Anti-trem2 antibodies and uses thereof

Abstract

The present disclosure provides an anti-TREM2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to TREM2, which is a hot target with potential to treat cancer and neurodegenerative diseases.

Description

[0001] This application is a divisional application of the patent application with the application date of December 21, 2023, the application number of 202311778506.0, and the invention name of Anti-TREM2 antibody and its use. TECHNICAL FIELD

[0002] The present disclosure belongs to the technical field of antibodies, and more particularly relates to an anti-TREM2 antibody or antigen-binding fragment thereof and uses thereof. BACKGROUND

[0003] Recent studies have shown that the deletion of mouse myeloid cell triggering receptor 2 (triggering receptor expressed on myeloid cells-2, TREM2) reduces tumor growth and is likely to achieve this by enhancing T cell killing function, while the genetic deletion of TREM2 leads to a significant decrease in the number of regulatory myeloid cells, and an increase in the immune response to tumors, such as an increase in natural killer cells (NK cells) and cytotoxic T cells; TREM2 deletion and anti-TREM2 mAb treatment both result in significant changes in the population of macrophages infiltrating tumors: a decrease in the infiltration of mannose receptor 1 (MRC1), chemokine receptors (CX3CR1), and immunosuppressive macrophages in the tumor microenvironment, and an expansion of myeloid subpopulations expressing immune stimulatory factors (promoting improved T cell response), while TREM2 deletion or combination with anti-TREM2 mAb can enhance anti-PD-1 therapy. Analysis of clinical tumor samples shows that TREM2 is highly expressed on the membranes of tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) of more than 200 cases of human primary and metastatic cancers (mediating immune suppression), and its high expression is negatively correlated with overall survival and prognosis of gastric cancer, colorectal cancer, and triple-negative breast cancer. Therefore, TREM2 is a selectable and high-quality target for immunotherapy of solid tumors from the perspective of remodeling the tumor microenvironment.

[0004] TREM2 is an emerging tumor immunotherapy target, and immune checkpoint therapy releases T cell control over tumors, but is disrupted by immunosuppressive myeloid cells. The anti-inflammatory and immunosuppressive activity of TREM2 promotes tumor growth and immune escape, and blocking TREM2 signaling or depleting TREM2+ myeloid cells in tumors to activate T cell-mediated anti-tumor immune responses is a new targeted therapy strategy that can be used in combination with pd-1 antibodies and enhance anti-pd-1 immunotherapy.

[0005] In Alzheimer's disease (AD), elevated risk of AD is associated with TREM2 mutations, such as R47H missense mutation of TREM2, causing microglial lipid recognition impairment. In preclinical models of AD, TREM2 is a lipid receptor expressed in microglia and other tissue macrophages, which binds to phospholipids, apolipoprotein (APOE) and lipoproteins, transmits intracellular activation signals through adaptor DAP12, promotes microglial activation, survival; formation of microglial barrier around Aβ plaques, formation of neuroprotection; at the same time, Aβ binding with APOE or clusterin (CLU) may help deliver these complexes to microglia, resulting in increased Aβ clearance and reduced neuronal damage.

[0006] In AD, agonistic antibodies targeting TREM2 can delay the onset and progression of Alzheimer's disease by promoting the activation of TREM2, activating microglia and inducing their response to neurodegeneration (including proliferation, survival, aggregation and phagocytosis).

[0007] Therefore, TREM2 is a hot target for treating cancer and neurodegenerative diseases. Due to the large number of unmet clinical medical needs of malignant tumors and neurodegenerative diseases, there is a need for other TREM2 antibodies with more desirable pharmaceutical characteristics. SUMMARY

[0008] In view of the above problems, the present disclosure provides antibodies, methods for preparing the same, compositions, etc. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies that can react with various targets. The present disclosure provides antibodies, preferably chimeric monoclonal antibodies or humanized monoclonal antibodies, that can specifically bind to TREM2.

[0009] In one aspect, the present disclosure provides an anti-TREM2 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to TREM2, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCDR) and a light chain variable region (LCDR), wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3:

[0010] The LCDR1 comprises the following sequence:

[0011] [K / R][S / A]S[Q / K / *][S / *][L / *][Q / L / V / E][D / H / N / S / Y][S / V / I][D / N / G][G / *][N / K / *]T[Y / N][L / V / I][N / H / S / Y];

[0012] the LCDR2 comprises the sequence of:

[0013] [L / K / G / H / R][V / A / M]S[K / N / E][L / R / S][D / F / Y / I / A][S / T];

[0014] the LCDR3 comprises the sequence of:

[0015] [W / S / G / Q / M]Q[G / S / H][T / Y / N / L][H / S / N / E][F / V / Y / W]P[Y / L / F]T;

[0016] the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3:

[0017] the HCDR1 comprises the sequence of:

[0018] [D / S][G / *]Y[G / W / Y / N][M / W][H / N];

[0019] the HCDR2 comprises the sequence of:

[0020] [A / R / Y / F]I[D / Y / S / N][P / *][E / G / Y / N][T / D / N / S]G[G / D / R / T][T / N / I][A / N / G / T][Y / F]N[Q / G / P][K / S / N][F / L]K[G / N];

[0021] the HCDR3 comprises the sequence of:

[0022] [E / K / S / P][L / G / P / S / *][W / H / T / F / Y / *][D / F / G / V / Y / *][G / Y / T / *][G / V / T / *][E / V / S / *][G / *][E / *][E / *][N / V / E / *][A / T / P / N / G / *][M / E / S / F / *][A / D]Y.

[0023] Further, the above isolated antibody or antigen binding fragment thereof, wherein,

[0024] the LCDR1 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 35, 38, 41, 44, 47, and 50; and / or,

[0025] the LCDR2 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 36, 39, 42, 45, 48, and 51; and / or,

[0026] the LCDR3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 37, 40, 43, 46, 49, and 52.

[0027] Further, the above-described isolated antibody or antigen-binding fragment thereof, wherein,

[0028] the HCDR1 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 56, 59, 62, 65, 68, and 71; and / or,

[0029] the HCDR2 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 57, 60, 63, 66, 69, and 72; and / or,

[0030] the HCDR3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 58, 61, 64, 67, 70, and 73.

[0031] In some embodiments of the application, the antibody or fragment, including the following combinations:

[0032] (a) HCDR1 of SEQ ID NO: 56, HCDR2 of SEQ ID NO: 57, and HCDR3 of SEQ ID NO: 58, LCDR1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37; or

[0033] (b) HCDR1 of SEQ ID NO: 59, HCDR2 of SEQ ID NO: 60, and HCDR3 of SEQ ID NO: 61, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; or

[0034] (c) HCDR1 of SEQ ID NO: 62, HCDR2 of SEQ ID NO: 63, and HCDR3 of SEQ ID NO: 64, LCDR1 of SEQ ID NO: 41, LCDR2 of SEQ ID NO: 42, and LCDR3 of SEQ ID NO: 43; or

[0035] (d) HCDR1 of SEQ ID NO: 65, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 67, LCDR1 of SEQ ID NO: 44, LCDR2 of SEQ ID NO: 45, and LCDR3 of SEQ ID NO: 46; or

[0036] (e) HCDR1 of SEQ ID NO: 68, HCDR2 of SEQ ID NO: 69, and HCDR3 of SEQ ID NO: 70, LCDR1 of SEQ ID NO: 47, LCDR2 of SEQ ID NO: 48, and LCDR3 of SEQ ID NO: 49; or

[0037] (f) HCDR1 of SEQ ID NO: 71, HCDR2 of SEQ ID NO: 72, and HCDR3 of SEQ ID NO: 73, LCDR1 of SEQ ID NO: 50, LCDR2 of SEQ ID NO: 51, and LCDR3 of SEQ ID NO: 52.

[0038] Further, the isolated antibody or antigen-binding fragment thereof described above, wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 20, 22, 24, 26, 28, and 30.

[0039] Further, the isolated antibody or antigen-binding fragment thereof described above, wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consists of, one of SEQ ID NOs: 20, 22, 24, 26, 28, and 30.

[0040] Further, the antibody or fragment described above, wherein,

[0041] (a) the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20, or consists of SEQ ID NO: 20;

[0042] wherein the light chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, or consists of SEQ ID NO: 19;

[0043] (b) the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22, or consists of SEQ ID NO: 22;

[0044] wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, or consists of SEQ ID NO: 24;

[0045] (c) the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, or consists of SEQ ID NO: 24;

[0046] wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, or consists of SEQ ID NO: 24;

[0047] (d) the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, or consists of SEQ ID NO: 26;

[0048] wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, or consists of SEQ ID NO: 24;

[0049] (e) the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, or consists of SEQ ID NO: 28;

[0050] wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, or consists of SEQ ID NO: 30;

[0051] wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, or consists of SEQ ID NO: 30;

[0052] wherein the heavy chain variable region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, or consists of SEQ ID NO: 30.

[0053] Further, the above-described isolated antibody or antigen-binding fragment thereof, further comprises a heavy chain constant region, wherein the heavy chain constant region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, or consists of SEQ ID NO: 34.

[0054] Further, the above-described isolated antibody or antigen-binding fragment thereof, further comprises a heavy chain constant region, wherein the heavy chain constant region comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, or consists of SEQ ID NO: 34.

[0055] Further, the above-described isolated antibody or antigen-binding fragment thereof, wherein comprises:

[0056] (a) a light chain comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 5, 7, 9, 11, 13, and 15 or consisting of one of SEQ ID NOs: 5, 7, 9, 11, 13, and 15;

[0057] (b) a heavy chain comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16.

[0058] Further, the above-described isolated antibody or antigen-binding fragment thereof, wherein the heavy chain comprises:

[0059] (a) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or consisting of SEQ ID NO: 5; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16;

[0060] (b) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or consisting of SEQ ID NO: 7; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16;

[0061] (c) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9 or consisting of SEQ ID NO: 9; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16;

[0062] (d) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 or consisting of SEQ ID NO: 11; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16;

[0063] (e) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13 or consisting of SEQ ID NO: 13; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16;

[0064] (f) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15 or consisting of SEQ ID NO: 15; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16 or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14, and 16.

[0065] Further, the above isolated antibody or antigen binding fragment thereof, wherein:

[0066] (a) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or consisting of SEQ ID NO: 5; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6 or consisting of SEQ ID NO: 6;

[0067] (b) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or consisting of SEQ ID NO: 7; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or consisting of SEQ ID NO: 8;

[0068] (c) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9 or consisting of SEQ ID NO: 9; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or consisting of SEQ ID NO: 10;

[0069] (d) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 or consisting of SEQ ID NO: 11; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12 or consisting of one of SEQ ID NO: 12;

[0070] (e) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13 or consisting of SEQ ID NO: 13; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14 or consisting of SEQ ID NO: 14;

[0071] (f) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15 or consisting of SEQ ID NO: 15; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16 or consisting of SEQ ID NO: 16.

[0072] Further, the antibody or antigen-binding fragment thereof of any one of the above claims, wherein the antibody is a monoclonal antibody.

[0073] Further, the antibody or antigen-binding fragment thereof of any one of the above claims, wherein the antibody is a chimeric antibody or a humanized antibody or an improved antibody such as an improved chimeric antibody.

[0074] Further, an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the above-described antibody or antigen-binding fragment thereof.

[0075] Further, a vector comprising the above-described nucleic acid molecule.

[0076] Further, a host cell comprising the above-described nucleic acid molecule or vector.

[0077] Further, a conjugate comprising the above-described antibody or antigen-binding fragment thereof coupled to at least one detectable label.

[0078] Further, an antibody drug conjugate comprising an antibody, wherein one or more drug moieties are covalently attached to the antibody or antigen binding fragment thereof described above, directly or via a linker.

[0079] Further, a multispecific molecule comprising the antibody or antigen binding fragment thereof described above; preferably, the multispecific molecule specifically binds to TREM2 and additionally specifically binds to one or more other targets; further preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

[0080] Further, a pharmaceutical composition or kit comprising the antibody or antigen binding fragment thereof, or nucleic acid molecule, or vector, or host cell, or conjugate, or antibody drug conjugate, or multispecific molecule, or pharmaceutically acceptable carrier described above.

[0081] Further, a method of making the antibody or antigen binding fragment thereof described above, comprising the steps of:

[0082] (i) expressing the antibody or antigen binding fragment thereof described above in the host cell described above; and optionally

[0083] (ii) isolating the antibody or antigen binding fragment thereof from the host cell.

[0084] Further, use of the antibody or antigen binding fragment thereof, or nucleic acid molecule, or vector, or host cell, or conjugate, or antibody drug conjugate, or multispecific molecule, or pharmaceutical composition, or kit described above in the manufacture of a kit for diagnosing, detecting or monitoring a disease associated with TREM2 expression.

[0085] Further, use of the antibody or antigen binding fragment thereof, or nucleic acid molecule, or vector, or host cell, or conjugate, or antibody drug conjugate, or multispecific molecule, or pharmaceutical composition, or kit described above in the manufacture of a medicament for treating or determining the prognosis of a disease associated with TREM2 expression.

[0086] Further, the use described above, in combination with PD-1.

[0087] Further, the use described above, wherein the disease associated with TREM2 expression is a cancer selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, brain glioma, ovarian cancer.

[0088] Further, the use as described above, wherein the disease associated with TREM2 expression is a neurodegenerative disease selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), autism spectrum disorders (ASDs), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS).

[0089] Further, the use as described above, wherein the disease associated with TREM2 expression is a metabolic syndrome selected from the group consisting of adipocyte hypertrophy, systemic hypercholesterolemia, inflammation, and glucose intolerance. BRIEF DESCRIPTION OF DRAWINGS

[0090] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, serve to explain the present disclosure, but are not intended to limit the present disclosure. In the drawings:

[0091] Figure 1A Results of the detection of the binding activity of anti-TREM2 antibodies C1 and G3 to cell surface huTREM2;

[0092] Figure 1B Results of the detection of the binding activity of anti-TREM2 antibodies 2A6 and 2A10 to cell surface huTREM2;

[0093] Figure 1C Results of the detection of the binding activity of anti-TREM2 antibodies mAb006c and mAb008c to cell surface huTREM2;

[0094] Figure 2A Results of the detection of the binding activity of anti-TREM2 antibodies C1 and G3 to monkey TREM2;

[0095] Figure 2B Results of the detection of the binding activity of anti-TREM2 antibodies 2A6 and 2A10 to monkey TREM2;

[0096] Figure 2C Results of the detection of the binding activity of anti-TREM2 antibodies mAb006c and mAb008c to monkey TREM2;

[0097] Figure 3A Results of the detection of the binding activity of anti-TREM2 antibodies C1 and G3 to mouse TREM2;

[0098] Figure 3B Results of the detection of the binding activity of anti-TREM2 antibodies 2A6 and 2A10 to mouse TREM2;

[0099] Figure 3C Results of the detection of the binding activity of anti-TREM2 antibodies mAb006c and mAb008c to mouse TREM2;

[0100] Figure 4 TREM2 reporter gene activity detection results, wherein the abscissa is the antibody concentration, and the ordinate RLU represents relative light units;

[0101] Figure 5 Mouse tumor growth curve, wherein G represents group, ip represents intraperitoneal injection, and BIW represents twice a week.

[0102] Figure 6 Survival curve of mice in each group, wherein Group represents group, ip represents intraperitoneal injection, and BIW represents twice a week. DETAILED DESCRIPTION

[0103] Hereinafter, only certain exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present disclosure. Therefore, the drawings and the description are considered to be exemplary in nature rather than limiting.

[0104] The preferred embodiments of the present disclosure are described below in conjunction with the accompanying drawings, it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0105] Unless otherwise defined herein, the scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by a person of ordinary skill in the art. In addition, unless otherwise required by the context, the singular forms of terms shall include the plural forms, and the plural forms of terms shall include the singular forms. More specifically, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting. In addition, the ranges provided in the specification and the appended claims include the endpoints and all values between the endpoints.

[0106] Definitions

[0107] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0108] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains can be referred to as “VH” and “VL,” respectively. The variable regions in both the heavy and light chains generally comprise three hypervariable loops, referred to as complementarity determining regions (CDRs) (light chain CDRs (LCDRs), including LCDR1, LCDR2, and LCDR3, heavy chain CDRs (HCDRs), including HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are flanked by regions known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified by the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, d, e, g, and m heavy chains, respectively. Several of the major antibody classes are divided into subclasses, e.g., lgG1 (g1 heavy chain), lgG2 (g2 heavy chain), lgG3 (g3 heavy chain), lgG4 (g4 heavy chain), lgA1 (a1 heavy chain), or lgA2 (a2 heavy chain).

[0109] As used herein, the term “antigen-binding fragment” refers to an antibody fragment, including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, a Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (dsdiabody), a single chain Fv (scFv), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelid single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise an entire antibody structure. An antigen-binding fragment is capable of binding to the same antigen bound by the parent antibody or parent antibody fragment (e.g., a parent scFv). In some embodiments, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted to framework regions from one or more different human antibodies.

[0110] As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites found within the variable region of both heavy and light chain polypeptides. These particular regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. Of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); Mac Callum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared to each other. However, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The columns listing the amino acid residues of the CDRs as defined by each of the above-cited references are set forth below in Table 1 for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated by reference herein in their entirety for use in the present disclosure and can be included in one or more claims herein. In some embodiments, the CDR sequences provided herein are based on the IMGT definition.For example, CDR sequences can be determined by the VBASE2 tool (http: / / www.vbase2.org / vbase2.php, see also Retter I, Althaus HH, Munch R, Muller W: VBASE2 an integrative V gene database. Nucleic Acids Res. 2005 Jan 1;33(Database issue):D671-4, which is incorporated by reference in its entirety). The recitation "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof, following the nomenclature of Honegger and Pluckthun, supra, refers to the numbering system used for the heavy chain variable domains or light chain variable domains in the antibody compilation of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids, which correspond to a shortening of, or insertions into, the FRs or hypervariable regions (HVRs) of the variable domain. For example, a heavy chain variable domain can include a single amino acid insertion (residue 52a according to Kabat) after residue 2 of H2 and insertions of residues (e.g. residues 82a, 82b and 82c according to Kabat, etc.) after residue 82 of the heavy chain FR. Kabat numbering of residues in a given antibody can be determined by alignment of the sequence of the antibody with the "standard" Kabat numbered sequence. Unless otherwise specified herein, numbering of amino acid residues in the immunoglobulin heavy chain is according to the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the numbering of the residues of the human IgGl EU antibody.

[0111] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as defined herein.

[0112] “Percent (%) amino acid sequence identity” or “homology” with respect to a polypeptide and antibody sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences and considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning a particular sequence, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R. C, Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R. C, BMC Bioinformatics 5(1): 113, 2004).

[0113] “Homologous” refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. A molecule is homologous at a position when the position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if the respective position in two protein molecules is occupied by lysine, or if the respective position in two DNA molecules is occupied by adenine. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if 6 of the 10 positions in two sequences are matched or homologous, then the two sequences have 60% homology. By way of example, the protein sequences SGTSTD and TGTSDA have 50% homology. Typically, the comparison is made over the full length of the two sequences to provide maximal homology. The term “constant domain” refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to other portions of the immunoglobulin that comprise the antigen binding site (the variable domain). Constant domains include the CH1, CH2 and CH3 domains of the heavy chain (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

[0114] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0115] The “CH1 domain” (also referred to as “C1” of the “H1” domain) generally extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0116] The "hinge region" is generally defined as the region of IgG corresponding to Glu216 to Pro230 of human IgGl (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge region of other IgG isotypes can be aligned with the IgGl sequence by placing the first and last cysteine residues that form inter-heavy chain S-S bonds in the same position. The "CH2 domain" (also referred to as the "C2" domain) of a human IgG Fc region generally extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is postulated that the carbohydrate can provide a substitute for domain-domain pairing and help to stabilize the CH2 domain. Burton, Molec. Immunol. 22:161-206 (1985).

[0117] The "CH3 domain" (also referred to as the "C2" domain) comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 of IgG).

[0118] The term "Fc region" or "fragment, crystallizable region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue Cys226, or from Pro230, to the carboxy-terminus. For example, the C-terminal lysine (residue 447 according to EU numbering system) of the Fc region can be removed, e.g., in the production or purification of an antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of an antibody. Accordingly, the composition of a whole antibody can comprise a population of antibodies with all K447 residues removed, a population of antibodies with no K447 residues removed, and a population of antibodies containing a mixture of antibodies with and without K447 residues. Native sequence Fc regions suitable for use with the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0119] “Fc receptor” or“FcR” describes a receptor that binds the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes FcyRI, FcyRII, FcRN, and FcyRIII subclasses of receptors including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an“activating receptor”) and FcyRIIB (an“inhibiting receptor”), which have similar amino acid sequences but differ in their cytoplasmic domains. The activating receptor FcyRIIA contains immunoreceptor tyrosine-based activation motifs (ITAM) in its cytoplasmic domains. The inhibiting receptor FcyRIIB contains immunoreceptor tyrosine-based inhibition motifs (ITIM) in its cytoplasmic domains. (See M. Annu. Rev. Immunol. 15:203-234 (1997)). FcRN is important for recycling antibodies from the blood into the blood and can extend the serum half-life of an antibody. Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immuno methods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995) review FcRs. Other FcRs, including those to be identified in the future, are encompassed by the term“FcR” herein.

[0120] As used herein, the term“epitope” refers to specific atoms or amino acid groups on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they can bind to the same epitope within the antigen.

[0121] As used herein, a first antibody or fragment thereof“competes” for binding to a target antigen with a second antibody or fragment thereof when the first antibody or fragment thereof inhibits target antigen binding by the second antibody or fragment thereof by at least about 50% (e.g., by at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of equimolar concentrations of the first antibody or fragment thereof. The high-throughput process for“binning” antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0122] As used herein, the terms "specifically binds," "specifically recognizes," and "is specific for" refer to a measurable and reproducible interaction such as binding of a target to an antibody or antibody portion, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody portion that specifically recognizes a target (which can be an epitope) is an antibody or antibody portion that binds that target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In some embodiments, the extent of binding of an antibody to an unrelated, non-target, is less than about 10% of the binding of the antibody to the target, for example as measured by a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target (or is specific for an antigen) means that the antibody binds to the target with a KDvalue of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of an antibody or antigen binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, BLI-, RIA-, ECL-, IRMA-, EIA-, BIACORE™-testing and peptide scanning.

[0123] An "isolated" or "purified" antibody (or construct) is one which has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from association with all other components of its production environment.

[0124] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its source of production. Preferably, an isolated nucleic acid is free from association with all components with which it is associated in its natural environment. The forms of the isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are different from those in which the nucleic acids are initially discovered. Thus, an isolated nucleic acid molecule encoding a polypeptide or antibody described herein is not the in situ, chromosomally-embedded state, nor is it the native state, e.g., associated with chromosome or chromosome fragments in the cell. The isolated nucleic acid molecule includes a nucleic acid molecule in the host cell's chromosome or a non-chromosomal state.

[0125] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are appropriate for a given organism can vary, but they always include at least promoter and ribosome binding site sequences. Appropriate transcription and translation control elements will be readily identified by one skilled in the art and will vary with the host organism.

[0126] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished over a distance of up to and including 25 nucleotides.

[0127] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been 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."

[0128] The term "transfected" or "transformed" or "transduced" as used herein refers to the process of introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. The cell includes the primary transformed cell and progeny of the primary transformed cell.

[0129] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny of the primary transformed cell that have the same functional or biological activity as screened or selected for in the initially transformed cell. Mutated progeny that have the same functional or biological activity as screened or selected for in the initially transformed cell are included herein.

[0130] The term "immunoconjugate" includes reference to the covalent attachment of a therapeutic agent or detectable marker to an antibody, e.g., an antibody moiety described herein. The linkage can be direct or indirect through a linker, e.g., a peptide linker.

[0131] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms resulting from the disease, diminishment of extent of disease, stabilization of the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying spread (e.g., metastasis) of the disease, preventing or delaying recurrence of the disease, delay or slowing of disease progression, amelioration of the disease state, providing a remission from the disease (partial or total), decreasing the dose of one or more other medications required to treat the disease, delay of disease progression, increasing or improving quality of life, increasing body weight, and / or prolonging survival. “Treatment” also includes reducing the pathological consequences of cancer (e.g., tumor volume). The methods of the present disclosure contemplate any one or more of these therapeutic aspects.

[0132] In the context of cancer, the term “treatment” includes any or all of: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0133] The terms “inhibition” or “inhibit” refer to a reduction or cessation of any phenotypic characteristic or a reduction or cessation in the incidence, extent, or likelihood of the characteristic. “Reducing” or “inhibiting” refers to a decrease, reduction, or prevention of an activity, function, and / or amount as compared to a reference. In certain embodiments, “reducing” or “inhibiting” refers to the ability to cause an overall reduction of 20% or more. In another embodiment, “reducing” or “inhibiting” refers to the ability to cause an overall reduction of 50% or more. In yet another embodiment, “reducing” or “inhibiting” refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more. As used herein, “reference” refers to any sample, standard, or level used for comparison purposes. The reference can be obtained from a healthy and / or non-diseased sample. In some examples, the reference can be obtained from an untreated sample. In some examples, the reference is obtained from a non-diseased or untreated sample of the individual. In some examples, the reference is obtained from one or more healthy individuals that are not the individual or patient.

[0134] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, inhibit, and / or postpone development of disease (e.g., cancer). This delay can be of varying lengths, depending on the history of the disease and / or individual being treated. As is evident to one of ordinary skill in the art, a sufficient or significant delay can, in effect, encompass prevention, i.e., the individual does not develop the disease. For example, development of a late stage cancer, such as metastasis, can be delayed.

[0135] As used herein, “preventing” includes providing prophylaxis of the onset or recurrence of a disease in an individual who can be predisposed to the disease but has not yet been diagnosed with the disease.

[0136] As used herein, “inhibiting” a function or activity refers to a decrease in the function or activity when compared to otherwise identical conditions except for the subject condition or parameter, or optionally, when compared to another condition. For example, an antibody that inhibits tumor growth decreases the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0137] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, a human, a bovine, an equine, a feline, a canine, a rodent, or a primate. In some embodiments, the individual is a human. An “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result. The specific dosage can vary according to one or more of the following: the particular agent selected; the dosing regimen to be followed; whether it is administered in combination with other compounds; the time of administration; the tissue to be imaged; and the physical delivery system in which the agent is carried.

[0138] A “therapeutically effective amount” of a substance / molecule, agonist, or antagonist of the present disclosure can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule, agonist, or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations.

[0139] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, a prophylactically effective amount will be less than a therapeutically effective amount.

[0140] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation which is in such form as to permit biological activity of an active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0141] A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation, formulation auxiliary, or vehicle which is commonly used in the art of pharmaceutical formulation for use with therapeutic agents, together with which it comprises a “pharmaceutical composition” for administration to an individual. A pharmaceutically acceptable carrier is non-toxic with the dosages and concentrations employed, and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is suitable for the formulation used.

[0142] A "sterile" formulation is free or essentially free of living microorganisms and their spores.

[0143] Administration "in combination" with one or more other therapeutic agents includes concurrent (synchronous) and consecutive or sequential administration in any order.

[0144] The term "synchronous" is used herein to refer to administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where administration of one therapeutic agent falls within a short period of time relative to administration of another therapeutic agent. For example, the two or more therapeutic agents are administered within a time interval of no more than about 60 minutes, for example no more than any of about 30, 15, 10, 5, or 1 minute.

[0145] The term "sequential" is used herein to refer to administration of two or more therapeutic agents where administration of one or more agents continues after administration of one or more other agents has ceased. For example, the two or more therapeutic agents are administered with a time interval of more than about 15 minutes, for example any of about 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month or more.

[0146] As used herein, "in combination" refers to administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in combination" refers to administration of one therapeutic modality prior to, during, or following administration of another therapeutic modality to an individual.

[0147] An "article of manufacture" is any manufacture (for example, a package or container) or kit containing at least one pharmaceutical agent (for example, a drug for treating a disease or disorder (for example, a cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing a method described herein.

[0148] In the present disclosure, the amino acid at a certain position can be allowed to vary, for example, [K / R] indicates that the amino acid at this position can be K or R; for another example, [S / *] indicates that the amino acid at this position can be S or absent.

[0149] It should be understood that the embodiments of the disclosure described herein include "consisting" and / or "consisting essentially of embodiments.

[0150] Unless otherwise indicated, the techniques and conditions in the examples are carried out in accordance with the techniques and conditions described in the literature (for example, refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, translated by Huang Peitang et al.) or in accordance with the product instructions. Unless otherwise indicated, the reagents or instruments used are all conventional products that can be obtained commercially.

[0151] Anti-TREM2 antibodies

[0152] In some aspects, the application includes an isolated antibody or antigen binding fragment thereof.

[0153] In the context of the present application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR- grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule that exhibits preferential association or binding to a TREM2 protein. Furthermore, unless otherwise specified by context, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or an improved chimeric monoclonal antibody.

[0154] The variable regions and CDRs in the antibody sequences can be identified according to general rules that have been developed in the art (as described above, for example, the Kabat numbering system or by aligning the sequences with a database of known variable regions.

[0155] In some embodiments, the isolated antibody or antigen-binding fragment thereof can comprise conservative substitutions or modifications or substitutions (e.g., conservative substitutions), deletions, or additions of amino acids in the variable region of the heavy chain and / or light chain (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; or up to 20, up to 15, up to 10, or up to 5 conservative substitutions) or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity to the antibody or antigen fragment thereof from which it is derived. Amino acid deletion variants comprising deletions at the N- and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncation variants. It is understood in the art that certain conservative sequence modifications can be made which do not abrogate antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O’Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43.

[0156] As described above, the term "conservative substitution / conservative replacement" as used herein refers to amino acid substitutions / replacements that do not adversely affect or alter the essential properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions / replacements can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a substitution of a physically or functionally similar residue (e.g., a residue having similar size, shape, charge, chemical properties including ability to form covalent or hydrogen bonds, etc.) for the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), uncharged polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a corresponding amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0157] According to the present application, if reference is made to an antibody comprising a specific antibody heavy chain and / or a specific antibody light chain (e.g., a chain comprising a specific CDR sequence), the two heavy chains and / or two light chains of the antibody are preferably each composed of the specific antibody heavy chain and / or the specific antibody light chain, respectively.

[0158] Regardless of how the antibody is produced, methods of testing the ability of an antibody to bind to an antigen (e.g., TREM2) are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assay (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competitive assays).

[0159] According to the present application, in a standard assay (e.g., an assay described in the present application), an antibody is capable of binding to a predetermined target (e.g., a TREM2 protein or a cell expressing TREM2) if the antibody has significant affinity for the predetermined target, and to test the binding of a monoclonal antibody to a live cell expressing TREM2, flow cytometry can be used. Preferably, in a flow cytometry analysis (FACS) assay, the binding of the antibody to a target expressed on the surface of a cell is assayed, and the antibody is capable of binding to the target if the antibody binds detectably to the target (TREM2 protein or cell expressing TREM2) with "affinity." Preferably, the antibody of the present application binds detectably to the target if the antibody is present at a concentration of 10 μg / mL or less, 5 μg / mL or less, 3 μg / mL or less, 2 μg / mL or less, 1 μg / mL or less, 0.5 μg / mL or less.

[0160] In some embodiments, the antibody or fragment has a binding property of binding human, murine, or monkey TREM2 with a KD of 1 x 10 -7 M or less; binding human, murine, or monkey TREM2 with a KD of 5 x 10 -8 M or less; binding human, murine, or monkey TREM2 with a KD of 1 x 10 -8 M or less; binding human, murine, or monkey TREM2 with a KD of 5 x 10 -9 M or less; binding human, murine, or monkey TREM2 with a KD of 1 x 10 - 9 M or less; binding human, murine, or monkey TREM2 with a KD of 1 x 10 -9 M or less.

[0161] In some embodiments, the antibody or fragment has an EC50 binding property of binding human TREM2 or monkey TREM2 with an EC50 of 5 μg / mL or less; binding human TREM2 or monkey TREM2 with an EC50 of 3 μg / mL or less; or binding human TREM2 or monkey TREM2 with an EC50 of 2 μg / mL or less; or binding human TREM2 or monkey TREM2 with an EC50 of 1 μg / mL or less; or binding human TREM2 or monkey TREM2 with an EC50 of 0.5 μg / mL or less; binding human TREM2 or monkey TREM2 with an EC50 of 0.3 μg / mL or less; or binding human TREM2 or monkey TREM2 with an EC50 of 0.1 μg / mL or less.

[0162] The TREM2 specificity described herein refers to the ability to bind to one or more TREM2 epitopes, in particular to a native conformation of a TREM2 epitope, in particular human TREM2 specificity.

[0163] In some embodiments, the antibody is a monoclonal antibody.

[0164] In some embodiments, the antibody is a chimeric antibody or a humanized antibody or an improved antibody such as an improved chimeric antibody.

[0165] In some embodiments, the isolated antibody or antigen binding fragment thereof comprises a constant region of an IgG. The constant region of an IgG is preferably selected from the group consisting of a constant region of an IgGl, IgG2, IgG3, or IgG4. The constant region of an IgG is more preferably selected from the group consisting of a constant region of an IgGl.

[0166] In the art, various means are employed to diversify the modification of an antibody without changing the desired properties of the antibody, such as the way the light chain and the heavy chain of an antibody are recombined, the substitution of amino acids, etc. as employed in the present disclosure. For example, the sequences in the present invention, including the chimeric antibody sequences or the humanized antibody sequences, can be subjected to conservative amino acid substitutions.

[0167] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences of the CDRs are more diverse between antibodies than other sequences. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express a recombinant antibody that mimics the properties of a particular naturally occurring antibody by constructing an expression vector that includes the CDR sequences from the particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germlines are sequences different from mature antibody gene sequences because they do not include a complete set of assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also have sequences different from a high affinity secondary repertoire antibody at individual places uniformly across the variable region.

[0168] Mouse antibodies are highly immunogenic in humans, leading to a decrease in therapeutic efficacy upon repeated applications, mediated primarily through the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided if each antibody is chimerized or humanized.

[0169] A chimeric antibody refers to an antibody having different portions from different animal species, for example, an antibody having variable regions from a mouse antibody and human immunoglobulin constant regions. The variable regions of the heavy and light chains of a mouse antibody are linked to the constant regions of the human heavy and light chains to obtain a chimeric antibody (e.g., as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by linking a human kappa light chain constant region to a mouse light chain variable region. In another preferred embodiment, a chimeric antibody can be produced by linking a human lambda light chain constant region to a mouse light chain variable region.

[0170] A humanized antibody, on the other hand, refers to an antibody in which CDR sequences / antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

[0171] To reduce the immunogenicity of the antibodies to humans, humanized anti-TREM2 antibodies are produced using the sequences of the TREM2 antibodies of the present disclosure, using the CDR regions of the murine anti-TREM2 antibodies in combination with human-derived framework regions (e.g., human immunoglobulin) to form the humanized anti-TREM2 antibodies of the present disclosure, which are expected to retain the function of binding to human TREM2 as well as the function of binding to monkey TREM2.

[0172] Preparation or production of antibodies

[0173] The antibodies of the present application can be produced by a variety of techniques, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although the hybridoma technique is preferred, in principle, other techniques for producing monoclonal antibodies can be employed, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using antibody gene libraries, somatic cell hybridization methods, and the like, or by recombinant techniques, e.g., by gene engineering. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells, and the transfected host cells are then cultured under specific conditions, and the antibodies of the present application are expressed.

[0174] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al.. Am. J. Clin. Pathol. 124:295 (2005)). Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0175] Monoclonal antibodies can be produced using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, or some combination thereof. For example, monoclonal antibodies can be produced using hybridoma and art-recognized biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed. 2009; Shire et. al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), each of which is hereby incorporated by reference in its entirety.

[0176] It will be appreciated that the selected binding sequences can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and antibodies comprising the altered target binding sequences are also antibodies of the application.

[0177] In some embodiments, the method of producing an antibody or fragment of the present disclosure comprises the steps of:

[0178] (i) expressing the antibody or fragment in a host cell; and optionally

[0179] (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

[0180] In a preferred embodiment, the anti-TREM2 monoclonal antibodies are prepared by using hybridomas.

[0181] To obtain hybridomas that produce antibodies of the application, e.g., human monoclonal antibodies of the application, spleen and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0182] Antibodies of the application can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In some embodiments, DNA encoding partial or full-length light and heavy chains will be inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector in such a way that transcriptional and translational control sequences within the vector

[0183] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to produce a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes a heavy chain constant region and a light chain constant region of the desired isotype, such that the V segment is operably linked to the CH segment and the VL segment is operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain(s) from a host cell. The antibody chain genes can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0184] To express the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected using standard techniques into host cells. The various forms of the term "transfect" are intended to encompass a variety of techniques for getting foreign DNA into a prokaryotic or eukaryotic host cell such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection and the like. The antibodies of the application can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete an appropriately folded and immunologically active antibody.

[0185] Mammalian host cells for expressing the recombinant antibodies of the application include Chinese hamster ovary cells (CHO cells) with a DHFR selection marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159:601-621) used in conjunction with a GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841, NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or to permit the secretion of the antibody into the culture medium in which the host cells are grown. Antibody can be recovered from the culture medium using standard protein purification methods.

[0186] In another preferred embodiment, transgenic or transchromosomal mice with a partial human immune system (rather than a mouse system) can be used to generate human monoclonal antibodies to TREM2.

[0187] Another strategy for generating monoclonal antibodies is to isolate the genes encoding the antibodies directly from antibody-producing lymphocytes of defined strategy, see for example Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering see also Welschof and Krau, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

[0188] To create a chimeric antibody, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). An isolated nucleic acid encoding a VHand can be converted to a full-length heavy chain gene by operably linking the VHand to another DNA molecule encoding a heavy chain constant region (CHI, CH2, and CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably is an IgGl or IgG4 constant region. An isolated nucleic acid encoding a VLregion can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VLto another DNA molecule encoding a light chain constant region, CL. Sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region, but a kappa constant region is generally preferred. Once DNA fragments encoding VHand VLsegments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, DNA fragments encoding VLor VHand another protein-encoding DNA fragment, such as an antibody constant region or a flexible linker, are operably linked. The term "operably linked," as used in this document, is intended to mean the joining together of two DNA fragments in such a way that the amino acid sequence encoded by the two DNA fragments remains in frame.

[0189] To prepare humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can also be utilized that are capable of producing a complete repertoire of human antibodies upon immunization and that are incapable of producing endogenous immunoglobulins. For example, it has been reported that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice completely inhibits endogenous antibody production from these mice, and that the subsequent transplantation of human germ-line immunoglobulin gene arrays allows the complete production of a human repertoire of antibodies (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of such transgenic animals include the HuMAb mouse (Medarex, Inc.), which contains human immunoglobulin gene miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474): 856-859); or the "KM mouse™" (see patent application WO 02 / 43478) which carries human heavy chain transgene and human light chain transchromosome. Other methods of humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0190] Nucleic acid molecules encoding antibodies of the invention

[0191] In some aspects, the present application relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an isolated antibody or fragment thereof as described above in the present disclosure.

[0192] Nucleic acids of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding such antibodies can be recovered from the gene library.

[0193] To make chimeric antibodies, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). An isolated nucleic acid encoding a VHand can be converted to a full-length heavy chain gene by operatively linking the nucleic acid encoding the VHand another DNA molecule encoding heavy chain constant regions (CHI, CH2, and CH3), and DNA fragments comprising these regions can be obtained by standard PCR amplification. An isolated nucleic acid encoding a VLcan be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the DNA encoding the VLto another DNA molecule encoding a light chain constant region, CL. Once DNA fragments encoding the VHand VLsegments have been obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as to convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragments encoding the VLor VHand another DNA fragment, such as an antibody constant region or a flexible linker, are operatively linked. An "operatively linked," as used herein, means that the two DNA fragments are connected in a functional relationship, i.e., the DNA fragments are ligated so that the amino acid sequences encoded by the DNA fragments are expressed in a single polypeptide.

[0194] In some embodiments, the present application relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof as described by the present disclosure.

[0195] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule described above.

[0196] In one aspect, the present disclosure provides a host cell comprising the nucleic acid molecule as described above or the vector as described above.

[0197] Conjugates

[0198] In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as previously described coupled to at least one detectable label. Detectable labels include, but are not limited to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g., FRET (Fluorescence Resonance Energy Transfer); (iii) affect mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape, or other physical parameter, or (iv) provide a capture moiety, e.g., an affinity, antibody / antigen, or ionic complex.

[0199] Suitable structures for labels are, for example, fluorescent labels, luminescent labels, chromophoric labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal particulate labels, magnetic particulate labels, polymeric particulate labels), small organic molecules (e.g., biotin, a ligand or binding molecule for a receptor (e.g., a cell adhesion protein or a phospholipid), a label sequence comprising nucleic acid and / or amino acid residues that can be detected using a binding agent, etc. Labels include, without limitation, barium sulfate, ioxitalamic acid, ipodate, calcium iodipamide, sodium amidotrizoate, meglumine amidotrizoate, meglumine metrizamide, sodium tyropanoate, and radiodiagnostic agents (including positron emitters (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131, and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium), luminous substances (e.g., isoluminol and acridinium ester), fluorescent substances (e.g., fluorescein and rhodamine), colored substances (e.g., latex particles and colloidal gold).

[0200] Detectable labels as described above can be detected by methods known in the art. For example, fluorescent labels can be detected using a light detector to detect emitted light. Enzymatic labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by action of the enzyme on the substrate. In certain embodiments, such labels can be adapted for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, detectable labels as described above can be linked to antibodies or antigen-binding fragments thereof of the present disclosure via linkers of varying lengths to reduce potential steric hindrance.

[0201] Antibody drug conjugates / immunoconjugates

[0202] In one aspect, the present disclosure provides an antibody drug conjugate comprising an antibody, which includes one or more drug moieties / therapeutic agents, the drug moieties being linked (e.g., covalently linked) to the antibody or fragment thereof as previously described, either directly or via a linker. The linker structure conjugating the anti-TREM2 antibody to the drug in the antibody-drug conjugates of the present application is not particularly limited as long as the resulting antibody-drug conjugate can be used.

[0203] Because the antibody-drug conjugates have the ability to selectively deliver one or more drugs to a target tissue (e.g., a regulatory myeloid cell), the antibody-drug conjugates can improve the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present application in treating a disease (e.g., cancer).

[0204] Multispecific molecules

[0205] The antibodies or antigen-binding fragments thereof of the present application can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present application can be part of a multispecific molecule (e.g., a bispecific molecule) that comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a different binding specificity than the antibodies or antigen-binding fragments thereof of the present application, thereby being able to bind at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present application can be linked to a second antibody or antigen-binding fragment thereof that is capable of specifically binding to any protein that can serve as a potential target for a combination therapy. To generate such bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present application can be linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

[0206] Thus, in some aspects, the present application provides a multispecific molecule comprising an antibody or antigen-binding fragment thereof of the present application.

[0207] In certain preferred embodiments, the multispecific molecule specifically binds to TREM2 (e.g., human TREM2 or monkey TREM2), and specifically binds to one or more other targets.

[0208] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.

[0209] In certain preferred embodiments, the multispecific molecule is a bispecific antibody.

[0210] Pharmaceutical compositions

[0211] In some aspects, the present application relates to pharmaceutical compositions, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or fragment as previously described, a nucleic acid molecule as previously described, a vector as previously described, a host cell as previously described, a conjugate as previously described, an antibody drug conjugate as previously described, a multispecific molecule as previously described; and a pharmaceutically acceptable carrier.

[0212] The pharmaceutical composition can optionally contain one or more additional pharmaceutically active ingredients, such as another anti

[0213] The pharmaceutical composition of the present application can also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-TREM2 antibody enhances the immune response to the vaccine. The pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending / dispersing agent, chelating agent, diluent, adjuvant, excipient or non-toxic auxiliary substance, combinations of various components known in the art or more.

[0214] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiothio sulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated hydroxyanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed herein, an antibody or antigen-binding fragment thereof comprising one or more antioxidants such as methionine can be oxidized in a solvent containing the antibody or antigen-binding fragment thereof of the present disclosure. Oxidation can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Accordingly, in some embodiments, the present application provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present application further provides methods in which an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine, such that the antibody or antigen-binding fragment thereof can be prevented from oxidizing, to extend its shelf life and / or increase activity.

[0215] For further explanation, the pharmaceutically acceptable carrier can include, for example, aqueous vehicles, such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer injection, non-aqueous vehicles such as fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial or antifungal agents, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate, antioxidants such as sodium bisulfate, local anesthetics such as procine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydropropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to a pharmaceutical composition in a multi-dose container comprising phenolics or cresylics, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0216] Administration, formulation, and dosage

[0217] The pharmaceutical compositions of the present application can be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present application can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. The appropriate preparation and administration route can be selected depending on the intended application and therapeutic regimen.

[0218] Suitable preparations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalers, and controlled release dosage forms thereof.

[0219] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). These liquids can additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulations isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, e.g., water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosage regimen (including dosing, timing, and repetition) will depend on the particular individual and the individual's medical history, and empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0220] The requirements for effective pharmaceutical carriers for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0221] The frequency of administration can be determined and adjusted during the course of therapy and is based on reducing the number of proliferating or tumorigenic cells, maintaining such reduction in tumor cells, reducing proliferation of tumor cells, or delaying development of metastases. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the application can be appropriate.

[0222] Those of skill will appreciate that an appropriate dosage can vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route, time and

[0223] Generally, the antibodies or antigen binding fragments thereof of the present application can be administered in a variety of ranges. These include about 4 μg / kg body weight to about 100 mg / kg body weight per dose; about 20 μg / kg body weight to about 50 mg / kg body weight per dose; about 50 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 15 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0224] In certain preferred embodiments, the course of treatment involving the antibodies or antigen binding fragments thereof of the present application will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen binding fragments thereof of the present application can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be appreciated that the dosage or interval can be altered based on patient response and clinical practice.

[0225] Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the antibodies or antigen binding fragments thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the antibodies or antigen binding fragments thereof will include 10 μg / mL, 20 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 800 μg / mL, 900 μg / mL, or 1 mg / mL. In other preferred embodiments, the antibody conjugate drug will include 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0226] The antibodies of the present application can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, antitumor agents, anti-angiogenic agents, or immunosuppressive agents) to reduce the induction of an immune response against the anti-TREM2 antibodies of the present application. The antibodies can be linked to the therapeutic agents (as an immunoconjugate) or can be administered separately from the therapeutic agents.

[0227] In the context of administering a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not restrict the order in which treatments or therapeutic agents are administered to a subject. A treatment or therapeutic agent can be administered prior to, simultaneously with, or following the administration of a second treatment or therapeutic agent to a patient. Preferably, the treatments or therapeutic agents are administered to a subject in an order, amount, and / or at an interval of time such that they act together. In a particular embodiment, the treatments or therapeutic agents are administered to a subject in an order, amount, and / or at an interval of time such that they provide an increased benefit than if administered otherwise, in particular independently of each other. Preferably, the increased benefit is a synergistic effect.

[0228] Medical uses

[0229] The antibodies, antibody compositions, and methods of the present application have a number of in vitro and in vivo uses, including, for example, detection of TREM2 or enhancement of an immune response. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to a human subject.

[0230] Preferred subjects include mammals, e.g., humans / patients. Mammals in the context of the present application are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as animals in zoos.

[0231] Treatment of disorders associated with TREM2 expression

[0232] In some aspects, the present application provides a method of treating a disorder in a mammal, comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0233] In some aspects, the present disclosure provides a method for treating or determining the prognosis of a disease associated with TREM2 expression in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0234] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method of treating or determining a prognosis of a disease associated with TREM2 expression in a subject.

[0235] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method of treating or determining a prognosis of a disease associated with TREM2 expression in a subject.

[0236] In one embodiment, the disease associated with TREM2 comprises a neoplastic disease, e.g., a cancer.

[0237] In some embodiments, the disease associated with TREM2 is a cancer, preferably selected from the group consisting of:

[0238] gastric cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, brain glioma, ovarian cancer.

[0239] The antibody or antigen-binding fragment thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiotherapy.

[0240] The antibody or antigen-binding fragment thereof can be used in combination with an anti-cancer agent, a cytotoxic agent or a chemotherapeutic agent.

[0241] The term "anti-cancer agent" or "anti-proliferative agent" means any agent useful in the treatment of a cellular proliferative disorder, such as a cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapies, radiation therapies, anti-metastatic agents, and immunotherapeutic agents. It will be appreciated that in selected embodiments as described above, such anti-cancer agents can comprise conjugates and can be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, a selected anti-cancer agent will be linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly contemplated within the scope of the present application. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

[0242] In some embodiments, the disease associated with TREM2 is a neurodegenerative disease selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), autism spectrum disorders (ASDs), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS).

[0243] In some embodiments, the disease associated with TREM2 is a metabolic syndrome selected from the group consisting of adipocyte hypertrophy, systemic hypercholesterolemia, inflammation, and glucose intolerance.

[0244] Diagnosis

[0245] The present disclosure provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, including contacting a patient or a sample obtained from a patient (in vivo or in vitro) with an antibody described herein and detecting the presence or absence or level of binding of the antibody to bound or free target molecules in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0246] In some aspects, the present disclosure provides a method of diagnosing, detecting or monitoring a disease associated with TREM2, comprising administering to a subject in need thereof an effective amount of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0247] In some aspects, the present disclosure provides a method of diagnosing, detecting or monitoring a disease associated with TREM2 in a subject, comprising administering to the subject an effective amount of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0248] In another aspect, the present disclosure provides use of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a medicament for diagnosing, detecting or monitoring a disease associated with TREM2 expression.

[0249] The sample can be analyzed by a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluoroimmunoassay, immunoblot assay, Western blot analysis, and flow cytometry assay. Compatible in vivo diagnostic or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.

[0250] The methods described herein for detecting or monitoring TREM2 expression or levels of TREM2-expressing cells in vitro can also be used for non-diagnostic purposes.

[0251] Preferred subjects include mammals, such as a human / patient in need.

[0252] The sample from the subject is blood, excretion (urine or feces), oral or nasal secretions, or alveolar lavage fluid, interstitial fluid, sweat, or an extract thereof from the subject.

[0253] Pharmaceutical packaging and kits

[0254] Also provided are pharmaceutical packs and kits comprising one or more containers containing one or more dosages of an antibody or antigen-binding fragment thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such unit doses are supplied as single-unit dosage preparations in a ready-to-use form in pre-charged, disposable syringes. In other embodiments, the composition contained in the unit dose can include saline, sucrose or the like; buffers such as phosphate, and / or other agents that are known or suspected to be compatible with the delivery of the conjugate composition. Alternatively, in certain embodiments, the conjugate composition can be supplied as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, such as sterile water or a saline solution. In certain preferred embodiments, the composition comprises one or more agents that inhibit protein aggregation, including but not limited to sucrose and arginine. Any labels on the container or associated with the container indicate that the conjugate composition contained therein is used for treating a selected neoplastic disease condition.

[0255] Such kits will typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anti-cancer agents or other agents in the same or different container(s). The kits can also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

[0256] More specifically, the kits can have a single container containing an antibody of the disclosure or antigen-binding fragment thereof, with or without additional components, or they can have separate containers for each of the desired reagents. Where a combination therapeutic is provided for conjugation, the single solution can be premixed in molar equivalents or one component in excess of the other. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers prior to administration to the patient. The kits can also include second / third container means for containing sterile pharmaceutical acceptable buffers or other diluents, e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

[0257] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit can be provided as dry powders. When the reagents or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent can also be provided in another container.

[0258] Examples

[0259] Example 1 Antigen preparation, mouse immunization and screening

[0260] A. DNA antigen preparation

[0261] The method for immunizing Balb / c mice with human TREM2 DNA antigen was as follows: a cDNA sequence encoding human TREM2 (Uniprot ID Q9NZC2) was synthesized (the sequence is shown as SEQ ID NO: 1), and the coding sequence of the above gene was cloned into an expression vector by enzyme digestion.

[0262] SEQ ID NO: 1 sequence:

[0263] ATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCCCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGCCCCTATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGAGAGAAGGGCCCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCAGTGCCAGAGCCTCCATGGCAGTGAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTGGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCACAGCATCTCCAGGAGCCTCTTGGAAGGAGAAATCCCCTTCCCACCCACTTCCATCCTTCTCCTCCTGGCCTGCATCTTTCTCATCAAGATTCTAGCAGCCAGCGCCCTCTGGGCTGCAGCCTGGCATGGACAGAAGCCAGGGACACATCCACCCAGTGAACTGGACTGTGGCCATGACCCAGGGTATCAGCTCCAAACTCTGCCAGGGCTGAGAGACACGTGA

[0264] B. Preparation of protein antigens

[0265] The preparation method of Human TREM2 ECD-hFc protein for immunizing Balb / c mice is as follows: a nucleotide fragment (the sequence is shown as SEQ ID NO: 2) encoding the Fc fusion protein of the extracellular region of human TREM2 (Uniprot ID Q9NZC2) is synthesized and loaded into an expression vector by a DNA ligase. The plasmid containing Human TREM2 ECD-hFc is transfected into ExpiCHO (Gibco, cat: A29127) cells by a PEI transfection method. Nine days after transfection, the culture medium supernatant containing the target protein is harvested by centrifugation and filtration, and the target protein is purified by using an affinity column (Cytiva, cat: 17549851). The purified protein is identified by SDS-PAGE and SEC-HPLC and the like.

[0266] SEQ ID NO:2 sequence:

[0267]

[0268] C. Preparation of reference antibody PY314

[0269] The reference antibody used in this study is anti-TREM2 monoclonal antibody developed by Pionyr Immunotherapeutics, the amino acid sequence is derived from 37012 sequence in patent US2020 / 0140546A1, the DNA sequence of the variable region amino acid is synthesized (the sequence is shown as SEQ ID NO:3), and the heavy chain and light chain variable region sequences are respectively loaded on pTT5 vector (HonorGene, cat: HG-VPW0888) by DNA ligase, wherein the heavy chain constant region subtype is hlgG1, and the light chain constant region subtype is kappa. The plasmids containing the heavy chain and light chain of the tool antibody are co-transfected into ExpiCHO cells by PEI transfection method. Nine days after transfection, the culture medium supernatant containing the target antibody is harvested by centrifugation and filtration, and the antibody protein is purified by Protein A column (Cytiva, cat: 17549851). The purified antibody is identified by SDS-PAGE and SEC-HPLC. The amino acid sequence of the reference antibody PY314 is shown as SEQ ID NO:17 for the full-length sequence of the light chain, SEQ ID NO:18 for the full-length sequence of the heavy chain, SEQ ID NO:31 for the variable region sequence of the light chain, SEQ ID NO:32 for the variable region sequence of the heavy chain, SEQ ID NO:53-55 for LCDR1-LCDR3, and SEQ ID NO:74-76 for HCDR1-HCDR3.

[0270] SEQ ID NO:3 sequence:

[0271] Heavy chain:

[0272] GAGGTGCAGCTGCTGGAAAGCGGAGGCGGACTGGTGCAACCCGGCGGCAGCCTGAGACTGAGCTGCGCTGCTTCTGGCTTTACATTCAGCAACTACTACATGGCCTGGGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCAGCCTCACCAACAGCGGCGGATCTACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTACAAGAGAGTGGGCCGGCAGCGGCTACTTCGACTACTGGGGCCAGGGCACCCTGGTCACAGTGTCCTCT

[0273] SEQ ID NO: 4 sequence:

[0274] Light chain:

[0275] GACATCCAGATGACCCAGAGCCCATCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATTACATGCAAGGCTTCTCAGAACGTGGGCAACAACCTGGCCTGGTACCAGCAGAAACCTGGCAAGGCCCCTAAGCTGCTGATCTACTATACATCTAATAGATTCACCGGCGTCCCCAGCAGATTCAGCGGATCTGGCAGCGGCACAGATTTTACCCTGACCATCAGCAGCCTGCAACCTGAGGACTTCGCCACCTACTACTGTCAGCGGATCTACAACAGCCCCTGGACCTTCGGCCAGGGAACAAAGCTGGAAATCAAG

[0276] D. Generation and screening of monoclonal antibodies

[0277] Balb / c mice (commercially available mice, purchased from RIKEN) were immunized with the human TREM2 expression vector prepared above and the human TREM2 ECD-hFc protein prepared above. The DNA antigen was mixed with gold powder and freeze-dried, and the mice were immunized on the abdomen by gene gun multiple times. The expression vector DNA was 4 ug each time, and the interval was two weeks. After two times of immunization, the mice were bled to measure the titer, and the mice with high serological titer were selected for booster immunization. Each mouse was injected intraperitoneally with 25 ug of human TREM2 ECD-hFc protein, and 3 days later, the spleen, lymph node and bone marrow cells of the target animals were collected, respectively. The immune cells were separated and enriched by mouse CD138 + plasma cell separation kit (Miltenyi, cat: 130-092-530), and the plasma cells were the main antibody-secreting cells. After adjusting the concentration of the plasma cells, they were introduced into the independent pen on the chip through the Beacon light guide system (Berkeley Lights, BLIBeacon® Optofluidic System), and positive B cells were screened. Single cells can be directly introduced into a 96-well plate after being lysed in the pen of Beacon. The RNA of the lysed single cells was collected by Agencourt RNA Clean XP Beads (Beckman Coulter, cat: A63987), and then cDNA synthesis was performed using SMARTer® RACE5’ / 3’ kit (Clontech, cat: 634859). cDNA purification was performed by Agencourt AMPure XP Beads (Beckman Coulter, cat: A63881). The cDNA was used as a template, and the mouse Ig-Primer Set (Novagen, 69831-3) was used as a gene-specific primer (Gene-Specific Primer, GSP) for PCR amplification of the heavy chain and light chain variable regions.

[0278] Example 2 Detection of the binding activity of anti-TREM2 antibodies to cell surface huTREM2

[0279] The plasmids containing the heavy chain and light chain of the TREM2 antibody were co-transfected into FUT8-KO ExpiCHO (RIKEN constructed) cells by PEI transfection method. Nine days after transfection, the culture medium supernatant containing the target antibody was harvested by centrifugation and filtration, and then the antibody protein was purified by Protein A column (Cytiva, cat: 17549851). The purified antibody was identified by SDS-PAGE and SEC-HPLC, etc. The antibody is C1.

[0280]

[0281] SEQ ID NO: 5 antibody C1 light chain full-length sequence

[0282] DIVMTQAAPSVSVTPGESVSVSCRSSKSLLYSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0283] SEQ ID NO: 6 antibody C1 heavy chain full-length sequence

[0284] EVQLQQSGPVLVKPGASVKMSCQASGYTFTDYYMNWVKQSHGKSLEWIGFINPYSGTTNYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARSLYYYGSEGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0285] The EC50 of the antibody molecule binding to cell surface huTREM2 at different concentrations was detected by FACS to evaluate the binding activity.

[0286] The stably transfected cell line CHOK1 -human TREM2-DAP12 expressing human TREM2 antigen was cultured and treated as described above for flow cytometry method. The harvested cells after digestion were centrifuged at 300 g for 5 minutes at room temperature, and after discarding the supernatant, the cells were washed twice with FACS buffer (PBS + 2% FBS). The cells were placed in a 96-well plate at 2-5E+05 / well, and after centrifugation and discarding the supernatant, the cells were resuspended with the diluted antibody solution, 100 uL / well, mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with FACS buffer, and goat anti-human IgG-Alexa fluro488 secondary antibody solution was added, 100 uL / well, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with FACS buffer, and finally resuspended with PBS, and the signal value was detected on the BD flow cytometer.

[0287] The results, as shown in Figure 1A , Figure 1B and Figure 1C , showed that C1, G3, 2A6, 2A10, mAb006c and mAb008c had strong binding activity with cell surface human TREM2. It was shown that C1, G3, 2A6, 2A10, mAb006c and mAb008c could effectively bind to the cell surface of CHOK1 -human TREM2-DAP12 cells overexpressing human TREM2, and had a certain dose-dependent, and the binding ability of C1, G3, 2A6, 2A10, mAb006c and mAb008c was significantly better than the reference antibody PY314.

[0288] Example 3 Species Cross-reactivity Detection

[0289] The EC50 of C1, G3, 2A6, 2A10, mAb006c and mAb008c binding to cell surface CynoTREM2 at different concentrations was detected by FACS to evaluate their species cross-reactivity.

[0290] The stably transfected cell line CHOK1-cyno TREM2-DAP12 expressing monkey TREM2 antigen was cultured and treated as described above for flow cytometry method. The harvested cells after digestion were centrifuged at 300 g for 5 minutes at room temperature, and after discarding the supernatant, the cells were washed twice with FACS buffer (PBS + 2% FBS). The cells were placed in a 96-well plate at 2-5E+05 / well, and after centrifugation and discarding the supernatant, the cells were resuspended with the diluted antibody solution, 100 uL / well, mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with FACS buffer, and a goat anti-human IgG-Alexa fluro 488 secondary antibody solution was added, 100 uL / well, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with FACS buffer, and finally resuspended with PBS, and the signal value was detected on a BD flow cytometer.

[0291] As shown in Figure 2A , Figure 2B and Figure 2C , the 6 antibodies all have good cyno TREM2 binding activity, and the binding activity of C1, G3, 2A6, 2A10, mAb006c and mAb008 to Cyno is significantly better than that of the reference antibody.

[0292] The EC50 of C1, G3, 2A6, 2A10, mAb006c and mAb008c binding to cell surface mouse TREM2 at different concentrations was detected by FACS to evaluate the species cross-reactivity.

[0293] The stably transfected cell line CHOK1-mouse TREM2-DAP12 expressing mouse TREM2 antigen was cultured and treated as described above for flow cytometry method. The harvested cells after digestion were centrifuged at 300 g for 5 minutes at room temperature, and after discarding the supernatant, the cells were washed twice with FACS buffer (PBS + 2% FBS). The cells were placed in a 96-well plate at 2-5E+05 / well, and after centrifugation and discarding the supernatant, the cells were resuspended with the diluted antibody solution, 100 uL / well, mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with FACS buffer, and a goat anti-human IgG-Alexa fluro 488 secondary antibody solution was added, 100 uL / well, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded, the cells were washed twice with FACS buffer, and finally resuspended with PBS, and the signal value was detected on a BD flow cytometer.

[0294] As shown in Figure 3A , Figure 3B and Figure 3C , all of the 6 antibodies can bind to mouse TREM2, in which the binding activity of mAb008c, C1 and G3 antibodies is significantly better than that of the reference antibody.

[0295] Example 4 TREM2 reporter assay

[0296] Using genetically engineered Jurkat cells as effector cells, Jurkat cells stably express TREM2 receptor and DAP12, and Luciferase gene driven by NFAT response element expression, the activity of luciferase produced by NFAT pathway activation can be used to quantitatively detect the activation or inhibition of therapeutic antibodies on TREM2 signaling pathway.

[0297] The antibody is coated or mixed with Crosslinker (anti-Fc) for crosslinking, and then incubated with Jurkat-NFAT-TREM2-DAP12 cells in a 37°C incubator for 6 hours. An equal volume of ONE-Glo (Promega, E6120) is added, mixed, and then incubated at room temperature in the dark for 10-15 minutes. The fluorescence signal is read with a microplate reader, and the data is analyzed.

[0298] The degree of aggregation of the antibody is increased using crosslinker (Abs: crosslinker = 1: 0.5), and the NFAT signal of the clone is detected after incubation with Jurkat-TREM2-DAP12-NFAT cells at 37°C for 6 h. As shown in Figure 4 , the effects of C1, G3 and mAb008c antibodies on cells are slightly inhibited, and the inhibition effect is comparable to that of the reference antibody PY314, while 2A6, 2A10 and mAb006c have stronger inhibition on cells than PY314.

[0299] Example 5 Affinity detection

[0300] The affinity of C1, G3, 2A6, 2A10, mAb006c and mAb008c is detected by Biacore.

[0301] SPR technology was used for capture method detection, CM5 chip coupled with Anti-mFc antibody was used to capture His-tagged recombinant hTREM2, cyno-TREM2 or mTREM2 protein as ligand (Ligand), different concentrations of TREM2 antibody were set as analyte (Analysis), and the kinetic detection was carried out with captured human recombinant hTREM2, cyno-TREM2 or mTREM2 protein, Biacore software was used for Kinetics fitting of the binding curve, and the affinity was obtained, and the experimental results are shown in Table 1.

[0302] Table 1 TREM2 antibody affinity

[0303]

[0304] Example 6 In vivo efficacy evaluation

[0305] The in vivo efficacy of C1, G3, 2A6, 2A10, mAb006c and mAb008c in combination with the same type of reference antibody PY314 tested PD-1 antibody in EMT6 cell subcutaneous transplantation mouse tumor model was evaluated.

[0306] 2x10 6 EMT6 cells at a concentration of 2x10 5 cells) were inoculated subcutaneously on the right side of the back of BALB / c mice. On the 5th day after inoculation, tumor-bearing mice with tumor volume ranging from 40 to 67 mm 3 (average tumor volume 55 mm 3 ) were selected, and were grouped according to tumor volume using stratified randomization method, with 8 mice in each group. Drug administration was started on the day of grouping (recorded as PG-D0), and was administered twice a week. The grouping of this experiment is shown in Table 2 (ip is intraperitoneal injection, BIW is twice a week):

[0307] Table 2 Efficacy grouping experiment

[0308]

[0309] Table 3 Tumor growth inhibition rate

[0310]

[0311] The tumor growth and inhibition rate results are shown in Figure 5As shown in Table 3, the tumor volumes of treatment group 2, treatment group 3, treatment group 4, treatment group 5, treatment group 6, treatment group 7, treatment group 8 and treatment group 9 were statistically significantly different from the solvent control group. The efficacy of 2A6+RMP1-14 and mAb006c+RMP1-14 was significantly better than that of the reference molecule PY314 in combination with the PD-1 drug.

[0312] The tumor volume reached 2000 mm 3 As a survival endpoint, the time from the start of administration to the euthanasia of the mice was considered as the survival time of the mice. The survival curve and survival analysis were as follows: Figure 6 As shown in Table 4, the median survival time (MST) of the tumor-bearing mice in the solvent control group was 17 days. The MST values of treatment group 2, treatment group 3, treatment group 4, treatment group 5, treatment group 6, treatment group 7, treatment group 8 and treatment group 9 were 18 days, 23 days, 19.5 days, 22 days, 29 days, 22 days, 24 days and 18 days, respectively. Compared with the solvent control group, treatment group 3, treatment group 5, treatment group 6, treatment group 7 and treatment group 8 could significantly prolong the survival of the mice (ILS>25%). The statistical Kaplan-Meier survival analysis showed that treatment group 5, treatment group 6, treatment group 7 and treatment group 8 were significantly different from the solvent control group.

[0313] Table 4. Survival analysis of mice in each group

[0314]

[0315] Finally, it should be noted that the above only describes the preferred embodiments of the present disclosure and is not intended to limit the present disclosure. Although the above embodiments of the present disclosure are described in detail, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent replacements to some technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to TREM2, wherein the isolated antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region and the heavy chain variable region comprise: HCDR1 shown in SEQ ID NO: 56, HCDR2 shown in SEQ ID NO: 57, and HCDR3 shown in SEQ ID NO: 58, LCDR1 shown in SEQ ID NO: 35, LCDR2 shown in SEQ ID NO: 36, and LCDR3 shown in SEQ ID NO:

37.

2. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein, The heavy chain variable region is shown in SEQ ID NO: 20; The variable region of the light chain is shown in SEQ ID NO:

19.

3. The isolated antibody or its antigen-binding fragment as described in claim 2, further comprising a heavy chain constant region, wherein the heavy chain constant region is as shown in SEQ ID NO: 34; It also includes a light chain constant region, as shown in SEQ ID NO:

33.

4. The isolated antibody or its antigen-binding fragment as described in claim 3, wherein, The antibody comprises: The light chain is shown in SEQ ID NO: 5; and the heavy chain is shown in SEQ ID NO:

6.

5. The antibody or antigen-binding fragment thereof as described in claim 4, wherein the antibody is a monoclonal antibody.

6. The antibody or antigen-binding fragment thereof as described in any one of claims 1-5, wherein the antibody is a chimeric antibody or a humanized antibody.

7. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-6.

8. A vector comprising the nucleic acid molecule as described in claim 7.

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8.

10. A conjugate comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6 conjugated to at least one detectable marker.

11. An antibody-drug conjugate comprising an antibody, including one or more drug portions, said drug portions being directly or covalently linked via a linker to an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-6.

12. A multispecific molecule comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-6.

13. The multispecific molecule of claim 12, wherein the multispecific molecule specifically binds to TREM2 and additionally specifically binds to one or more other targets.

14. The multispecific molecule of claim 13, wherein the multispecific molecule further comprises at least one molecule having a second binding specificity against a second target.

15. A pharmaceutical composition or kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6, or a nucleic acid molecule as claimed in claim 7, or a carrier as claimed in claim 8, or a host cell as claimed in claim 9, or a conjugate as claimed in claim 10, or an antibody-drug conjugate as claimed in claim 11, or a multispecific molecule as claimed in any one of claims 12-14; and a pharmaceutically acceptable carrier.

16. A method for preparing an antibody or antigen-binding fragment thereof as described in any one of claims 1-6, comprising the following steps: (i) Expressing the antibody or antigen-binding fragment thereof as described in any one of claims 1-6 in the host cell as described in claim 9; (ii) Isolate the antibody or its antigen-binding fragment from the host cell.

17. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the nucleic acid molecule as described in claim 7, the vector as described in claim 8, the host cell as described in claim 9, the conjugate as described in claim 10, the antibody-drug conjugate as described in claim 11, or the multispecific molecule as described in any one of claims 12-14 in the preparation of a kit for diagnosing, detecting, or monitoring diseases associated with TREM2 expression, wherein the diseases associated with TREM2 expression are cancers selected from the group consisting of: gastric cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, glioma, and ovarian cancer.

18. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the nucleic acid molecule as described in claim 7, the vector as described in claim 8, the host cell as described in claim 9, the conjugate as described in claim 10, the antibody-drug conjugate as described in claim 11, or the multispecific molecule as described in any one of claims 12-14 in the preparation of a medicament for treating or determining the prognosis of a disease associated with TREM2 expression, wherein the disease associated with TREM2 expression is cancer selected from the group consisting of: gastric cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, glioma, and ovarian cancer.

19. The use as described in claim 17 or 18, in combination with PD-1.

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