Anti-TREM2 antibodies and their uses

Abstract

This disclosure provides an anti-TREM2 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to TREM2, which is a promising target for the treatment of cancer and neurodegenerative diseases.

Description

[0001] This application is a divisional application of the invention patent application filed on December 21, 2023, with application number 202311778506.0 and invention title: Anti-TREM2 antibody and its use. Technical Field

[0002] This disclosure belongs to the field of antibody technology, and more specifically relates to an anti-TREM2 antibody or its antigen-binding fragment and its uses. Background Technology

[0003] The International Agency for Research on Cancer (IARC) released its latest global cancer burden data for 2020. The data shows that in 2019, the total output value of anti-tumor drugs in China reached 115.62 billion yuan, a year-on-year increase of 16%. Based on the average annual compound growth rate over the past five years, the total output value of anti-tumor drugs in China reached 134.12 billion yuan in 2020, while the global oncology market reached 111.9 billion US dollars.

[0004] According to statistics from the World Health Organization, there are currently 52 million people worldwide suffering from Alzheimer's disease (AD), and the number is growing explosively, projected to reach 150 million by 2050.

[0005] Recent research shows that the absence of triggering receptor expressed on myeloid cells-2 (TREM2) in mouse myeloid cells reduces tumor growth, most likely by enhancing T cell killing function. TREM2 gene deletion also leads to a significant reduction 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. Both TREM2 deficiency and anti-TREM2 monoclonal antibody therapy result in significant changes in the macrophage population infiltrating the tumor: reduced infiltration of mannose receptor 1 (MRC1), chemokine receptors (CX3CR1), and immunosuppressive macrophages in the tumor microenvironment, and an expansion of myeloid subsets expressing immunostimulatory factors (promoting improved T cell responses). Furthermore, TREM2 deficiency or the combination of TREM2 and anti-TREM2 mAb can enhance anti-PD-1 therapy. Analysis of clinical tumor samples showed that TREM2 was highly expressed on the membranes of tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) of more than 200 human primary and metastatic cancer cases (mediating immunosuppression). Its high expression was negatively correlated with overall survival and prognosis in gastric cancer, colorectal cancer, and triple-negative breast cancer. Therefore, TREM2 is a potentially high-quality target for immunotherapy of solid tumors from the perspective of reshaping the tumor microenvironment.

[0006] TREM2 is an emerging target for tumor immunotherapy. Immune checkpoint therapy releases T-cell control over tumors, but this control is disrupted by immunosuppressive myeloid cells. The anti-inflammatory and immunosuppressive activities of TREM2 promote tumor growth and immune escape. Blocking TREM2 signaling or depleting TREM2+ myeloid cells in the tumor to activate T-cell-mediated anti-tumor immune responses is a novel targeted therapy strategy that can be used in combination with PD-1 antibodies to enhance anti-PD-1 immunotherapy.

[0007] In Alzheimer's disease (AD), an increased risk of AD is associated with TREM2 mutations, such as the R47H missense mutation in TREM2, which causes lipid recognition impairment in microglia. In preclinical AD models, TREM2 is a lipid receptor expressed in microglia and other tissue macrophages. Phospholipids, apolipoproteins (APOE), and lipoproteins bind to TREM2, transmitting intracellular activation signals through the adapter DAP12, promoting microglia activation and survival; forming a microglia barrier around Aβ plaques, thus providing neuroprotection; and Aβ binding to APOE-containing or clustering proteins (CLUs) may facilitate the delivery of these complexes to microglia, thereby increasing Aβ clearance and reducing neuronal damage.

[0008] In AD, agonist antibodies targeting TREM2 can activate microglia by promoting TREM2 activation and inducing their response to neurodegenerative changes (including proliferation, survival, aggregation, and phagocytosis), thus delaying the onset and progression of Alzheimer's disease.

[0009] Therefore, TREM2 is a promising target for the treatment of cancer and neurodegenerative diseases. Due to the significant unmet clinical needs in malignant tumors and neurodegenerative diseases, there is a demand for other TREM2 antibodies with more desirable pharmaceutical characteristics. Summary of the Invention

[0010] To address the aforementioned problems, this disclosure provides antibodies, methods for their preparation, compositions, etc. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with antibodies that respond to various targets. This invention provides antibodies capable of specifically binding to TREM2, preferably chimeric monoclonal antibodies or humanized monoclonal antibodies.

[0011] On one hand, this 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:

[0012] The LCDR1 includes the following sequence:

[0013] [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];

[0014] The LCDR2 includes the following sequence:

[0015] [L / K / G / H / R][V / A / M]S[K / N / E][L / R / S][D / F / Y / I / A][S / T];

[0016] The LCDR3 includes the following sequence:

[0017] [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;

[0018] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3:

[0019] The HCDR1 contains the following sequence:

[0020] [D / S][G / *]Y[G / W / Y / N][M / W][H / N];

[0021] The HCDR2 contains the following sequence:

[0022] [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];

[0023] The HCDR3 contains the following sequence:

[0024] [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.

[0025] Furthermore, the isolated antibody or its antigen-binding fragment, wherein,

[0026] 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 with one of SEQ ID NO: 35, 38, 41, 44, 47, and 50; and / or,

[0027] 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 with one of SEQ ID NO: 36, 39, 42, 45, 48, and 51; and / or,

[0028] 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 with one of SEQ ID NO: 37, 40, 43, 46, 49, and 52, or consists of one of SEQ ID NO: 37, 40, 43, 46, 49, and 52.

[0029] Furthermore, the isolated antibody or its antigen-binding fragment, wherein,

[0030] 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 with one of SEQ ID NO: 56, 59, 62, 65, 68, and 71; and / or,

[0031] 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 with one of SEQ ID NO: 57, 60, 63, 66, 69, and 72; and / or,

[0032] 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 with one of SEQ ID NO: 58, 61, 64, 67, 70, and 73, or consists of one of SEQ ID NO: 58, 61, 64, 67, 70, and 73.

[0033] In some embodiments of the present invention, the antibody or fragment includes combinations of the following:

[0034] (a) 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; or

[0035] (b) HCDR1 shown in SEQ ID NO: 59, HCDR2 shown in SEQ ID NO: 60, and HCDR3 shown in SEQ ID NO: 61, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40; or

[0036] (c) HCDR1 shown in SEQ ID NO: 62, HCDR2 shown in SEQ ID NO: 63, and HCDR3 shown in SEQ ID NO: 64, LCDR1 shown in SEQ ID NO: 41, LCDR2 shown in SEQ ID NO: 42, and LCDR3 shown in SEQ ID NO: 43; or

[0037] (d) HCDR1 shown in SEQ ID NO: 65, HCDR2 shown in SEQ ID NO: 66, and HCDR3 shown in SEQ ID NO: 67, LCDR1 shown in SEQ ID NO: 44, LCDR2 shown in SEQ ID NO: 45, and LCDR3 shown in SEQ ID NO: 46; or

[0038] (e) HCDR1 shown in SEQ ID NO: 68, HCDR2 shown in SEQ ID NO: 69, and HCDR3 shown in SEQ ID NO: 70, LCDR1 shown in SEQ ID NO: 47, LCDR2 shown in SEQ ID NO: 48, and LCDR3 shown in SEQ ID NO: 49; or

[0039] (f) HCDR1 shown in SEQ ID NO: 71, HCDR2 shown in SEQ ID NO: 72, and HCDR3 shown in SEQ ID NO: 73, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 51, and LCDR3 shown in SEQ ID NO: 52.

[0040] Furthermore, the isolated antibody or its antigen-binding fragment described above, wherein the heavy chain variable region 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 with one of SEQ ID NO: 20, 22, 24, 26, 28, and 30, or is composed of one of SEQ ID NO: 20, 22, 24, 26, 28, and 30.

[0041] Furthermore, the isolated antibody or its antigen-binding fragment described above, wherein the light chain variable region 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 with SEQ ID NO: 19, 21, 23, 25, 27, and 29, or is composed of one of SEQ ID NO: 19, 21, 23, 25, 27, and 29.

[0042] Furthermore, the antibody or fragment, wherein,

[0043] (a) The heavy chain variable region 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 with SEQ ID NO: 20, or is composed of SEQ ID NO: 20;

[0044] The light chain variable region 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 with SEQ ID NO: 19, or is composed of SEQ ID NO: 19;

[0045] (b) The heavy chain variable region 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 with SEQ ID NO: 22, or is composed of SEQ ID NO: 22;

[0046] The light chain variable region 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 with SEQ ID NO: 21, or is composed of SEQ ID NO: 21;

[0047] (c) The heavy chain variable region 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 with SEQ ID NO: 24, or is composed of SEQ ID NO: 24;

[0048] The light chain variable region 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 with SEQ ID NO: 23, or is composed of SEQ ID NO: 23;

[0049] (d) The heavy chain variable region 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 with SEQ ID NO: 26, or is composed of SEQ ID NO: 26;

[0050] The light chain variable region 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 with SEQ ID NO: 25, or is composed of SEQ ID NO: 25;

[0051] (e) The heavy chain variable region 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 with SEQ ID NO: 28, or is composed of SEQ ID NO: 28;

[0052] The light chain variable region 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 with SEQ ID NO: 27, or is composed of SEQ ID NO: 27;

[0053] (f) The heavy chain variable region 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 with SEQ ID NO: 30, or is composed of SEQ ID NO: 30;

[0054] The light chain variable region 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 with SEQ ID NO: 29, or is composed of SEQ ID NO: 29.

[0055] Furthermore, the isolated antibody or its antigen-binding fragment also includes a heavy chain constant region, wherein the heavy chain constant region contains 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 with SEQ ID NO: 34, or is composed of SEQ ID NO: 34.

[0056] Furthermore, the isolated antibody or its antigen-binding fragment also includes a light chain constant region, which contains 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 with SEQ ID NO: 33, or is composed of SEQ ID NO: 33.

[0057] Furthermore, the isolated antibody or its antigen-binding fragment described above includes:

[0058] (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 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 with one of SEQ ID NO: 5, 7, 9, 11, 13, and 15;

[0059] (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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16, or consisting of one of SEQ ID NO: 6, 8, 10, 12, 14, and 16.

[0060] Furthermore, the isolated antibody or its antigen-binding fragment described above includes:

[0061] (a) A light chain or a light chain consisting of SEQ ID NO: 5 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 with SEQ ID NO: 5; and a heavy chain or a heavy chain consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0062] (b) A light chain or a light chain consisting of SEQ ID NO: 7 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 with SEQ ID NO: 7; and a heavy chain or a heavy chain consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0063] (c) A light chain or a light chain consisting of SEQ ID NO: 9 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 with SEQ ID NO: 9; and a heavy chain or a heavy chain consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0064] (d) A light chain or a light chain consisting of SEQ ID NO: 11 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 with SEQ ID NO: 11; and a heavy chain or a heavy chain consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0065] (e) A light chain or a light chain consisting of SEQ ID NO: 13 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 with SEQ ID NO: 13; and a heavy chain or a heavy chain consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0066] (f) A light chain or a light chain consisting of SEQ ID NO: 15 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16 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 with one of SEQ ID NO: 6, 8, 10, 12, 14, and 16.

[0067] Furthermore, the isolated antibody or its antigen-binding fragment, wherein:

[0068] (a) A light chain or a light chain consisting of SEQ ID NO: 5 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 with SEQ ID NO: 5; and a heavy chain or a heavy chain consisting of SEQ ID NO: 6 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 with SEQ ID NO: 6;

[0069] (b) A light chain or a light chain consisting of SEQ ID NO: 7 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 with SEQ ID NO: 7; and a heavy chain or a heavy chain consisting of SEQ ID NO: 8 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 with SEQ ID NO: 8;

[0070] (c) A light chain or a light chain consisting of SEQ ID NO: 9 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 with SEQ ID NO: 9; and a heavy chain or a heavy chain consisting of SEQ ID NO: 10 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 with SEQ ID NO: 10;

[0071] (d) A light chain or a light chain consisting of SEQ ID NO: 11 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 with SEQ ID NO: 11; and a heavy chain or a heavy chain consisting of one of SEQ ID NO: 12 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 with SEQ ID NO: 12;

[0072] (e) A light chain or a light chain consisting of SEQ ID NO: 13 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 with SEQ ID NO: 13; and a heavy chain or a heavy chain consisting of SEQ ID NO: 14 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 with SEQ ID NO: 14;

[0073] (f) A light chain or a light chain consisting of SEQ ID NO: 15 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 with SEQ ID NO: 15; and a heavy chain or a heavy chain consisting of SEQ ID NO: 16 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 with SEQ ID NO: 16.

[0074] Furthermore, the antibody or antigen-binding fragment thereof as described in any of the preceding claims, wherein the antibody is a monoclonal antibody.

[0075] Furthermore, the antibody or antigen-binding fragment thereof as described in any one of the preceding claims, wherein the antibody is a chimeric antibody or a humanized antibody or a modified antibody, such as a modified chimeric antibody.

[0076] Furthermore, an isolated nucleic acid molecule containing a nucleic acid sequence encoding the aforementioned antibody or its antigen-binding fragment.

[0077] Furthermore, a carrier containing the aforementioned nucleic acid molecules.

[0078] Furthermore, a host cell containing the aforementioned nucleic acid molecules or vectors.

[0079] Furthermore, a conjugate comprising the aforementioned antibody or its antigen-binding fragment conjugated to at least one detectable marker.

[0080] Furthermore, an antibody-drug conjugate comprising an antibody includes one or more drug portions which are directly or covalently linked to the antibody or its antigen-binding fragment via a linker.

[0081] Furthermore, a multispecific molecule comprising the antibody or its antigen-binding fragment described above; preferably, the multispecific molecule specifically binds to TREM2 and additionally specifically binds to one or more other targets; more preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity against a second target.

[0082] Furthermore, a pharmaceutical composition or kit may contain an antibody or its antigen-binding fragment, or a nucleic acid molecule, or a carrier, or a host cell, or a conjugate, or an antibody-drug conjugate, or a multispecific molecule, or a pharmaceutically acceptable carrier, as described above.

[0083] Furthermore, a method for preparing the above-mentioned antibody or its antigen-binding fragment includes the following steps:

[0084] (i) Expressing the antibody or its antigen-binding fragment in the host cells described above; and optionally...

[0085] (ii) Isolate the antibody or its antigen-binding fragment from the host cell.

[0086] Furthermore, the use of antibodies or their antigen-binding fragments, nucleic acid molecules, vectors, host cells, conjugates, antibody-drug conjugates, multispecific molecules, pharmaceutical compositions, or kits, as described above, in the preparation of kits for the diagnosis, detection, or monitoring of diseases related to TREM2 expression.

[0087] Furthermore, the use of antibodies or their antigen-binding fragments, nucleic acid molecules, vectors, host cells, conjugates, antibody-drug conjugates, multispecific molecules, pharmaceutical compositions, or kits, as described above, in the preparation of medicaments for treating diseases associated with TREM2 expression or for determining their prognosis.

[0088] Furthermore, as described above, it is used in combination with PD-1.

[0089] Furthermore, as described above, 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.

[0090] Furthermore, as described above, the diseases associated with TREM2 expression are neurodegenerative diseases selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), autism (ASDs), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS).

[0091] Furthermore, as described above, the TREM2 expression-related disease is a metabolic syndrome selected from the group consisting of: adipocyte hypertrophy, systemic hypercholesterolemia, inflammation, and glucose intolerance. Attached Figure Description

[0092] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the embodiments of the present disclosure to explain the disclosure and do not constitute a limitation thereof. In the drawings:

[0093] Figure 1A Results of assay for the binding activity of anti-TREM2 antibodies C1 and G3 to huTREM2 on the cell surface;

[0094] Figure 1B Results of assay for the binding activity of anti-TREM2 antibodies 2A6 and 2A10 to huTREM2 on the cell surface;

[0095] Figure 1C Results of assay for the binding activity of anti-TREM2 antibodies mAb006c and mAb008c to huTREM2 on the cell surface;

[0096] Figure 2A Results of assay for the binding activity of anti-TREM2 antibodies C1 and G3 with monkey TREM2;

[0097] Figure 2B Results of assay for the binding activity of anti-TREM2 antibodies 2A6 and 2A10 with TREM2 in monkeys;

[0098] Figure 2C Results of assay for the binding activity of anti-TREM2 antibodies mAb006c and mAb008c with TREM2 in monkeys;

[0099] Figure 3A Results of assay for the binding activity of anti-TREM2 antibodies C1 and G3 with mouse TREM2;

[0100] Figure 3B Results of assay for the binding activity of anti-TREM2 antibodies 2A6 and 2A10 with mouse TREM2;

[0101] Figure 3C Results of assay for the binding activity of anti-TREM2 antibodies mAb006c and mAb008c with mouse TREM2;

[0102] Figure 4 The TREM2 reporter gene activity assay results show that the horizontal axis represents antibody concentration and the vertical axis RLU represents relative light units.

[0103] Figure 5 Mouse tumor growth curves, where G represents the group, ip represents intraperitoneal injection, and BIW represents administration twice a week;

[0104] Figure 6 Survival curves for mice in each group. Group represents the group, ip represents intraperitoneal injection, and BIW represents administration twice a week. Detailed Implementation

[0105] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this disclosure. Therefore, the drawings and description are to be considered exemplary in nature and not restrictive.

[0106] The preferred embodiments of this disclosure are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0107] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints.

[0108] definition

[0109] The term "antibody" is used in its broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, provided they exhibit the desired antigen-binding activity. The term "antibody moiety" refers to a full-length antibody or its antigen-binding fragment.

[0110] Full-length antibodies consist of two heavy chains and two light chains. Variable regions of both the light and heavy chains are responsible for antigen binding. These variable domains can be referred to as “VH” and “VL”, respectively. The variable regions in both chains typically contain three highly variable loops called 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 antibody-antigen binding fragments disclosed herein can be defined or identified according to 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 either the heavy or light chain are situated between flanks called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0111] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, bispecific antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bispecific antibodies (dsdiabody), single-chain Fv (scFv), scFv dimers (bivalent bispecific antibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelidoid single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen bound by a parent antibody or a parent antibody fragment (e.g., a parental scFv). In some embodiments, the antigen-binding fragment may contain one or more CDRs from a specific human antibody grafted into a frame region from one or more different human antibodies.

[0112] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable region of heavy and light chain polypeptides. These specific regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. 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 MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlaps or subsets of amino acid residues when compared with each other. However, the application of any definition to refer to an antibody or transplanted antibody or its variants is intended to be within the scope of the terminology defined and used herein. The amino acid residues covering the CDRs defined in each of the references cited above are listed in Table 1 below 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 references cited in this paragraph are incorporated herein by reference in their entirety for use in this disclosure and may be included in one or more claims herein. In some embodiments, the CDR sequence provided herein is based on the IMGT definition.For example, the CDR sequence can be determined using the VBASE2 tool (http: / / www.vbase2.org / vbase2.php, see also Retter I, Althaus HH, Münch R, Müller W: VBASE2 an integrative V gene database. Nucleic Acids Res. 2005 Jan 1; 33 (Database issue): D671-4, which is incorporated herein by reference in its entirety). The residue numbering follows the nomenclature of Honegger and Plückthun above, expressed as "as in Kabat's variable domain residue numbering" or "as in Kabat's amino acid position numbering," and its variations, referring to the numbering system used for the heavy chain variable domain or light chain variable domain in the antibody compilation by Kabat et al. above. Using this numbering system, the actual linear amino acid sequence can contain fewer or more amino acids, corresponding to the shortening or insertion of the FR or hypervariable region (HVR) of the variable domain. For example, the variable domain of the heavy chain may include a single amino acid insertion following the H2 residue (according to Kabat residue 52a) and an insertion of residues following the heavy chain FR residue 82 (e.g., according to Kabat residues 82a, 82b, and 82c, etc.). The Kabat number of residues in a given antibody can be determined by aligning the homologous region of the antibody sequence to a “standard” Kabat numbered sequence. Unless otherwise stated herein, the residue numbers in the immunoglobulin heavy chain are those of the EU index as described above by Kabat et al. “EU index as described in Kabat” refers to the residue number of the human IgG1 EU antibody.

[0113] “Frame” or “FR” residues are those variable domain residues other than the CDR residues defined in this paper.

[0114] The "percentage (%) amino acid sequence identity" or "homology" of the peptide and antibody sequences identified in this paper is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the peptide being compared, after sequence alignment, taking into account any conserved substitutions as part of sequence identity. For example, alignments for determining the percentage of amino acid sequence identity can be performed in various ways within the scope of the art 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 measuring alignments, including any algorithms required to achieve maximum alignment over the full length of the sequence being compared. However, for the purposes of this paper, the % amino acid sequence identity value is generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32 (5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5 (1): 113, 2004).

[0115] "Homology" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. Molecules are homologous at that position when positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if each position in two protein molecules is occupied by a lysine, or if each position in two DNA molecules is occupied by an adenine. The percentage of 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 and multiplied by 100. For example, if six out of ten positions in two sequences are matching or homologous, the two sequences have 60% homology. For instance, the protein sequences SGTSTD and TGTSDA have 50% homology. Typically, comparisons are performed when the two sequences are aligned to provide maximum homology. The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to other parts of the immunoglobulin containing the antigen-binding site (variable domains). The 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.

[0116] The "light chain" of antibodies (immunoglobulins) in any mammalian species can be assigned to one of two distinct types, called "κ" and "λ," based on the amino acid sequence of their constant domains.

[0117] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0118] The "hinge region" is generally defined in IgG as the region from Glu216 to Pro230 corresponding to human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues forming the inter-heavy chain SS bond in the same position. The "CH2 domain" (also known as the "C2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the carbohydrates provide a substitute for domain-domain pairing and contribute to the stability of the CH2 domain. (Burton, Molec. Immunol. 22:161-206 (1985)).

[0119] The “CH3 domain” (also known as the “C2 domain”) contains the extension of the Fc region from the C-terminus of the residues to the CH2 domain (i.e. from about amino acid residue 341 to the C-terminus of the antibody sequence, usually located at amino acid residues 446 or 447 of IgG).

[0120] The term "Fc region" or "fragment crystallizable region" used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. For example, during antibody production or purification, or by recombinant modification of the nucleic acid encoding the antibody heavy chain, the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed. Therefore, compositions of complete antibodies may comprise antibody groups with all K447 residues removed, antibody groups with K447 residues not removed, and antibody groups containing a mixture of antibodies with and without K447 residues. Native sequence Fc regions suitable for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0121] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are those that bind to IgG antibodies (γ receptors) and include subclasses of FcγRI, FcγRII, FcRN, and FcγRIII, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. (See M. Annu. Rev. Immunol. 15:203-234 (1997)). FcRNs are essential for antibody circulation into the bloodstream and can prolong the serum half-life of antibodies. 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) reviewed FcR. Other FcRs, including those to be identified in the future, are included in the term "FcR" in this paper.

[0122] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen that an antibody or antibody moiety binds to. If two antibodies or antibody moiety exhibit competitive binding to an antigen, they can bind to the same epitope within the antigen.

[0123] As used herein, in the presence of an equimolar concentration of a first antibody or a fragment thereof, when the first antibody or a fragment thereof inhibits the binding of a second antibody or a fragment thereof to the target antigen by at least about 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), the first antibody or a fragment thereof “competes” with the second antibody or a fragment thereof to bind to the target antigen, and vice versa. A high-throughput process for “binding” antibodies based on their cross-competition is described in PCT Publication No. WO03 / 48731.

[0124] As used herein, the terms “specific binding,” “specific recognition,” and “specific to” refer to measurable and reproducible interactions, such as the binding between a target and an antibody or antibody moiety, which determines the presence of the target in the presence of a heterogeneous group of molecules, including biomolecules. For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) is an antibody or antibody moiety that binds to the target with higher affinity, stronger affinity, easier binding, and / or longer duration of binding compared to binding to other targets. In some embodiments, such as as measured by radioimmunoassay (RIA), the degree of binding of an antibody to an unrelated target is less than about 10% of the antibody binding to the target. In some embodiments, an antibody that specifically binds to a target (or is specific to an antigen) means that the antibody binds to the target with less than about 10% affinity. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 A KD value of M or smaller binds to the target. In some embodiments, the antibody specifically binds to an epitope on a conserved protein from different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding. The binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, BLI, RIA, ECL, IRMA, EIA, BIACORE™ assays, and peptide scanning.

[0125] "Isolated" or "purified" antibodies (or constructs) are antibodies that have been identified, isolated, and / or recovered from components in their production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide does not bind to any other components in its production environment.

[0126] An "isolated" nucleic acid molecule encoding the constructs, antibodies, or antigen-binding fragments thereof described herein is a nucleic acid molecule identified and isolated from at least one contaminated nucleic acid molecule that normally binds to it in the environment in which it is produced. Preferably, the isolated nucleic acid does not bind to any components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are in a form different from their form or environment found in nature. Therefore, the isolated nucleic acid molecules are different from nucleic acids encoding the polypeptides and antibodies described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules that are normally contained in cells containing nucleic acid molecules, but which are present outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0127] The term "control sequence" refers to the DNA sequence necessary for the expression of an operable coding sequence in a specific host organism. Control sequences applicable to prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0128] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably ligated." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, the DNA of the pre-sequence or secretory leader sequence is operably ligated to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably ligated to the coding sequence; or if a ribosome binding site is located to facilitate translation, the ribosome binding site is operably ligated to the coding sequence. Generally, "operably ligated" means that the ligated DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and contiguous within the reading frame. However, enhancers do not need to be contiguous. Ligation is accomplished by connecting to a convenient restriction site. If such a site is not available, synthetic oligonucleotide aptamers or adapters are used as a standard practice.

[0129] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell that has been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0130] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary test cells and their progeny.

[0131] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of progeny cells may not be exactly the same as that of the parent cells, and they may contain mutations. This article includes mutant progeny cells with the same function or biological activity as those screened or selected in the initially transformed cells.

[0132] The term "immunoconjugate" includes references to the covalent linking of a therapeutic agent or detectable marker to an antibody (such as the antibody portion described herein). The linking can be direct or indirect, via a linker (such as a peptide linker).

[0133] As used herein, “treatment / treating” is a method of achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease, reduction of the severity of the disease, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, provision of remission (partial or complete) of the disease, reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, increase or improvement of quality of life, weight gain, and / or prolongation of survival. “Treatment” also includes reduction of the pathological consequences of cancer (e.g., tumor volume). The methods of this disclosure consider any one or more of these treatment aspects.

[0134] In the case of cancer, the term "treatment" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and improving one or more symptoms associated with the disease.

[0135] The term “inhibition” or “inhibit” refers to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the incidence, extent, or likelihood of that trait. “Reduction” or “inhibition” means a reduction, decrease, or cessation of activity, function, and / or quantity compared to a reference. In some embodiments, “reduction” or “inhibition” means the ability to result in a reduction of 20% or more overall. In another embodiment, “reduction” or “inhibition” means the ability to result in a reduction of 50% or more overall. In yet another embodiment, “reduction” or “inhibition” means the ability to result in a reduction of 75%, 85%, 90%, 95%, or more overall. As used herein, “reference” refers to any sample, standard, or level used for comparative purposes. A reference may be obtained from healthy and / or disease-free samples. In some instances, a reference may be obtained from untreated samples. In some instances, a reference is obtained from a disease-free or untreated sample of an individual. In some instances, a reference is obtained from one or more healthy individuals who are not the individual or a patient.

[0136] As used herein, “delaying disease progression” means slowing, hindering, mitigating, blocking, stabilizing, inhibiting, and / or delaying the development of a disease (e.g., cancer). Such delay can have varying durations, depending on the individual’s medical history and / or treatment received. As will be apparent to those skilled in the art, sufficient or significant delay can indeed include prevention, i.e., the individual does not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed.

[0137] As used in this article, “prevention” includes providing prevention of the onset or recurrence of the disease in individuals who may be susceptible to the disease but have not yet been diagnosed with it.

[0138] As used herein, "inhibitory" function or activity refers to a reduction in function or activity when compared to the same conditions other than the target condition or parameter, or optionally, compared to another condition. For example, an antibody that inhibits tumor growth reduces the tumor growth rate compared to the tumor growth rate in the absence of the antibody.

[0139] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to mammals, including but not limited to humans, cattle, horses, felines, canines, rodents, or primates. In some embodiments, an individual is a human. An “effective amount” of a drug refers to the amount that effectively achieves the desired therapeutic or preventative outcome within the necessary dosage and time period. The specific dosage may vary depending on one or more of the following: the particular drug selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the drug.

[0140] The "therapeutic effective amount" of the substance / molecule, agonist, or antagonist disclosed herein may vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the individual. Therapeutic effective amount is also the amount by which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are offset by the beneficial therapeutic effect. Therapeutic effective amounts may be delivered in one or more administrations.

[0141] "Prophylactic effective dose" refers to the amount that effectively achieves the desired preventive outcome within the necessary dosage and time period. It is usually, but not always, less necessary because the preventive dose is administered to the subject before or in the early stages of the disease; therefore, the preventive effective dose will be less than the therapeutic effective dose.

[0142] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation whose form allows for the effective biological activity of the active ingredient and which does not contain any additional ingredients that would have unacceptable toxicity to the individual to whom the formulation is administered. Such formulations may be sterile.

[0143] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or carrier conventionally used in the art for use with therapeutic agents, which together constitute a "pharmaceutical composition" for individual administration. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used.

[0144] "Sterile" preparations are sterile or substantially free of live microorganisms and their spores.

[0145] Administering in combination with one or more other therapeutic agents includes simultaneous (synchronous) and sequential or sequential administration in any order.

[0146] The term “synchronization” is used herein to refer to the administration of two or more therapeutic agents, wherein at least part of the administration overlaps in time, or the administration of one therapeutic agent falls within a short period of time relative to the administration of another therapeutic agent. For example, the time interval between the administration of two or more therapeutic agents does not exceed about 60 minutes, such as not exceeding any one of about 30, 15, 10, 5, or 1 minute.

[0147] The term “in sequence” is used in this document to refer to the administration of two or more therapeutic agents, wherein the administration of one or more agents continues after the administration of one or more other agents has been discontinued. For example, two or more therapeutic agents are administered at intervals of more than about 15 minutes, such as about 20, 30, 40, 50 or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks or 1 month or longer.

[0148] As used in this article, “combination” means applying a treatment in addition to another treatment. Therefore, “combination” means applying a treatment before, during, or after applying another treatment to an individual.

[0149] "Article" is any manufactured article (e.g., packaging or container) or kit that contains at least one pharmaceutical agent (e.g., a medicine for treating a disease or condition (e.g., cancer)) or a probe for specifically detecting the biomarkers described herein. In some embodiments, the manufactured article or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0150] In this disclosure, the amino acid at a certain position can be varied. For example, [K / R] indicates that the amino acid at that position can be either K or R; another example is [S / *], where the * sign indicates a deletion, meaning that the amino acid at that position can be either S or missing.

[0151] It should be understood that the embodiments described in this disclosure include embodiments that are “composed of” and / or “substantially comprised of”.

[0152] Unless otherwise specified in the examples, the techniques or conditions described in the literature in this field (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press) or the product instructions should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0153] Anti-TREM2 antibody

[0154] In some aspects, the present invention includes isolated antibodies or antigen-binding fragments thereof.

[0155] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate-derived antibodies, CDR transplantation antibodies, human antibodies, recombinant antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies, including mutant proteins and variants thereof, modified 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 the TREM2 protein. Furthermore, unless the context otherwise requires, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, 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 a modified chimeric monoclonal antibody.

[0156] Variable regions and CDRs in antibody sequences can be identified according to general rules already developed in the art (as described above, such as the Kabat numbering system or by comparing the sequence with a database of known variable regions).

[0157] In some embodiments, the isolated antibody or its antigen-binding fragment may contain conserved substitutions or modifications or replacements (e.g., conserved substitutions), deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids; or conserved substitutions of up to 20, 15, 10, or 5 amino acids) in the variable regions of the heavy and / or light chains, or have sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% with the antibody or antigen fragment from which it is derived. Amino acid deletion variants containing missing amino acids at the N-terminus and / or C-terminus of a protein are also referred to as N-terminal and / or C-terminal truncation variants. It is understood in the art that certain modifications to the conserved sequences that do not eliminate antigen binding can be made. See, for example, 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.

[0158] As described above, the term "conservative substitution / replacement" as used herein refers to an amino acid substitution / replacement that does not adversely affect or alter the fundamental properties of a protein / peptide comprising an amino acid sequence. For example, conservative substitution / replacement can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution includes substitution in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as substitution of physically or functionally similar residues (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, 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).

[0159] According to the present invention, when referring to a specific antibody heavy chain and / or a specific antibody light chain (e.g., a chain containing a specific CDR sequence), the two heavy chains and / or the two light chains of the antibody are preferably composed of the specific antibody heavy chain and / or the specific antibody light chain, respectively.

[0160] Regardless of how the antibody is produced, methods for 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 blotting, immunoprecipitation, SPR, and competitive inhibition assays (see, for example, Janeway et al., below and U.S. Patent Application Publication No. 2002 / 0197266 and the above sections concerning competitive assays).

[0161] According to the present invention, in a standard assay (e.g., the assay described herein), if an antibody has a significant affinity for a predetermined target (e.g., the TREM2 protein or cells expressing TREM2), then the antibody is capable of binding to the predetermined target. Flow cytometry can be used to test the binding of a monoclonal antibody to live cells expressing TREM2. Preferably, in a flow cytometry (FACS) analysis, the binding of the antibody to a target expressed on the cell surface is measured. If the antibody detectably binds to the target (TREM2 protein or cells expressing TREM2), then the antibody is capable of binding to the target and has "affinity". Preferably, if the antibody of the present invention 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, or 0.5 μg / mL or less, the antibody detectably binds to the target.

[0162] In some embodiments, the antibody or fragment may have binding properties of 1×10-1. -7 M or lower KD binding to human, mouse, or monkey TREM2; at 5 × 10 -8 M or lower KD binding to human, mouse, or monkey TREM2; at 1×10 -8 M or lower KD binding to human, mouse, or monkey TREM2; at 5 × 10 -9 M or lower KD binding to human, mouse, or monkey TREM2; at 1×10 - 9 M or lower KD binding to human, mouse, or monkey TREM2; or at 1×10 -9 M or lower KD binds to TREM2 in humans, mice, or monkeys.

[0163] In some embodiments, the EC50 binding property of the antibody or fragment may be: binding to human TREM2 or monkey TREM2 with an EC50 of 5 μg / mL or less; binding to human TREM2 or monkey TREM2 with an EC50 of 3 μg / mL or less; binding to human TREM2 or monkey TREM2 with an EC50 of 2 μg / mL or less; binding to human TREM2 or monkey TREM2 with an EC50 of 1 μg / mL or less; binding to human TREM2 or monkey TREM2 with an EC50 of 0.5 μg / mL or less; binding to human TREM2 or monkey TREM2 with an EC50 of 0.3 μg / mL or less; or binding to human TREM2 or monkey TREM2 with an EC50 of 0.1 μg / mL or less.

[0164] The TREM2 specificity described in this invention refers to the ability to bind to one or more TREM2 epitopes, especially TREM2 epitopes in their natural conformation, particularly human TREM2 specificity.

[0165] In some implementations, the antibody is a monoclonal antibody.

[0166] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a modified antibody, such as a modified chimeric antibody.

[0167] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a constant region of IgG. The constant region of IgG is preferably selected from the constant regions of IgG1, IgG2, IgG3, or IgG4. More preferably, the constant region of IgG is selected from the constant region of IgG1.

[0168] In this field, various methods are employed to modify antibodies without altering their desired properties, such as the recombination of the light and heavy chains of the antibody or the substitution of amino acids, as used in this disclosure. For example, the sequences in this invention, including chimeric antibody sequences or humanized antibody sequences, can be modified by making conserved amino acid substitutions.

[0169] Antibodies primarily interact with target antigens through amino acid residues located in the complementarity-determining regions (CDRs) of the six heavy and light chains. For this reason, the amino acid sequences of the CDRs are more diverse among antibodies than other sequences. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors containing the CDR sequence from that specific naturally occurring antibody, which are then grafted onto frame sequences from different antibodies with different properties (see, for example, 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. USA 86: 10029-10033). Such framework sequences are available from public DNA databases containing germline antibody gene sequences. These germlines are sequences that differ from mature antibody gene sequences because they do not contain fully assembled variable genes, which are formed during B cell maturation via V(D)J linkages. Germline gene sequences will also have sequences that differ from those of the high-affinity secondary repertoire antibody at individual locations that uniformly traverse the variable region.

[0170] Mouse antibodies exhibit high immunogenicity in humans, leading to decreased therapeutic efficacy with repeated administration. The primary immunogenicity is mediated by the heavy chain constant region. However, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimeric or humanized versions of the antibodies.

[0171] Chimeric antibodies are antibodies whose different parts originate from different animal species; for example, antibodies having a variable region derived from a mouse antibody and a constant region derived from a human immunoglobulin. Chimeric antibodies are obtained by linking the variable regions of the mouse antibody heavy and light chains to the constant regions of the human heavy and light chains (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are generated by linking the constant region of the human κ light chain to the variable region of the mouse light chain. In another preferred embodiment, chimeric antibodies are generated by linking the constant region of the human λ light chain to the variable region of the mouse light chain.

[0172] Humanized antibodies are antibodies that have had their CDR sequences / antigen-binding portions or sites derived from another mammalian species, such as mice, transplanted onto a human frame sequence.

[0173] To reduce the immunogenicity of the antibody to humans, a humanized anti-TREM2 antibody is produced using the sequence of the TREM2 antibody disclosed herein. The CDR region of a mouse-derived anti-TREM2 antibody is combined with a human-derived framework region (e.g., human immunoglobulin) to form the humanized anti-TREM2 antibody of this disclosure. The humanized antibody is expected to retain the function of binding to human TREM2 as well as the function of binding to monkey TREM2.

[0174] Antibody preparation or production

[0175] The antibodies of this invention can be generated using various techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While hybridoma techniques are preferred, other techniques for generating monoclonal antibodies can be used in principle, such as viral or oncogene transformation of B lymphocytes or phage display using antibody gene libraries, somatic cell hybridization, and, for example, genetic engineering recombination techniques. For instance, DNA molecules encoding the heavy and light chain genes of the antibodies of this invention can be obtained through chemical synthesis or PCR amplification, the resulting DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of this invention.

[0176] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995), see also Rossie 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 spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0177] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, or combinations thereof. For example, monoclonal antibodies can be produced using hybridomas and well-established 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, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0178] It should be understood that the selected binding sequence can be further modified, for example, to increase the affinity for the target, humanize the target binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention.

[0179] In some embodiments, a method for producing the antibodies or fragments described in this disclosure includes the following steps:

[0180] (i) Expressing the antibody or fragment in host cells; and optionally

[0181] (ii) Isolate the antibody or its antigen-binding fragment from the host cell.

[0182] In a preferred embodiment, the anti-TREM2 monoclonal antibody is prepared by using a hybridoma.

[0183] To obtain hybridomas that produce the antibodies of the present invention, such as the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused into a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0184] The antibodies of the present invention can also be generated in host cells transfected with tumors 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 a partial or full-length light and heavy chain, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the gene is operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.

[0185] 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 generate a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, such that the VH segment is operatively linked to the CH segment within the vector and the VL segment is operatively linked to the CL segment within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the N-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 derived from a non-immunoglobulin protein).

[0186] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete antibodies with appropriate folding and immunological activity.

[0187] Mammalian host cells used to express the recombinant antibodies of the present invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with DHFR selection markers (e.g., as described in RJ Kaufman and PASharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. Specifically, 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 the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow antibody expression in the host cells or by secreting the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

[0188] 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 against TREM2.

[0189] Another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding gene from the antibody-producing lymphocytes of a defined strategy, for example, see Babcocketal, 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of a defined strategy. For details on recombinant antibody engineering, see Welschof and Krau, Recombinant Antibodies for Cancer Therapy ISBN-0-89603-918-8 and Benny KCLo Antibody Engineering ISBN 1-58829-092-1.

[0190] To prepare chimeric antibodies, the variable region of mouse immunoglobulin can be ligated to the constant region of human immunoglobulin using methods known in the art (see, for example, US Patent 4,816,567 to Cabilly et al.). The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably ligating the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDA Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region. By operatively linking DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a κ or λ constant region, but is generally preferred to be a κ constant region. Once the DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used herein is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by both DNA fragments remain within the frame.

[0191] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter's US 5,225,539; Queen et al.'s US 5,530,101; US ​​5,585,089; US 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used. For example, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice can completely suppress the production of endogenous antibodies. Then, the transfer of human germline immunoglobulin gene arrays into said germline mutant mice will cause the mice to produce human antibodies upon exposure to antigens (see, for example, 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 the aforementioned transgenic animals include the HuMAb mouse (Medarex, Inc.) containing a miniloci of the human immunoglobulin gene encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, coupled with targeted mutations that inactivate the endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474): 856-859); or the “KM mouse™” carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods of antibody humanization 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).

[0192] Nucleic acid molecules encoding the antibodies of this invention

[0193] In some aspects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding isolated antibodies or fragments thereof as disclosed herein.

[0194] The nucleic acids of this invention can be obtained using standard molecular biology techniques. For antibodies expressed in hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), the light and heavy chains of the antibody prepared via hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acid encoding such antibody can be recovered from the gene library.

[0195] To prepare chimeric antibodies, methods known in the art can be used to ligate the variable region of mouse immunoglobulins to the constant region of human immunoglobulins (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). By operatively ligating a nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated nucleic acids encoding the VH region can be converted into a full-length heavy chain gene, and DNA fragments containing these regions can be obtained by standard PCR amplification. By operatively ligating DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). Once DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these operations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker.

[0196] In some embodiments, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding antibodies or antigen-binding fragments thereof as described in this disclosure.

[0197] On the one hand, this disclosure provides a carrier containing the aforementioned nucleic acid molecules.

[0198] On the one hand, this disclosure provides a host cell that contains the nucleic acid molecules or the vectors described above.

[0199] Conjugate

[0200] On one hand, this disclosure provides a conjugate comprising an antibody or fragment thereof as described above, conjugated to at least one detectable marker. The detectable marker includes, but is not limited to: (i) providing a detectable signal; (ii) interacting with a second marker to modify the detectable signal provided by the first or second marker, such as FRET (Fluorescence Resonance Energy Transfer); (iii) influencing mobility (e.g., electrophoretic mobility) through charge, hydrophobicity, shape, or other physical parameters; or (iv) providing a trapping component, such as affinity, antibody / antigen, or ion complexation.

[0201] Suitable structures for labeling include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal particulate labels, magnetic particulate labels, polymer particulate labels), and small organic molecules (e.g., biotin, receptor ligands or binding molecules (e.g., cell adhesion proteins or lecithin), which can be bound by... The reagents detect marker sequences containing nucleic acid and / or amino acid residues. The markers, without limitation, include barium sulfate, iodopic acid, iodopic acid, calcium amiodarone propionate, sodium diatrizoate, meglumine diatrizoate, meglumine methyl diatrizoate, sodium caseinate, 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 isotopes (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridine ester), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0202] The detectable markers described above can be detected by methods known in the art. For example, fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention using linkers of varying lengths to reduce potential steric hindrance.

[0203] Antibody drug conjugates / immunoconjugates

[0204] On one hand, this disclosure provides an antibody-drug conjugate comprising one or more drug portions / therapeutic agents, said drug portions being directly or via a linker (e.g., covalently linked) to the antibody or fragment thereof as described above. In the antibody-drug conjugates of this application, there are no particular limitations on the linker structure for conjugating the anti-TREM2 antibody to the drug, as long as the resulting antibody-drug conjugate can be used.

[0205] Because antibody-drug conjugates have the ability to selectively deliver one or more drugs to target tissues (e.g., regulatory myeloid cells), antibody-drug conjugates can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments of the present invention in treating diseases (e.g., cancer).

[0206] Multispecific molecules

[0207] The antibodies or antigen-binding fragments of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments of the present invention can be part of a multispecific molecule (e.g., a bispecific molecule) comprising a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments of the present invention, thereby enabling binding to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments of the present invention can be linked to a second antibody or antigen-binding fragment capable of specifically binding to any protein that can be used as a potential target for combination therapy. To generate said bispecific or multispecific molecules, the antibodies or antigen-binding fragments of the present invention can be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0208] Therefore, in some aspects, the present invention provides a multispecific molecule comprising the antibody or antigen-binding fragment of the present invention.

[0209] In some 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.

[0210] In some preferred embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity against a second target.

[0211] In some preferred embodiments, the multispecific molecule is a bispecific antibody.

[0212] Pharmaceutical Composition

[0213] In some aspects, the present invention relates to pharmaceutical compositions, and this disclosure provides a pharmaceutical composition or kit comprising, as described above, an antibody or fragment, a nucleic acid molecule, a carrier, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, as described above; and a pharmaceutically acceptable carrier.

[0214] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another anti-inflammatory agent.

[0215] The pharmaceutical compositions of the present invention may 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. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, combinations or more of various components known in the art.

[0216] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed in this invention, antibodies containing compositions disclosed herein can be oxidized in solvents containing one or more antioxidants such as methionine, which are reducing antibodies or antigen-binding fragments thereof. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this invention provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing the antibody or its antigen-binding fragment from oxidation, thereby extending its shelf life and / or increasing its activity.

[0217] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous media such as non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating agents 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 pharmaceutical compositions containing phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride in multi-dose containers. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, or cyclodextrin.

[0218] Application, formulation and dosage

[0219] The pharmaceutical compositions of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or implantation or inhalation. The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0220] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers and their controlled-release formulations.

[0221] 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 liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Similarly, specific dosing regimens (including dose, time, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetic parameters (e.g., half-life, clearance, etc.).

[0222] The requirements for effective drug carriers for injectable formulations / compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, edited by Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)).

[0223] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition of the present invention may be suitable.

[0224] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the specific compound, administration, timing of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, the patient's sex, age, weight, condition, general health status, and medical history. However, a dosage is generally chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0225] Typically, the antibodies or antigen-binding fragments of the present invention 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; and 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 some embodiments, the dose 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, and at least about 10 mg / kg body weight.

[0226] In some preferred embodiments, the treatment process involving the antibody or antigen-binding fragment thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding fragment thereof of the present invention may 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 is understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0227] Compatible formulations intended for parenteral administration (e.g., intravenous injection) will contain an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL. In some selected embodiments, the concentration of the antibody or antigen-binding fragment thereof will include 10 μg / mL, 20 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 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-drug conjugate 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.

[0228] The antibody of the present invention can be co-administered with one or more therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, antitumor agents, antiangiogenic agents, or immunosuppressants) to reduce the induction of an immune response against the antibody of the present invention. The antibody can be conjugated to the therapeutic agent (as an immune complex) or administered separately from the therapeutic agent.

[0229] In the context of treatment administration, the terms "combination" or "co-administration" as used herein refer to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which treatments or therapeutic agents are administered to the subject. Treatments or therapeutic agents may be administered before, simultaneously with, or after administering a second treatment or therapeutic agent to the patient. Preferably, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that the treatments or therapeutic agents can work together. In one specific embodiment, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that they provide an increased benefit compared to if administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0230] Medical Use

[0231] The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo uses, including, for example, the detection of TREM2 or the enhancement of immune responses. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects.

[0232] Preferred subjects include mammals, such as humans / patients. In the context of this invention, mammals include humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, Guinea pigs, etc., and captive animals, such as zoo animals.

[0233] Treatment of conditions related to TREM2 expression

[0234] In some aspects, the present invention provides a method for treating a disease in mammals, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject (e.g., a human) requiring treatment.

[0235] In some aspects, this disclosure provides a method for treating a disease associated with TREM2 expression or determining its prognosis in a subject, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to the desired subject.

[0236] In some aspects, this disclosure provides an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, or a pharmaceutical composition or kit in a method for treating a disease associated with TREM2 expression or determining its prognosis in a subject.

[0237] In some aspects, this disclosure provides the use of the antibody or its antigen-binding fragment, 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 preparation of reagents (or drugs) for treating diseases associated with TREM2 expression or determining their prognosis.

[0238] In one implementation, diseases associated with TREM2 include oncological diseases such as cancer.

[0239] In some implementations, the TREM2-related disease is cancer, preferably selected from the group consisting of:

[0240] Stomach cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, glioma, ovarian cancer.

[0241] Antibodies or their antigen-binding fragments can be used alone as a monotherapy or in combination with chemotherapy or radiotherapy.

[0242] Antibodies or their antigen-binding fragments can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

[0243] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cell-proliferating conditions such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, anti-angiogenic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, antimetastatic agents, and immunotherapy agents. It should be understood that, in selected embodiments as described above, such anticancer agents may comprise conjugates and may be bound to a disclosed site-specific antibody prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of this invention. In other embodiments, the disclosed anticancer agent is administered in combination with a site-specific conjugate comprising the various therapeutic agents described above.

[0244] In some implementations, the TREM2-related disease is a neurodegenerative disease selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), autism (ASDs), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS).

[0245] In some implementations, the TREM2-related disease is a metabolic syndrome selected from the group consisting of: adipocyte hypertrophy, systemic hypercholesterolemia, inflammation, and glucose intolerance.

[0246] diagnosis

[0247] This invention provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative disorders, as well as methods for screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying an individual with cancer for treatment or monitoring cancer progression, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein and detecting the presence or absence of the antibody in the sample, or the binding level, of a bound or free target molecule. In some embodiments, the antibody will comprise a detectable marker or reported molecule as described herein.

[0248] In some aspects, this disclosure provides a method for diagnosing, detecting, or monitoring TREM2-related diseases, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a desired subject.

[0249] In some aspects, this disclosure provides an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, or a pharmaceutical composition or kit in a method for diagnosing, detecting, or monitoring TREM2-related diseases in a subject.

[0250] In another aspect, this disclosure provides the use of the antibody or its antigen-binding fragment, 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 preparation of reagents (or drugs) for diagnosing, detecting or monitoring diseases associated with TREM2 expression.

[0251] Samples can be analyzed using a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, immunoblotting, Western blot analysis, and flow cytometry. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., as known to those skilled in the art.

[0252] The method described in this invention for detecting or monitoring TREM2 expression or the level of TREM2-expressing cells in vitro can also be used for non-diagnostic purposes.

[0253] Preferred subjects include mammals, such as people / patients in need.

[0254] The subject's samples are blood, excrement (urine or feces), oral or nasal secretions, or bronchoalveolar lavage fluid, tissue fluid, sweat, or extracts thereof.

[0255] Drug packaging and reagent kits

[0256] Pharmaceutical packages and kits containing one or more containers of an antibody or its antigen-binding fragment thereof are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding fragment, with or without one or more other reagents. For other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; buffers, such as phosphates; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the packaged conjugate composition is intended for the treatment of selected oncological conditions.

[0257] Such kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally contain one or more anticancer agents or other pharmaceutical agents in the same or different containers. The kits may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

[0258] More specifically, the kits may have a single container containing the antibody or antigen-binding fragment of the present disclosure, with or without additional components, or they may have different containers for each desired reagent. In the case of provided combination therapeutic agents for conjugation, a single solution may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the conjugates and any optional anticancer agents in the kit may be stored separately in different containers prior to administration to the patient. The kits may also contain a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0259] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0260] Example

[0261] Example 1 Antigen preparation, mouse immunization and screening

[0262] A. DNA antigen preparation

[0263] The method for preparing human TREM2 DNA antigen for immunizing Balb / c mice is as follows: synthesize the cDNA sequence encoding human TREM2 (Uniprot IDQ9NZC2) (as shown in SEQ ID NO:1), and clone the coding sequence of the above gene into the expression vector by enzyme digestion.

[0264] SEQ ID NO:1 sequence:

[0265] ATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCCCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGCCCCTATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGAGAGAAGGGCCCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCAGTGCCAGAGCCTCCATGGCAGTGAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTGGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCACAGCATCTCCAGGAGCCTCTTGGAAGGAGAAATCCCCTTCCCACCCACTTCCATCCTTCTCCTCCTGGCCTGCATCTTTCTCATCAAGATTCTAGCAGCCAGCGCCCTCTGGGCTGCAGCCTGGCATGGACAGAAGCCAGGGACACATCCACCCAGTGAACTGGACTGTGGCCATGACCCAGGGTATCAGCTCCAAACTCTGCCAGGGCTGAGAGACACGTGA

[0266] B. Protein antigen preparation

[0267] The preparation method of human TREM2 ECD-hFc protein immunized with Balb / c mice was as follows: A nucleotide fragment encoding the extracellular Fc fusion protein of human TREM2 (Uniprot ID Q9NZC2) (sequence shown in SEQ ID NO:2) was synthesized and loaded into an expression vector using DNA ligase. The plasmid containing Human TREM2 ECD-hFc was transfected into ExpiCHO (Gibco, cat: A29127) cells using PEI transfection. Nine days after transfection, the culture supernatant containing the target protein was harvested by centrifugation and filtration, and then purified using an affinity column (Cytiva, cat: 17549851). The purified protein was identified by SDS-PAGE and SEC-HPLC.

[0268] SEQ ID NO:2 sequence:

[0269]

[0270] C. Preparation of reference antibody PY314

[0271] This study used the anti-TREM2 monoclonal antibody developed by Pionyr Immunotherapeutics as a reference antibody. The amino acid sequence was derived from sequence 37012 in patent US2020 / 0140546A1. The DNA sequence of the variable region amino acids was synthesized (as shown in SEQ ID NO:3). The heavy and light chain variable region sequences were loaded into the pTT5 vector (HonorGene, cat: HG-VPW0888) using DNA ligase. The heavy chain constant region isotype was hIgG1, and the light chain constant region isotype was kappa. The plasmids containing the heavy and light chains of the tool antibody were co-transfected into ExpiCHO cells using PEI transfection. Nine days after transfection, the culture supernatant containing the target antibody was harvested by centrifugation and filtration, and the antibody protein was purified using a Protein A column (Cytiva, cat: 17549851). The purified antibody was identified by SDS-PAGE and SEC-HPLC. The amino acid sequences of the reference antibody PY314 are as follows: the full-length light chain sequence is shown in SEQ ID NO:17, the full-length heavy chain sequence is shown in SEQ ID NO:18, the light chain variable region sequence is shown in SEQ ID NO:31, the heavy chain variable region sequence is shown in SEQ ID NO:32, LCDR1-LCDR3 are shown in SEQ ID NO:53-55, and HCDR1-HCDR3 are shown in SEQ ID NO:74-76.

[0272] SEQ ID NO:3 sequence:

[0273] Heavy chain:

[0274] GAGGTGCAGCTGCTGGAAAGCGGAGGCGGACTGGTGCAACCCGGCGGCAGCCTGAGACTGAGCTGCGCTGCTTCTGGCTTTACATTCAGCAACTACTACATGGCCTGGGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCAGCCTCACCAACAGCGGCGGATCTACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTACAAGAGAGTGGGCCGGCAGCGGCTACTTCGACTACTGGGGCCAGGGCACCCTGGTCACAGTGTCCTCT

[0275] SEQ ID NO:4 sequence:

[0276] Light chain:

[0277] GACATCCAGATGACCCAGAGCCCATCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATTACATGCAAGGCTTCTCAGAACGTGGGCAACAACCTGGCCTGGTACCAGCAGAAACCTGGCAAGGCCCCTAAGCTGCTGATCTACTATACATCTAATAGATTCACCGGCGTCCCCAGCAGATTCAGCGGATCTGGCAGCGGCACAGATTTTACCCTGACCATCAGCAGCCTGCAACCTGAGGACTTCGCCACCTACTACTGTCAGCGGATCTACAACAGCCCCTGGACCTTCGGCCAGGGAACAAAGCTGGAAATCAAG

[0278] D. Generation and screening of monoclonal antibodies

[0279] Balb / c mice (commercially purchased mice from Ruizhi Chemical) were immunized with the human TREM2 expression vector and the human TREM2 ECD-hFc protein prepared above. The DNA antigen was mixed with gold powder, lyophilized, and then used to immunize the mice multiple times via a gene gun. Each immunization with the expression vector DNA was 4 μg, with a two-week interval between immunizations. After two immunizations, blood was collected from the mice to measure titers. Mice with high serum titers were selected for booster immunization, with each mouse receiving an intraperitoneal injection of 25 μg of human TREM2 ECD-hFc protein. Three days later, spleen, lymph nodes, and bone marrow cells were collected from the target animals. Plasma cells, the main antibody-secreting cells, were isolated and enriched using a mouse CD138+ plasma cell isolation kit (Miltenyi, cat: 130-092-530). After adjusting the plasma cells to an appropriate concentration, they were introduced into individual pen on the chip via a Beacon light guiding system (Berkeley Lights, BLIBeacon® Optofluidic System) for screening to obtain positive B cells. Single cells could be lysed in a Beacon pen and directly exported to a 96-well plate. RNA from the lysed cells was collected using AgencourtRNAClean XP Beads (Beckman Coulter, cat: A63987) and subsequently synthesized as cDNA using the SMARTer® RACE5' / 3' kit (Clontech, cat: 634859). cDNA purification was performed using Agencourt AMPure XP Beads (Beckman Coulter, cat: A63881). Using the cDNA as a template and mouse Ig-Primer Set (Novagen, 69831-3) as gene-specific primers (GSP), PCR amplification was performed on the variable regions of the heavy and light chains.

[0280] Example 2: Detection of the binding activity of anti-TREM2 antibody to huTREM2 on the cell surface

[0281] Plasmids containing both the heavy and light chains of the TREM2 antibody were co-transfected into FUT8-KO ExpiCHO (constructed by WiseChem) cells using PEI transfection. Nine days post-transfection, the culture supernatant containing the target antibody was harvested by centrifugation and filtration, and the antibody protein was then purified using a Protein A column (Cytiva, cat:17549851). The purified antibody was identified by SDS-PAGE and SEC-HPLC. The antibody was identified as mAb008c.

[0282]

[0283] SEQ ID NO:15 Antibody mAb008c light chain full-length sequence

[0284] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPLTFGAGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0285] SEQ ID NO:16 Antibody mAb008c Heavy Chain Full-Length Sequence

[0286] EVQLQQSGPELVKPGASVKMSCKAPGYTFTDYNMHWVKQSHGKNLEWIGYINPNNGGTTYNQKFKGKAALTVNKSSSTAYMELRSLTSEDSAVYYCVHSNSDYWGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0287] The binding activity of antibody molecules at different concentrations was evaluated by measuring the EC50 of huTREM2 on the cell surface using FACS.

[0288] As described above, the stable cell line CHOK1-human TREM2-DAP12 expressing human TREM2 antigen was cultured and processed using flow cytometry. After digestion, the harvested cells were centrifuged at 300 g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). 2-5E+05 cells / well were placed in a 96-well plate, centrifuged, and the supernatant was discarded. The cells were resuspended in diluted antibody solution (100 μL / well), mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded, and the cells were washed twice with FACS buffer. Goat anti-human IgG-Alexa fluro488 secondary antibody solution (100 μL / well) was added, and the cells were incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded, and the cells were washed twice with FACS buffer. Finally, the cells were resuspended in PBS, and the signal values ​​were detected using a BD flow cytometer.

[0289] The results are as follows Figure 1A , Figure 1B and Figure 1C As shown, C1, G3, 2A6, 2A10, mAb006c, and mAb008c exhibit strong binding activity to human TREM2 on the cell surface. This indicates that C1, G3, 2A6, 2A10, mAb006c, and mAb008c can effectively bind to the surface of CHOK1-human TREM2-DAP12 cells overexpressing human TREM2, and this binding activity is dose-dependent. The binding abilities of C1, G3, 2A6, 2A10, mAb006c, and mAb008c are significantly superior to those of the reference antibody PY314.

[0290] Example 3: Species Cross-Activity Detection

[0291] The EC50 of C1, G3, 2A6, 2A10, mAb006c, and mAb008c bound to CynoTREM2 on the cell surface at different concentrations was measured by FACS to evaluate their species cross-reactivity.

[0292] As described above, the stable cell line CHOK1-cyno TREM2-DAP12 expressing the monkey TREM2 antigen was cultured and processed using flow cytometry. After digestion, the harvested cells were centrifuged at 300 g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). 2-5E+05 cells / well were placed in a 96-well plate, centrifuged, and the supernatant was discarded. The cells were resuspended in diluted antibody solution (100 μL / well), mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded, and the cells were washed twice with FACS buffer. Goat anti-human IgG-Alexa fluro488 secondary antibody solution (100 μL / well) was added, and the cells were incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded, and the cells were washed twice with FACS buffer. Finally, the cells were resuspended in PBS, and the signal values ​​were detected using a BD flow cytometer.

[0293] Species cross-reactivity results with monkeys, such as Figure 2A , Figure 2B and Figure 2C As shown, all six antibodies exhibited good cynoTREM2 binding activity, with C1, G3, 2A6, 2A10, mAb006c, and mAb008 showing significantly better binding activity to Cyno than the reference antibody.

[0294] The EC50 of C1, G3, 2A6, 2A10, mAb006c, and mAb008c bound to mouseTREM2 on the cell surface at different concentrations was measured by FACS to evaluate their species cross-reactivity.

[0295] As described above, the stable cell line CHOK1-mouse TREM2-DAP12 expressing mouse TREM2 antigen was cultured and treated using flow cytometry. After digestion, the harvested cells were centrifuged at 300 g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). 2-5E+05 cells / well were placed in a 96-well plate, centrifuged, and the supernatant was discarded. The cells were resuspended in diluted antibody solution (100 μL / well), mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded, and the cells were washed twice with FACS buffer. Goat anti-human IgG-Alexa fluro488 secondary antibody solution (100 μL / well) was added, and the cells were incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded, and the cells were washed twice with FACS buffer. Finally, the cells were resuspended in PBS, and the signal values ​​were detected using a BD flow cytometer.

[0296] Species cross-reactivity results with mice, such as Figure 3A , Figure 3B and Figure 3C As shown, all six antibodies were able to bind to mouseTREM2, with mAb008c, C1, and G3 antibodies exhibiting significantly better binding activity to mouse than the reference antibody.

[0297] Example 4: TREM2 Reporter Gene Experiment

[0298] Using genetically engineered Jurkat cells as effector cells, the TREM2 receptor and DAP12, as well as the Luciferase gene driven by the NFAT response element, are stably expressed on Jurkat cells. The activity of luciferase generated by NFAT pathway activation can be used to quantitatively detect the activation or inhibition of the TREM2 signaling pathway by therapeutic antibodies.

[0299] The antibody was coated or cross-linked with Crosslinker (anti-Fc), and then incubated with Jurkat-NFAT-TREM2-DAP12 cells at 37 degrees Celsius for 6 hours. An equal volume of ONE-Glo (Promega, E6120) was added, and the mixture was incubated at room temperature in the dark for 10-15 minutes. The fluorescence signal was read using a microplate reader, and the data were analyzed.

[0300] Antibody aggregation was increased using crosslinker (Abs: crosslinker = 1: 0.5). After incubation at 37°C for 6 h with Jurkat-TREM2-DAP12-NFAT cells, clonal NFAT signaling was detected. The results are as follows: Figure 4 As shown, the C1, G3, and mAb008c antibodies exhibited mild inhibitory effects on cells, comparable to the reference antibody PY314, while 2A6, 2A10, and mAb006c showed stronger inhibitory effects on cells, superior to PY314.

[0301] Example 5 Affinity Detection

[0302] The affinity of C1, G3, 2A6, 2A10, mAb006c, and mAb008c was tested using Biacore.

[0303] The capture method was performed using SPR technology. A CM5 chip coupled with an Anti-mFc antibody was used to capture recombinant hTREM2, cyno-TREM2, or mTREM2 proteins with a His tag as ligands. Different concentrations of TREM2 antibody were set as analytes, and the kinetics of the captured human recombinant hTREM2, cyno-TREM2, or mTREM2 proteins were detected. The binding curves were fitted with Biacore software using kinetics to obtain the affinity. The experimental results are shown in Table 1.

[0304] Table 1. TREM2 antibody affinity

[0305]

[0306] Example 6: In vivo efficacy evaluation

[0307] The in vivo efficacy of C1, G3, 2A6, 2A10, mAb006c, and mAb008c in combination with the same type of reference antibody PY314 and PD-1 antibody in a mouse model of EMT6 cell subcutaneous allogeneic tumor transplantation was evaluated.

[0308] 2×10 6 / mL of EMT6 cells were subcutaneously injected into the right side of each mouse with 0.1 mL of cell suspension (containing 2 x 10⁶ cells). 5 (1 cell) was injected into the right back of BALB / c mice. On day 5 post-inoculation, tumors ranging from 40 to 67 mm in volume were selected. 3 (mean tumor volume 55 mm) 3 Mice with tumors were randomly assigned to groups of 8 mice each using a stratified randomization method based on tumor volume. Drug administration began on the day of grouping (denoted as PG-D0) and was administered twice weekly. The groupings for this experiment are shown in Table 2 (ip means intraperitoneal injection, BIW means twice weekly):

[0309] Table 2. Drug Efficacy Grouping Experiment

[0310]

[0311] Table 3 Tumor growth inhibition rate

[0312]

[0313] Tumor growth and inhibition rate results are as follows Figure 5As shown in Table 3, compared with the solvent control group, statistical analysis revealed significant differences in tumor volume in treatment groups 2, 3, 4, 5, 6, 7, 8, and 9. Compared with the reference molecule PY314 combined with PD-1 drugs, 2A6+ RMP1-14 and mAb006c+ RMP1-14 showed significantly better efficacy than the reference molecule.

[0314] With a tumor volume reaching 2000 mm 3 As the survival endpoint, the time from the start of drug administration to the euthanasia of the mice was considered the survival time. Survival curves and survival analyses are as follows: Figure 6 As shown in Table 4, the median survival time (MST) of tumor-bearing mice in the solvent control group was 17 days. The MST values ​​for treatment groups 2, 3, 4, 5, 6, 7, 8, and 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 groups 3, 5, 6, 7, and 8 significantly prolonged the survival time of mice (ILS > 25%). Statistical Kaplan-Meier survival analysis showed significant differences in treatment groups 5, 6, 7, and 8 compared with the solvent control group.

[0315] Table 4. Survival analysis of mice in each group

[0316]

[0317] Finally, it should be noted that the above descriptions are merely preferred embodiments of this disclosure and are not intended to limit this disclosure. Although this disclosure has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. An isolated antibody or 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 and a light chain variable region, wherein the light chain variable region and heavy chain variable region comprise: HCDR1 as set forth in SEQ ID NO: 71, HCDR2 as set forth in SEQ ID NO: 72, and HCDR3 as set forth in SEQ ID NO: 73, LCDR1 as set forth in SEQ ID NO: 50, LCDR2 as set forth in SEQ ID NO: 51, and LCDR3 as set forth in SEQ ID NO:

52.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the heavy chain variable region is as set forth in SEQ ID NO: 30; the light chain variable region is as set forth in SEQ ID NO:

29.

3. The isolated antibody or antigen-binding fragment thereof of claim 2, further comprising a heavy chain constant region, wherein the heavy chain constant region is as set forth in SEQ ID NO: 34; further comprising a light chain constant region, the light chain constant region is as set forth in SEQ ID NO:

33.

4. The isolated antibody or antigen-binding fragment thereof of claim 3, wherein, wherein the antibody comprises: a light chain as set forth in SEQ ID NO: 15; and a heavy chain as set forth in SEQ ID NO:

16.

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

6. The antibody or antigen-binding fragment thereof of 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 the antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. A vector comprising the nucleic acid molecule of claim 7.

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

10. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6 coupled to at least one detectable label.

11. A multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6.

12. The multispecific molecule of claim 11, which specifically binds to TREM2 and additionally specifically binds to one or more other targets.

13. The multispecific molecule of claim 12, further comprising at least one molecule having a second binding specificity for a second target.

14. A pharmaceutical composition or kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the conjugate of claim 10, or the multispecific molecule of any one of claims 11-13; and a pharmaceutically acceptable carrier.

15. A method of preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising the steps of: (i) expressing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in a host cell according to claim 9; (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a nucleic acid molecule according to claim 7, or a vector according to claim 8, or a host cell according to claim 9, or a conjugate according to claim 10, or a multispecific molecule according to any one of claims 11 to 13, in the manufacture of a kit for diagnosing, detecting or monitoring a disease associated with TREM2 expression, which is a cancer selected from the group consisting of breast cancer.

17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a nucleic acid molecule according to claim 7, or a vector according to claim 8, or a host cell according to claim 9, or a conjugate according to claim 10, or a multispecific molecule according to any one of claims 11 to 13, in the manufacture of a medicament for treating or determining the prognosis of a disease associated with TREM2 expression, which is a cancer selected from the group consisting of breast cancer.

18. The use according to claim 16 or 17, in combination with PD-1.

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