-157, and Ile-159 (residues identified by scanning mutagenesis are underlined). Antibody 3.50G1 was also affected by the E156A and S160A mutations, whereas 3.10C2 was not. Another antibody clone, 3.47B1, according to the present disclosure, had a more limited set of mutations affecting binding to TREM2, with only H157A and I159A having a detectable effect on binding (Figure 5). Antibody Para.09, which comprises the Para.09 heavy chain variable region paired with the light chain variable region of antibody 3.27H7 (and thus also referred to herein as Para.09-L27H7), had a similar pattern to 3.10C2, except that no effect on binding was observed for mutations H154A and S158A (Figure 5).Further examination of the crystal structures shows that 3.10C2 and Para.09 are very similar in complex structure, with differences in the angle and number of contacts and the depth of the hydrophobic pockets likely resulting from the different CDR H3 lengths of both antibodies which may explain the observed differences.

[0273] Several previously described anti-TREM2 antibodies were also evaluated for binding to the stalk domain of TREM2 using mutation scanning. For antibodies 14D3 and 14D8 (e.g., as described in WO2018015573), the A153G mutation affected binding, whereas the D152A and S158A mutations did not (Figure 5), indicating differences in epitope specificity between the 3.10C2 group and Para.09 compared to the 14D3 / 14D8 antibodies. The previously described 9F5 and AL2p-31 antibodies (described in WO2017062672 and / or WO201928292), the latter of which is a humanized and affinity matured version of 9F5, had a different pattern of sensitivity to mutations compared to the 3.10C2, 3.50G1 and Para.09-L27H7 antibodies, which extend from residue 150 to residues 156 or 157 just before the cleavage site (Figure 5). In contrast to 3.10C2, 3.50G1 and Para.09-L27H7, an alanine mutation from residue 158 to 161 did not affect binding of the 9F5 and AL2p-31 Fabs. Thus, these previously described antibodies appear distinct in that they do not interact with residues on either side of the cleavage site, but rather only with residues found in sTREM2 upon cleavage.

[0274] Example 7: Humanized antibody 3.10C2 A. Materials and Methods Antibody 3.10C2 was humanized in the VK2 and VH3 frameworks by CDR grafting. Briefly, the CDR regions of the rat antibody were grafted into the light chain IGKV2-28*01 and heavy chain IGKV3-73*01 frameworks, which are the closest human germlines of these antibodies (Figures 6A-6B). The CDR regions included Kabat positions 24-34 (CDR L1), 50-56 (CDR L2) and 89-97 (CDR L3) for the light chain and Kabat positions 26-35 (CDR H1), 50-65 (CDR H2) and 93-102 (CDR H3) for the heavy chain. As is commonly done in CDR-graft humanization (see, e.g., U.S. Patent No. 8,426,147), rat framework residues known as "Vernier" positions and domain interfaces that differ between rat antibodies and human germline were added to the CDR graft to rescue binding to the antigen. These included, in this case, light chain residues 2, 4, and 68 and heavy chain framework residues 24, 48, 49, 76, and 78 (Figures 6A-B). Mutants with different sets of human and mouse framework residues were expressed as human IgG1 in Expi293 cells and purified by Protein A chromatography.

[0275] The humanized heavy chain variants were affinity tested by combining them with the 3.10C2-L1 light chain. These variants were expressed as Fab fragments, purified by Protein A chromatography, and tested for affinity to TREM2-Fc coated onto a Protein A chip using a Biacore™ T200 instrument.

[0276] B. Results Purification yields of humanized variants with the 3.10C2-L1 light chain variant, which has all the rat framework Bernier residues, were consistently low (Figure 7A). Another light chain variant, 3.10C2-L5, which has rat framework residue Thr-2 and human residues Met-4 and Gly-68, also had consistently low yields in Expi293 cells (Figure 7A).

[0277] Two humanized variants, the 3.10C2-H3 / 3.10C2-L1 and 3.10C2-H5 / 3.10C2-L1 Fab fragments, had comparable dissociation rates to the rat 3.10C2 Fab fragment, but significantly reduced association rates (Table 2). The other variants had significantly lower dissociation rates (Table 2). [Table 3]

[0278] The low expression of the humanized variants, coupled with the unexpectedly low association rates of the Fab variants tested in solution, suggested expression and product quality deficiencies. Substitution of Pro-100 (rat residue) with Gln-100 (human residue) in the light chains L9 and L10 (corresponding to light chains 1 and 5 with Pro-100, respectively), a framework position 4 not typically included in humanization, restored IgG expression in the context of several humanized heavy chain variants (Figure 7A), but retained the elution peak corresponding to the heavy chain (HH) dimer.

[0279] The results for the light chain Pro-100 mutant suggested that the poor expression was due to a defect in the light chain. We confirmed this by expressing hybrid Fab fragments composed of the 3.10C2-H1 heavy chain (a humanized variant with framework positions 24, 48, 49, 76 and 78 as the 3.10C2 rat residues) combined with either the humanized 3.10C2-L1 or the rat 3.10C2 light chain. The 3.10C2-H1 / 3.10C2-L1 humanized variant was poorly expressed, whereas the hybrid Fab fragment with the 3.10C2-H1 heavy chain and the rat 3.10C2 light chain was robustly expressed (Figure 7B), confirming that the humanized 3.10C2 light chain is defective in expression. We investigated whether other light chains in the 3.10C2 antibody family are more amenable to humanization and expression.

[0280] We tested whether a humanized light chain variant of the anti-TREM2 antibody 3.27H7 paired with a humanized 3.10C2 heavy chain, a clonal variant of 3.10C2, would result in better expression and product quality of the humanized variant. The 3.27H7 light chain was humanized within the same framework as the 3.10C2 light chain, including light chain framework residues 2, 4, 58 and 68 within the graft (Figures 8A-8B). The humanized 3.10C2 heavy chain variant was combined with the humanized 3.27H7 light chain variant to produce a human IgG1 with the N297G mutation.

[0281] Four humanized 3.10C2 / 3.27H7 hybrid variants were expressed and purified. The 3.10C2-H1 / 3.27H7-L1 clone was well expressed at 246 mg / L in transiently transfected CHO cells after purification by Protein A chromatography, and showed a monodisperse peak for 99.5% of the material.

[0282] The mutants 3.10C2-H3 / 3.27H7-L1 IgG and 3.10C2-H3 / 3.27H7-L6 IgG were also well expressed in CHO cells at 364 and 408 mg / L, although some heavy-heavy chain (HH) dimers were detectable by mass spectrometry in purified samples.

[0283] The mutant 3.10C2-H1 / 3.27H7-L6 IgG was fully expressed in CHO cells at 356 mg / L, showed a monodisperse peak by size exclusion chromatography, and showed no detectable HH dimers by mass spectrometry under non-reducing conditions. This mutant was renamed h3.10C2.v1 and its sequence is shown in Figure 9A-B.

[0284] The affinity of h3.10C2.v1 (Figures 9A-9B) for human and cynomolgus TREM2 was measured in two formats by SPR on a Biacore™ T200 instrument, both at 37°C. In one format, TREM2-Fc was immobilized on a Protein A chip and a soluble anti-TREM2 Fab fragment was used as the ligand. In the second format, anti-TREM2 IgG was immobilized on an anti-CH1 Biacore™ chip and a soluble monomeric TREM2 was used as the ligand. The affinity of humanized antibody h3.10C2.v1 for hTREM2 ranged from 390 to 420 pM, while the affinity for cynomolgus TREM2 was similar at 430 pM (Table 3). Thus, humanized anti-TREM2 h3.10C2.v1 was found to express well and retain high affinity for both human and cynomolgus TREM2.

[0285] Example 8: Affinity Maturation of Antibody 3.10C2 Heavy Chain by Saturation Mutagenesis A. Materials and Methods A library of mutants based on the 3.10C2 antibody heavy chain was constructed by overlapping recombinant PCR with the aim of further improving the antibody affinity. DNA fragments encoding the heavy and light chains were mixed one for each mutant, used to transfect Expi293 cells at a 1 ml scale, and purified by protein A chromatography as previously described (Bos et al., Biotechnol. Bioeng. (2015); Luan et al., Mabs (2018)).

[0286] The resulting library consisted of individual IgG clones, each with one mutation per molecule in the heavy chain variable region. Clones were screened for off-rates by SPR on a Biacore™ 8K instrument using an anti-human Fab chip to capture mutant IgG and soluble monomeric TREM2 as ligands.

[0287] B. Results Only two of the hundreds of mutant clones generated, including mutations T93E and T93H, had significant but relatively small (1.4-2 fold) reductions in off-rates compared to the parental 3.10C2 clone (Figure 10). Interestingly, mutations G94D and D101E, the respective CDR H3 residues of Para.09, compared to the same positions in antibody 3.10C2, either reduced the affinity of 3.10C2 for TREM2 (G94D) or abolished binding (D101E) (Figure 10), indicating that the CDR H3 of Para.09 is not structurally equivalent to the CDR H3 of 3.10C2. Furthermore, scanning mutagenesis (Figure 10) showed that clone 3.10C2 affinity was near maximal and could not be easily improved by mutagenesis of the heavy chain CDRs. Thus, the substantially higher affinity achieved with clone Para.09 for the same epitope as the 3.10C2 group required substantial alterations in the CDR H3, including insertions that were not readily accessible with traditional affinity optimization methods that explore mutations in the same structural CDR H3 sequence but do not explore structurally distinct solutions for the CDR H3.

[0288] Example 9: Humanized antibody Para.09 A. Materials and Methods Antibody Para.09, which contains the Para.09 heavy chain variable region and the 3.27H7 light chain variable region (also called Para.09-27H7), was humanized in the VK2 and VH3 frameworks by CDR grafting using the same framework as antibody 3.10C2. Briefly, the CDR regions of the rat Para.09 VH sequence were grafted onto the heavy chain IGKV3-73*01 framework, which is the closest human germline of this antibody (Figure 9B). The light chain for Para.09 humanization used the same humanized light chain variant of antibody 3.27H7 described above (Figure 9A). As described in Example 7 of 3.10C2 antibody humanization, rat framework residues known as "Bernier" positions and domain interfaces that differ between rat antibodies and human germline were added to the CDR graft to rescue binding to the antigen. A mutant called Para.09-H1 / 3.27H7-L1, which has all framework Bernier positions, was expressed as a human IgG1 with the N297G mutation in CHO cells.

[0289] B. Results Purification yields of humanized Para.09-H1 / 3.27H7-L1 were lower compared to chimeric antibodies with either mouse IgG2a or human IgG1 constant regions (Figure 11). However, expression of humanized Para.09-H1 heavy chain paired with 3.27H7-L6, the same light chain as h3.10C2.v1, in CHO cells produced high levels of IgG (314 mg / L of culture), no HH dimers, and relatively few other by-products of IgG assembly. Further analysis of heavy chain variants with fewer rat framework residues identified Para.09-H5 as the variant with the fewest rat residues, which when combined with light chain 3.27H7-L6, produced high levels of IgG expression in CHO cells (544 mg / L of culture) without detectable HH dimers. This variant was designated hPara.09.v2, and its sequence is shown in Figures 12A-B. The monovalent affinities of this variant, renamed hPara.09.v2 (Figures 12A-B), for TREM2, determined as described above for the h3.10C2.v1 IgG and Fab fragments, ranged from 11-21 pM for hTREM2 and 18 to 22 pM for cynomolgus TREM2 (Table 3). Table 3: Binding affinity of hPara.09.v2 and h3.10C2.v1 for human TREM2 and cynomolgus TREM2 [Table 4]

[0290] In summary, the TREM2 binding epitopes of a series of rat anti-TREM2 antibodies were determined and the binding affinities were measured. See Examples 1-6. Candidate antibodies were engineered and humanized to improve TREM2 affinity and expression yield. See Examples 7-9. Antibodies identified included: (1) rat Para.09 (which comprises a 3.27H7 light chain and a Para.09 heavy chain, i.e., Para.09-L27H7, sequence shown in Figures 3A-3B), (2) humanized Para.09.v2 (i.e., hPar.09.v2; sequence shown in Figures 12A-12B), (3) rat 3.10C2 (sequence shown in Figures 6A-6B), (4) humanized 3.10C2.v1 (i.e., h.310.C2.v1; sequence shown in Figures 9A-9B).

[0291] Example 10: Determination of off-target binding A. Materials and Methods The potential for the rat and humanized antibodies to bind "off-targets" (i.e. targets other than the intended TREM2) was tested in an ELISA assay using baculovirus particles as antigen (BV ELISA; Hotzel et al., Landes Bioscience dx.doi.org / 10.4161 / mabs.22189(2012)). Normalization was performed by dividing the ELISA absorbance readings of the antibody samples by the absorbance readings of blank wells containing no test antibody as previously described (Id). Control antibodies previously shown to have high (score >5), moderate (score ≈5) and low (undetectable, score <1) off-target binding in this assay were tested in parallel. Four antibodies were tested: a chimeric clone with rat variable regions and a human IgG1 constant region and humanized variants h3.10C2.v1 and hPara.09.v2 with a human IgG1 constant region carrying the N297G mutation.

[0292] B. Results A chimeric rat antibody with a human IgG1 constant region had very low reactivity in the assay, scoring less than 1 (Figure 15). Similarly, the humanized anti-TREM2 antibodies h3.10C2.v1 and hPara.09.v2, both human IgG1 N297G, had normalized BV ELISA scores less than 1 (Figure 15), showing no detectable propensity for off-target binding. Thus, the humanized h3.10C2.v1 and hPara.09.v2 antibodies have high binding affinity for TREM2 in the mid- to low-picomolar range, but retain favorable low or undetectable off-target binding potential.

[0293] Example 11: Determination of binding to soluble TREM2 A. Materials and Methods A human TREM2 ELISA assay was used to measure binding of over 40 antibodies to soluble TREM2 (sTREM2) as follows: Test anti-hTREM2 antibodies were coated onto plate wells as capture reagents, and biotinylated IgV-reactive monoclonal antibody 3.17A9 was used as detection reagent. Antibody 1.16B8 was used as a control antibody to establish a standard for sTREM2 binding. Plates coated with test or control antibodies were incubated with diluted culture supernatants from bone marrow-derived macrophage (BMDM) cultures from transgenic mice expressing hTREM2. sTREM2 is constitutively produced by BMDM cultures expressing hTREM2 and excreted into the culture supernatant. Measurements were recorded at 450 nm for detection and 570 nm for background. The 3.17A9 / 1.16B8 antibody pair was chosen over the reagents provided in the R&D Systems kit because commercially available reagents were not compatible with sTREM2 detection for some of the antibodies tested. The light chain sequence of the control antibody 1.16B8 is shown in SEQ ID NO: 170 and its heavy chain sequence is shown in SEQ ID NO: 171. The light chain sequence of the detection antibody 3.17A9 is shown in SEQ ID NO: 168 and the heavy chain sequence is shown in SEQ ID NO:169.

[0294] B. Results Figure 16 shows the sTREM2 binding values ​​for the antibodies. Binding was normalized to the control (CTL) antibody 1.16B8, which was set to 1. Normalized sTREM2 binding is shown for: rat Para.09-LC 3.27H7 mIgG2a LALAPG (Para09), rat 3.10C2 mIgG2a LALAPG (3.10C2), rat 3.18E5 mIgG2a LALAPG (3.18E5), rat 3.50G1 mIgG2a LALAPG (3.50G1), rat 3.27H7 mIgG2a LALAPG (3.27H7), rat 3.36F5 mIgG2a LALAPG (3.36F5), A.9F5, AL2p-12, AL2p-31, AL2p-58, BM.3D3, BM.42E8, BM.RS9, BM.14D3, and BM.14D8. The antibodies tested, rat Para.09-LC 3.27H7 mIgG2a LALAPG (Para09), rat 3.10C2 mIgG2a LALAPG (3.10C2), rat 3.18E5 mIgG2a LALAPG (3.18E5), rat 3.50G1 mIgG2a LALAPG (3.50G1), rat 3.27H7 mIgG2a LALAPG (3.27H7), and rat 3.36F5 mIgG2a LALAPG (3.36F5), contain the mIgG2a LALAPG heavy chain constant region. Of the antibodies tested, only 3.10C2, Para.09 and the clonally related antibodies 3.18E5, 3.50G1, 3.27H7, and 3.36F5 did not show any binding to sTREM2 (see FIG. 16, which shows near zero normalized sTREM2 binding for these antibodies in contrast to the 1.116B8 control). The humanized antibody Para.09.v2 antibody with the hIgG1 N297G constant region was also tested for sTREM2 binding and showed no binding to sTREM2, as did the rat Para.09 with mIgG2 LALAPG.

[0295] Additionally, nine antibodies previously described in the literature for binding to stalk hTREM2 were tested in this experiment, including A.9F5 (also referred to as 9F5), AL2p-12, AL2p-31, AL2p-58, BM.3D3, BM.42E8, BM.RS9, BM.14D3, and BM.14D8, e.g., as described in WO2018015573, WO201955841, WO2017062672 and / or WO201928292. Unlike the 3.10C2, Para.09, 3.18E5, 3.50G1, 3.27H7, and 3.36F5 antibodies, the nine previously described antibodies tested here bound to sTREM2 in the assay (see FIG. 16, showing normalized sTREM2 binding of 0.5 to 1.5 for those antibodies). This result is surprising when considered in conjunction with the binding profiles of the tested antibodies, since the tested antibodies have epitopes in the TREM2 stalk domain, and the results may reflect unique epitopes of 3.10C2, Para.09, 3.18E5, 3.50G1, 3.27H7, and 3.36F5 compared to the previously described antibodies (see, e.g., FIG. 5).

[0296] The peptide mapping and mutational scanning results described in the previous examples may provide the basis for the observed differences in binding to soluble TREM2 between 3.10C2 / Para.09 / 3.50G1 and antibody 9F5 and its derivatives. The epitopes of Para.09, 3.10C2 and 3.50G1 span the ADAM10 cleavage site between His-157 and Ser-158, with important contact sites on either side of the cleavage site as determined by mutational scanning and structural analysis. (See FIG. 5). In contrast, as noted in Example 6 above, the epitope of A.9F5 (9F9 in FIG. 5) and its derivative AL2p-31 is located entirely on the N-terminal side of the TREM2 cleavage site. (See FIG. 5). This difference in TREM2 contact points may explain the observed differences in binding to sTREM2 and, consequently, binding of these previously described antibodies to soluble TREM2.

[0297] Surprisingly, however, antibodies 14D3 and 14D8 have a binding pattern to the alanine mutant peptide that appears similar to antibodies 3.10C2, 3.50G1 and Para.09 (Figure 5), but still show robust binding to sTREM2 in a monovalent and physiologically relevant manner (Figure 16). This was highly surprising, as 3.10C2 and Para.09, which have very high affinity, would be expected to retain binding to partial epitopes in soluble TREM2, whereas the lower affinity (3-5 nM) 14D3 and 14D8 antibodies may more easily lose binding to these partial epitopes. This indicates that the binding profiles described in the previous examples cannot account for all interactions between anti-TREM2 antibodies and TREM2 that are necessary for the anti-TREM2 antibodies to distinguish between full-length membrane-bound and soluble TREM2 forms, further establishing the uniqueness of the antibodies of the present disclosure.

[0298] Thus, as these results show, antibodies with the binding profile of the 3.10C2, Para.09, 3.18E5 and 3.50G1 antibodies are unique. These antibodies bind effectively to the TREM2 stalk domain but not to soluble TREM2. The specificity of these antibodies may have several potential advantages, including allowing soluble TREM2 in the periphery and brain following in vivo dosing to remain free of the administered antibody. The high affinity of these antibodies for epitopes spanning the cleavage site of intact TREM2 and lack of binding to cleaved soluble TREM2 may be beneficial in vivo in any of several ways, from allowing more of the administered anti-TREM2 antibody to reach the desired target of TREM2 on the surface of the cell to reducing the amount of sTREM2 released from the cell surface.

[0299] Example 12: Anti-TREM2 antibodies induce TREM2-dependent and NFAT-driven luciferase activity A. Materials and Methods A Jurkat-based luciferase reporter cell line was used to test the ability of full-length anti-hTREM2 antibodies to induce human TREM2-associated signaling. TREM2 activation and its interaction with DAP12 (DNAX-activating protein of 12 kilodaltons) at the cell membrane can induce phosphorylation of Syk kinase (a spleen tyrosine kinase whose phosphorylated form is called pSYK or phospho-SYK) in the cytoplasm and other cell signaling events, ultimately resulting in activation of nuclear factor of activated T cells (NFAT)-regulated gene expression. Thus, both phosphorylation of Syk kinase and NFAT-regulated gene expression can be used to determine the effect of antibodies on TREM2 activity and whether the antibodies agonize TREM2. In this assay, Jurkat cells were engineered to express a luciferase reporter under the control of an NFAT response element and to co-express human TREM2 and DAP12 to test whether the addition of antibodies induces reporter gene expression through activation of NFAT.

[0300] A parental Jurkat luciferase reporter cell line (human T lymphocytes; Signosis) was engineered to stably express a firefly luciferase reporter gene under the control of an NFAT response element, and was then transduced with an MSCV-based retroviral vector to co-express TREM2 (wild type or mutant) and DAP12 to generate the Jurkat-NFAT luciferase TREM2 reporter cell line.

[0301] The Jurkat reporter assay was used to test the agonist effect of anti-human TREM2 antibodies compared to an isotype control antibody. Each antibody and isotype control was added in soluble form at 10ug / mL to Jurkat-NFAT luciferase reporter cells and incubated at 37°C for 24 hours. Luciferase activity was measured by adding Bright-Glo™ Substrate (Promega Cat# E2610) substrate. After 3 minutes of incubation at room temperature, luminescence readings were recorded using the M1000 program on a Tecan plate reader.

[0302] To determine the time-dependent reporter activity of anti-human TREM2 antibodies, Jurkat-NFAT luciferase reporter cells were cultured in the presence of 10 μg / mL of anti-TREM2 antibodies for 3, 6 or 24 hours, respectively, followed by the addition of Bright-Glo™ substrate to measure luciferase activity.

[0303] B. Results Fifty-six rat anti-human TREM2 antibodies (in the form of recombinant mIgG2a) identified in the screening of Example 1 were screened in the Jurkat reporter assay. Of these 56, only eight antibodies, including four "3.10C2 group" antibodies: 3.10C2, 3.18E5, 3.27H7 and 3.50G1, induced luciferase reporter activity in Luc RLU (luciferase relative light units) compared to the isotype control, suggesting that these anti-TREM2 antibodies can mimic the natural ligand activity in Jurkat-NFAT luciferase reporter cells (Figure 17). Of those eight antibodies, four "3.10C2 group" antibodies: 3.10C2, 3.18E5, 3.27H7 and 3.50G1 showed equally strong agonism, while the four other tested antibodies showed only moderate agonist activity (Figure 17). The remaining 48 tested antibodies had lower Luc RLU values ​​than the isotype control. These data indicate that the 3.10C2 group of antibodies that specifically bind to a specific epitope at positions 151-165 of the TREM2 stalk have relatively strong agonistic activity compared to antibodies that bind outside of that region of the stalk.

[0304] The same 56 antibodies were also tested in a time course assay using Jurkat-NFAT reporter cells. Of the antibodies tested, the "3.10C2 group" antibodies 3.10C2, 3.18E5, 3.27H7, and 3.50G1 also showed strong time-dependent agonist activity (Luc RLU fold change) compared to the isotype.

[0305] Example 13: Anti-TREM2 antibodies in mIgG2a LALAPG or hIgG1 N297G formats induce luciferase activity in Jurkat reporter cells A. Materials and Methods The rat anti-human antibodies Para.09 and 3.10C2 were engineered to contain a mouse IgG2a LALAPG Fc region (to reduce effector function of the Fc) and further tested for agonist activity in Jurkat-NFAT luciferase reporter cells. The humanized antibodies Para.09.v2 and 3.10C2 were similarly engineered and tested in a human IgG1 N297G format to reduce effector function. Jurkat-based luciferase reporter assays were performed as described above in Example 12. To test the dose-dependent activity of anti-human TREM2 antibodies, Jurkat-NFAT luciferase reporter cells were cultured in the presence of serially diluted anti-human TREM2 antibodies for 24 hours.

[0306] We also evaluated anti-hTREM2 antibody activity against common hTREM2 mutations: we constructed Jurkat-NFAT luciferase reporter cell lines expressing three hTREM2 mutants: R47H, R62H and H157Y, the presence of which may correlate with increased risk of Alzheimer's disease.

[0307] Supernatant samples were collected for TREM2 shedding analysis before adding Bright-Glo (Promega catalogue no. E2610) substrate for measurement of luciferase-driven luminescence.

[0308] B. Results Both the rat and humanized antibodies exhibited agonist activity at 24 hours as indicated by induction of luciferase reporter activity. See Figure 18A (rat antibody) and Figure 18B (humanized antibody). These data further demonstrate that both the rat and humanized Para.09 and 3.10C2 antibodies have agonist activity and that such activity is not related to antibody effector function.

[0309] Four "3.10C2 group" antibodies (mIgG2a LALAPG format), 3.10C2, 3.18E5, 3.27H7 and 3.50G1, also induced luciferase activity in Jurkat-NFAT reporter cells engineered to express three hTREM2 mutants, R47H (Figure 19A), R62H (Figure 19B) and H157Y (Figure 19C), respectively. These results demonstrate that anti-hTREM2 antibodies have agonistic activity against both wild-type hTREM2 and these hTREM2 mutations and therefore may be useful in patients with these mutant TREM2 proteins.

[0310] Example 14: Anti-TREM2 antibodies in mIgG2a LALAPG or hIgG1 N297G formats block TREM2 shedding in Jurkat reporter cells A. Materials and Methods TREM2 shedding, which releases soluble TREM2, can occur in Alzheimer's disease patients decades before disease onset. Binding of soluble TREM2 to amyloid plaques can interfere with neuroprotective plaque compaction by microglia. Therefore, both rat and humanized Para.09 and 3.10C2 antibodies herein were tested in Jurkat-NFAT luciferase reporter cells described in Examples 12 and 13 to determine whether they can also inhibit TREM2 shedding. To determine the TREM2 shedding inhibitory activity of soluble anti-human TREM2 antibodies in Jurkat-NFAT luciferase reporter cells, supernatants were harvested from the reporter assay plates after 24 hours and sTREM2 was measured using an ELISA assay as described above in Example 11.

[0311] Next, the activity of the humanized Para.09 variants, hPara.09 v2, hPara.09 v2.Q100P / V104L and hPara.09.v2 Q100P, in the Jurkat-NFAT reporter and TREM2 shedding inhibition assays was compared to the activity of the humanized 3.10C2.v1 antibody. Jurkat-NFAT luciferase reporter cells were cultured in the presence of serial dilutions of humanized antibodies for 24 hours and tested as described above.

[0312] B. Results In the ELISA assay, Para.09 in mIgG2a LALAPG format showed the strongest TREM2 shedding inhibition activity compared to other tested antibodies in the same format. Humanized antibody hPara.09.v2 in hIgG1 N297G format showed stronger TREM 2 shedding inhibition than humanized antibody h3.10C2 v1 in hIgG1 N297G format (see FIG. 20B). Inhibition of sTREM2 shedding by the rat antibody is shown in contrast to another antibody (1.20A2 mIgG2a LALAPG) that showed little shedding inhibition in the assay (see FIG. 20A).

[0313] The variants hPara.09 v2.Q100P / V104L and hPara.09.v2 Q100P are identical to hPara.09v2, except for the Q100P / V104L and Q100P mutations, respectively (FIGS. 13A-B). As shown in FIG. 20C-D, hPara.09 v2, hPara.09 v2.Q100P / V104L and hPara.09.v2 Q100P in the hIgG1 N297G format exhibited comparable activity. In addition, consistent with the results shown in Example 13, the activity of the three Para.09 humanized antibodies was higher than that of the humanized 3.10C2.v1 antibody.

[0314] Jurkat NFAT luciferase reporter cells (EC 50 ) and TREM2 shedding inhibition (IC 50 The measured potencies of TREM2 antibodies in ) are summarized in Table 4 below. Table 4: Potency of TREM2 antibodies (EC 50 ) and TREM2 shedding inhibition (IC 50 ) [Table 5] (NA=unavailable)

[0315] Example 15: Soluble anti-TREM2 antibodies block TREM2 shedding in human iPSC-derived microglia A. Materials and Methods Since TREM2 is expressed in microglial cells in vivo, inhibition of TREM2 shedding was next tested in human iPSC-derived microglial cells. To determine TREM2 shedding inhibition by anti-human TREM2 antibody in human iPSC-derived microglia (Cellular Dynamics), iPSC-microglia were cultured with maintenance medium (Cellular Dynamics) in the presence of 10ug / mL soluble anti-TREM2 mIgG2a LALAPG antibody. ADAM10 protease inhibitor GI254023X (Sigma-Aldrich) was used as a positive control to block cleavage and shedding of sTREM2. Culture supernatant samples were taken on d...

Claims

1. 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, 19 or 62, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, 20, 55, 63, 65 or 73, and a CDR-H3 comprising the amino acid sequence of: １ -X ２ -X ３ -Y (wherein, X １ and X ２ are both either IL or L, and X ３ and a heavy chain variable region (VH) comprising:

2. 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, 19 or 62, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, 20, 55, 63, 65 or 73, and a CDR-H3 comprising the amino acid sequence of: １ -X ２ -X ３ -Y (wherein, X １ and X ２ are both either IL or L, and X ３ and a light chain variable region (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, 27, 37, 47, 57 or 67; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29, 39, 49, 59 or 69. An isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), comprising a heavy chain variable region (VH) comprising: CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 27, 37, 47, 57 or 67; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, 29, 39, 49, 59 or 69.

3. 1. An isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), the antibody comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 9, 11, or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 10, 12, or 20, and a CDR-H3 comprising the amino acid sequence of: １ -X ２ -X ３ -Y (wherein, X １ and X ２ are both either IL or L, and X ３ and a light chain variable region (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

4. The antibody has the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with greater affinity to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO:96) or 151-165 (SEQ ID NO:97) than to a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO:92) and / or 159-175 (SEQ ID NO:94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibits a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions, as compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interference assays); (f) specifically binds to human and cynomolgus TREM2 with a KD of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM at 37° C. by surface plasmon resonance (SPR); and (g) a K of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM at 37° C. by surface plasmon resonance (SPR). Ｄ specifically binds to human and cynomolgus monkey TREM2 at The isolated antibody of any one of claims 1 to 3, having one or more of the following:

5. 4. The isolated antibody of any one of claims 1 to 3, wherein the antibody has a VH sequence derived from a rat IGHV6-8 germline segment and / or the antibody has a VL sequence derived from a rat IGKV2S11 germline segment.

6. An antibody described in any one of claims 1 to 3, wherein the light chain variable region (VL) comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 4, 27 or 67, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, 29 or 69.

7. The antibody according to any one of claims 1 to 3, wherein the light chain variable region (VL) comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

8. (a) the heavy chain variable region (VH) comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and the light chain variable region (VL) comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or (b) the heavy chain variable region (VH) comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 or 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and the light chain variable region (VL) comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; The antibody according to any one of claims 1 to 3.

9. The antibody of any one of claims 1 to 3, wherein the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 30, 40, 50, 60, 70, 76, 77, 78, 81, 82, 83, 133, 135, 137, 139, 146, 148, 150, or 152.

10. The antibody of any one of claims 1 to 3, wherein the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 133, 135, 137, 139, 146, 148, 150, or 152.

11. The antibody of any one of claims 1 to 3, wherein the antibody comprises a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8, 18, 31, 41, 51, 61, 71, 79, 80, 84, 85, 132, 134, 136, 138, 145, 147, 149, or 151.

12. The antibody of any one of claims 1 to 3, wherein the antibody comprises a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8, 18, 132, 134, 136, 138, 145, 147, 149, or 151.

13. The antibody of any one of claims 1 to 3, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, 17, 133, 135, 137, 139, 146, 148, 150, or 152.

14. The antibody of any one of claims 1 to 3, wherein the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, 18, 132, 134, 136, 138, 145, 147, 149, or 151.

15. 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising: (a) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (c) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (d) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (e) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 24, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29; (f) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (g) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 47, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 49; (h) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 57, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 58, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59; or (i) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66, and a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 69; 2. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), comprising:

16. The antibody, (a) or a VH comprising the CDRs of claim 15(a) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7; (b) or a VH comprising the CDRs of claim 15(b) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:7; (c) or a VH comprising the CDRs of claim 15(c) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17; (d) or a VH comprising the CDRs of claim 15(d) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17; (e) or a VH comprising the CDRs of claim 15(e) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 30; (f) or a VH comprising the CDRs of claim 15(f) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 40; (g) or a VH comprising the CDRs of claim 15(g) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:50; (h) or a VH comprising the CDRs of claim 15(h) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 60; or (i) 15(i) comprising the CDRs of claim 15(i) and further comprising a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:

70. The antibody described in claim 15.

17. The antibody, (a) or a VL comprising the CDRs of claim 15(a) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:8; (b) or a VL comprising the CDRs of claim 15(b) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:8; (c) or a VL comprising the CDRs of claim 15(c) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18; (d) or a VL comprising the CDRs of claim 15(d) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18; (e) or a VL comprising the CDRs of claim 15(e) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31; (f) or a VL comprising the CDRs of claim 15(f) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 41; (g) or a VL comprising the CDRs of claim 15(g) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:51; (h) or a VL comprising the CDRs of claim 15(h) and which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 61; or (i) 15(i) comprising the CDRs of claim 15(i) and further comprising a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:

71.

17. The antibody of claim 15 or 16.

18. The antibody, (a) or a VH comprising the CDRs according to claim 15(a) and the amino acid sequence of SEQ ID NO:7; (b) or a VH comprising the CDRs according to claim 15(b) and the amino acid sequence of SEQ ID NO:7; (c) or a VH comprising the CDRs according to claim 15(c) and the amino acid sequence of SEQ ID NO: 17; (d) or a VH comprising the CDRs according to claim 15(d) and the amino acid sequence of SEQ ID NO: 17; (e) or a VH comprising the CDRs according to claim 15(e) and the amino acid sequence of SEQ ID NO: 30; (f) or a VH comprising the CDRs according to claim 15(f) and the amino acid sequence of SEQ ID NO: 40; (g) or a VH comprising the CDRs according to claim 15(g) and the amino acid sequence of SEQ ID NO: 50; (h) or a VH comprising the CDRs of claim 15(h) and comprising the amino acid sequence of SEQ ID NO: 60; or (i) 15(i) comprising the CDRs of claim 15(i) and further comprising a VH comprising the amino acid sequence of SEQ ID NO:

70.

17. The antibody of claim 15 or 16.

19. The antibody, (a) or a VL comprising the CDRs according to claim 15(a) and the amino acid sequence of SEQ ID NO:8; (b) or a VL comprising the CDRs according to claim 15(b) and the amino acid sequence of SEQ ID NO:8; (c) or a VL comprising the CDRs according to claim 15(c) and the amino acid sequence of SEQ ID NO: 18; (d) or a VL comprising the CDRs according to claim 15(d) and the amino acid sequence of SEQ ID NO: 18; (e) or a VL comprising the CDRs according to claim 15(e) and the amino acid sequence of SEQ ID NO: 31; (f) or a VL comprising the CDRs according to claim 15(f) and the amino acid sequence of SEQ ID NO: 41; (g) or a VL comprising the CDRs according to claim 15(g) and the amino acid sequence of SEQ ID NO: 51; (h) or a VL comprising the CDRs according to claim 15(h) and the amino acid sequence of SEQ ID NO: 61; or (i) 15(i) comprising the CDRs according to claim 15(i) and further comprising a VL comprising the amino acid sequence of SEQ ID NO:

71.

17. The antibody of claim 15 or 16.

20. The antibody of claim 15 or 16, comprising a VH having the amino acid sequence of SEQ ID NO: 7, and a VL having the amino acid sequence of SEQ ID NO:

8.

21. The antibody of claim 15 or 16, comprising a VH having the amino acid sequence of SEQ ID NO: 17, and a VL having the amino acid sequence of SEQ ID NO:

18.

22. The antibody of claim 15 or 16, wherein the antibody comprises a VL comprising 1 to 5 amino acid substitutions in the framework regions compared to human IGKV2-28*01 germline, optionally wherein the amino acid substitutions comprise Q100P and / or V104L.

23. The antibody, (a) or a VL comprising the CDRs according to claim 15(a) and the amino acid sequence of SEQ ID NO: 145; (b) or a VL comprising the CDRs according to claim 15(b) and the amino acid sequence of SEQ ID NO: 145; (c) or a VL comprising the CDRs according to claim 15(a) and the amino acid sequence of SEQ ID NO: 147; (d) or a VL comprising the CDRs according to claim 15(b) and the amino acid sequence of SEQ ID NO: 147; (e) or a VL comprising the CDRs according to claim 15(a) and the amino acid sequence of SEQ ID NO: 149; (f) or a VL comprising the CDRs according to claim 15(b) and the amino acid sequence of SEQ ID NO: 149; (g) or a VL comprising the CDRs according to claim 15(a) and the amino acid sequence of SEQ ID NO: 151; (h) or a VL comprising the CDRs according to claim 15(b) and the amino acid sequence of SEQ ID NO: 151; (i) or a VL comprising the CDRs according to claim 15(c) and the amino acid sequence of SEQ ID NO: 132; (j) or a VL comprising the CDRs according to claim 15(d) and the amino acid sequence of SEQ ID NO: 132; (k) or a VL comprising the CDRs according to claim 15(c) and the amino acid sequence of SEQ ID NO: 134; (l) or a VL comprising the CDRs according to claim 15(d) and the amino acid sequence of SEQ ID NO: 134; (m) or a VL comprising the CDRs according to claim 15(c) and the amino acid sequence of SEQ ID NO: 136; (n) or a VL comprising the CDRs according to claim 15(d) and the amino acid sequence of SEQ ID NO: 136; (o) or a VL comprising the CDRs according to claim 15(c) and the amino acid sequence of SEQ ID NO: 138; or (p) 15(d) comprising the CDRs according to claim 15(d) and further comprising a VL comprising the amino acid sequence of SEQ ID NO:

138.

17. The antibody of claim 15 or 16.

24. The antibody has the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with greater affinity to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO:96) or 151-165 (SEQ ID NO:97) than to a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO:92) and / or 159-175 (SEQ ID NO:94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibits a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions, as compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interference assays); (f) a K by surface plasmon resonance (SPR) at 37° C. of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or between 100 and 500 pM, or between 100 and 200 pM Ｄ specifically binds to human and cynomolgus monkey TREM2 at (g) a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM, or between 10 and 25 pM Ｄ specifically binds to human and cynomolgus monkey TREM2 at 17. The isolated antibody of claim 15 or 16, having one or more of the following:

25. 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising: (a) a VH comprising the amino acid sequence of SEQ ID NO: 146, and a VL comprising the amino acid sequence of SEQ ID NO: 145; (b) a VH comprising the amino acid sequence of SEQ ID NO: 148, and a VL comprising the amino acid sequence of SEQ ID NO: 147; (c) a VH comprising the amino acid sequence of SEQ ID NO: 150, and a VL comprising the amino acid sequence of SEQ ID NO: 149; or (d) a VH comprising the amino acid sequence of SEQ ID NO: 152, and a VL comprising the amino acid sequence of SEQ ID NO: 151; 2. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), comprising:

26. The antibody has the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with greater affinity to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO:96) or 151-165 (SEQ ID NO:97) than to a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO:92) and / or 159-175 (SEQ ID NO:94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibits a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry); and (f) a K by surface plasmon resonance (SPR) at 37° C. of less than 1 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM, or between 100 and 500 pM, or between 100 and 200 pM Ｄ specifically binds to human and cynomolgus monkey TREM2 at 26. The isolated antibody of claim 25, having one or more of the following:

27. 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising: (a) a VH comprising the amino acid sequence of SEQ ID NO: 133, and a VL comprising the amino acid sequence of SEQ ID NO: 132; (b) a VH comprising the amino acid sequence of SEQ ID NO: 135, and a VL comprising the amino acid sequence of SEQ ID NO: 134; (c) a VH comprising the amino acid sequence of SEQ ID NO: 137, and a VL comprising the amino acid sequence of SEQ ID NO: 136; or (d) a VH comprising the amino acid sequence of SEQ ID NO: 139, and a VL comprising the amino acid sequence of SEQ ID NO: 138; 2. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), comprising:

28. The antibody has the following characteristics: (a) specifically binds to the stalk domain of TREM2; (b) does not bind to soluble TREM2 (sTREM2); (c) specifically binds with greater affinity to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO:96) or 151-165 (SEQ ID NO:97) than to a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO:92) and / or 159-175 (SEQ ID NO:94); (d) specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site; (e) exhibits a reduced binding affinity for a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry); and (f) a K by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10 and 50 pM, or between 10 and 25 pM. Ｄ specifically binds to human and cynomolgus monkey TREM2 at 28. The isolated antibody of claim 27, having one or more of the following:

29. The antibody has the following characteristics: (a) induces luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (b) reducing the level of sTREM2 in plasma in vivo; (c) inhibiting sTREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1; (g) inhibiting sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and (i) increasing total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia (e.g., as described in the assay of Example 17 herein); 30. The antibody of any one of claims 1, 15, 25, and 27, having one or more of the following:

30. 30. The antibody of any one of claims 1, 15, 25, and 27, wherein the antibody has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (e.g., as described in Example 10 herein).

31. Fv, single chain Fv (scFv), Fab, Fab', or (Fab') ２ 30. The antibody of any one of claims 1, 15, 25, and 27, which is an antibody fragment such as

32. 30. The antibody of any one of claims 1, 15, 25, and 27, which is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody.

33. The antibody of claim 32 , wherein the antibody comprises a wild-type human IgG1 or IgG4 Fc region.

34. 34. The antibody of claim 33, wherein the antibody comprises a human IgGl Fc region comprising: (a) an N297G substitution; (b) an L234A, L235A, and P329G substitution (LALAPG substitution); or (c) an N297G, M428L, and N434S substitution.

35. The antibody of claim 33, wherein the antibody has reduced effector function or is effectorless, or does not bind to an FcγR.

36. The antibody of any one of claims 1, 15, 25, and 27, wherein the antibody comprises a human IgGl Fc region comprising an N297G substitution.

37. The antibody of any one of claims 1, 15, 25, and 27, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 144 and / or a light chain comprising the amino acid sequence of SEQ ID NO:

176.

38. 30. The antibody of any one of claims 1, 15, 25, and 27, wherein the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 144, but lacking the C-terminal lysine of SEQ ID NO: 144 or lacking the C-terminal glycine and lysine of SEQ ID NO: 144, and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

176.

39. 30. The antibody of any one of claims 1, 15, 25, and 27, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 144 and / or a light chain consisting of the amino acid sequence of SEQ ID NO:

176.

40. 34. The antibody of claim 33, wherein the antibody comprises a human IgG4 Fc region comprising an S228P substitution or comprising S228P, M252Y, S254T and T256E substitutions.

41. 30. The antibody of any one of claims 1, 15, 25, and 27, which is a full-length antibody.

42. 30. The antibody of any one of claims 1, 15, 25, and 27, which is an IgG antibody lacking a C-terminal lysine in the heavy chain constant region.

43. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:17 and / or a light chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:

18.

44. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), wherein the antibody comprises a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:144 and / or a light chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:

176.

45. 1. An isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), said antibody comprising a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:144, but lacking the C-terminal lysine of SEQ ID NO:144 or lacking the C-terminal glycine and lysine of SEQ ID NO:144, and / or a light chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:

176.

46. An isolated antibody that specifically binds to a triggering receptor expressed on myeloid cells-2 (TREM2), the antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO:144 and / or a light chain consisting of the amino acid sequence of SEQ ID NO:

176.

47. 47. The isolated antibody of any one of claims 43-46, wherein the antibody specifically binds to a TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), the antibody specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site, and / or specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO:96) or 151-165 (SEQ ID NO:97) with greater affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO:92) and / or 159-175 (SEQ ID NO:94).

48. 47. The isolated antibody of any one of claims 43-46, wherein the antibody specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), and wherein the antibody is a TREM2 agonist.

49. 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 or 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, the antibody further comprising a CDR-H2 domain that specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), the antibody specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), and the antibody ... and the antibody specifically binds to the TREM2 stalk domain and does not bind to soluble TREM2 (sTREM2), 1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-2 (TREM2), which is an agonist, wherein the antibody specifically binds to a TREM2 epitope spanning the H157-S158 cleavage site and / or specifically binds to a TREM2 polypeptide consisting of amino acids 146-161 (SEQ ID NO:96) or 151-165 (SEQ ID NO:97) with greater affinity than a TREM2 polypeptide consisting of amino acids 139-158 (SEQ ID NO:92) or / or 159-175 (SEQ ID NO:94).

50. the antibody exhibits reduced binding affinity to a TREM2 stalk domain polypeptide comprising the D152A, H157A and I159A substitutions compared to a wild-type TREM2 stalk domain polypeptide (e.g., as measured by bilayer interferometry), and / or has a K for human and cynomolgus TREM2 by surface plasmon resonance (SPR) at 37° C. of less than 100 pM, less than 50 pM, less than 10 pM, less than 7 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM, or between 10-50 pM or 10-25 pM. Ｄ 50. The isolated antibody of any one of claims 43 to 46 and 49, which specifically binds to

51. The antibody has the following characteristics: (a) induces luciferase reporter activity in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (b) reducing the level of sTREM2 in plasma in vivo; (c) inhibiting sTREM2 shedding in Jurkat-NFAT luciferase reporter cells expressing human TREM2; (d) induces tyrosine phosphorylation in human MDM cells; (e) induces SYK phosphorylation in human MDM cells; (f) enhancing survival of human iPSC-derived microglia in the absence of IL-34 and CSF-1; (g) inhibiting sTREM2 shedding in human iPSC-derived microglia; (h) induces SYK phosphorylation in human iPSC-derived microglia; and (i) increasing total Aβ plaque intensity and / or average X04 plaque intensity in the presence of Aβ oligomers in human iPSC-derived microglia (e.g., as described in the assay of Example 17 herein); 50. The isolated antibody of any one of claims 43 to 46 and 49, having one or more of:

52. 50. The antibody of any one of claims 43-46 and 49, wherein the antibody has a low off-target binding score (e.g., a score of less than 1) in an off-target binding assay (e.g., as described in Example 10 herein).

53. 48. The antibody of claim 47, wherein the VH comprises the amino acid sequence of SEQ ID NO: 17, 133, 135, 137 or 139, or the antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:

144.

54. 48. The antibody of claim 47, wherein the antibody comprises a human IgGl Fc region comprising: (a) an N297G substitution; (b) an L234A, L235A, and P329G substitution (LALAPG substitution); or (c) an N297G, M428L, and N434S substitution.

55. 48. The antibody of claim 47, wherein the antibody has reduced effector function or is effectorless, or does not bind to an FcγR.

56. 50. The antibody of any one of claims 1, 15, 25, 27, 43-46, and 49, which is a bispecific or multispecific antibody or is covalently or non-covalently conjugated to at least one other molecule.

57. 57. The antibody of claim 56, wherein the antibody is covalently or non-covalently conjugated to at least one other molecule, the at least one other molecule comprising a detectable label and / or a drug.

58. A pharmaceutical composition comprising an antibody according to any one of claims 1, 15, 25, 27, 43 to 46, and 49, and a pharma- ceutically acceptable carrier.

59. An isolated nucleic acid or set of two or more nucleic acids encoding the antibody of any one of claims 1, 15, 25, 27, 43-46, and 49.

60. 50. An isolated vector comprising one or more nucleic acids encoding the heavy and light chains of an antibody according to any one of claims 1, 15, 25, 27, 43-46, and 49.

61. 60. An isolated host cell comprising the nucleic acid of claim 59.

62. A method for producing an antibody that specifically binds to TREM2, comprising culturing a host cell described in claim 61 under conditions suitable for expression of the antibody.

63. 63. The method of claim 62, further comprising recovering the antibody from the host cell.

64. 63. An antibody produced by the method of claim 62.

65. A method of treating a condition associated with loss of TREM2 function in a subject in need of such treatment, comprising administering an antibody described in any one of claims 1, 15, 25, 27, 43-46, and 49.

66. A method of reducing levels of sTREM2 in a subject in need thereof, comprising administering an antibody of any one of claims 1, 15, 25, 27, 43-46, and 49.

67. 66. The method of claim 65, wherein the condition is a neuroinflammatory or neurodegenerative disease or the subject is afflicted with a neuroinflammatory or neurodegenerative disease.

68. 68. The method of claim 67, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury.

69. 69. The method of claim 68, wherein the disease is Alzheimer's disease.

70. 69. The method of claim 68, wherein the disease is MS.

71. 50. The antibody of any one of claims 1, 15, 25, 27, 43-46 and 49 for use in treating a condition associated with loss of TREM2 function in a subject in need of such treatment.

72. 50. The antibody of any one of claims 1, 15, 25, 27, 43-46 and 49 for use in reducing levels of sTREM2 in a subject in need thereof.

73. 72. The antibody for use according to claim 71, wherein the condition is a neuroinflammatory or neurodegenerative disease or the subject is suffering from a neuroinflammatory or neurodegenerative disease.

74. 74. The antibody for use according to claim 73, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury.

75. 75. The antibody for use according to claim 74, wherein the disease is Alzheimer's disease.

76. 75. The antibody for use according to claim 74, wherein the disease is MS.

77. 50. Use of an antibody according to any one of claims 1, 15, 25, 27, 43-46 and 49 in the preparation of a medicament for treating a condition associated with loss of TREM2 function in a subject in need of such treatment.

78. 50. Use of the antibody of any one of claims 1, 15, 25, 27, 43-46 and 49 in the preparation of a medicament for reducing levels of sTREM2 in a subject in need thereof.

79. 78. The use of claim 77, wherein the condition is a neuroinflammatory or neurodegenerative disease or the subject is suffering from a neuroinflammatory or neurodegenerative disease.

80. 80. The use of claim 79, wherein the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Nath-Hakola disease, Guillain-Barre syndrome (GBS), lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury.

81. 81. The use of claim 80, wherein the disease is Alzheimer's disease.

82. 81. The use according to claim 80, wherein the disease is MS.

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