Antitrem2 antibodies and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of the invention patent application for anti-TREM2 antibodies and their uses, with the application date of December 21, 2023; application number: 202311778506.0; and the invention name: Technical Field

[0002] The present disclosure belongs to the field of antibody technology, and more specifically relates to an anti-TREM2 antibody or an antigen-binding fragment thereof and uses thereof. Background Art

[0005] The latest studies have shown that the loss of triggering receptor expressed on myeloid cells-2 (TREM2) in mice reduces tumor growth, most likely by enhancing the killing function of T cells. At the same time, the genetic loss of TREM2 leads to a significant decrease in the number of regulatory myeloid cells and an increase in the immune response to tumors, such as an increase in natural killer cells (NK cells) and cytotoxic T cells. Both TREM2 loss and anti-TREM2 monoclonal antibody treatment lead to significant changes in the macrophage population infiltrating the tumor: the infiltration of mannose receptor 1 (MRC1), chemokine receptors (CX3CR1) and immunosuppressive macrophages in the tumor microenvironment is reduced, and the myeloid subsets expressing immunostimulatory factors (promoting improved T cell responses) are expanded. At the same time, TREM2 loss or combined use of anti-TREM2 mAb can enhance anti-PD-1 treatment. Clinical tumor sample analysis shows that TREM2 is highly expressed (mediating immunosuppression) on the membrane of tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) of more than 200 human primary and metastatic cancer cases, and its high expression is negatively correlated with the overall survival rate and prognosis of gastric cancer, colorectal cancer, and triple-negative breast cancer. Therefore, TREM2 is an optional high-quality target for solid tumor immunotherapy from the perspective of reshaping the tumor microenvironment.

[0006] TREM2 is an emerging tumor immune target. Immune checkpoint therapy releases T cell control of tumors, but is destroyed by immunosuppressive myeloid cells. TREM2's anti-inflammatory and immunosuppressive activities promote tumor growth and immune escape. Blocking TREM2 signal transduction or depleting TREM2+ myeloid cells in tumors to activate T cell-mediated anti-tumor immune responses is a new targeted drug treatment strategy that can be combined with PD-1 antibodies and enhance anti-PD-1 immunotherapy.

[0007] In Alzheimer's disease (AD), the increased risk of AD is associated with TREM2 mutations, such as the R47H missense mutation of TREM2, which causes lipid recognition disorders 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, and transmit intracellular activation signals through the adapter DAP12, promoting the activation and survival of microglia; the formation of microglial barriers around Aβ plaques, forming neuroprotection; at the same time, Aβ binding to proteins containing APOE or clusterin (CLU) may help deliver these complexes to microglia, thereby increasing Aβ clearance and reducing neuronal damage.

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

[0009] Therefore, TREM2 is a hot target with potential for treating cancer and neurodegenerative diseases. Due to the large number of unmet clinical medical needs for malignant tumors and neurodegenerative diseases, there is a need for other TREM2 antibodies with more desirable pharmaceutical characteristics. Summary of the invention

[0010] In view of the above problems, the present disclosure provides antibodies, preparation methods, compositions, etc. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies that react with various targets. The present invention provides antibodies that can specifically bind to TREM2, preferably chimeric monoclonal antibodies or humanized monoclonal antibodies.

[0011] In one aspect, the present disclosure provides an anti-TREM2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds 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 comprises 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 comprises 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 comprises 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 comprises HCDR1, HCDR2 and HCDR3:

[0019] The HCDR1 comprises the following sequence:

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

[0021] The HCDR2 comprises 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 comprises 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 above-mentioned isolated antibody or antigen-binding fragment thereof, wherein:

[0026] The LCDR1 comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 35, 38, 41, 44, 47 and 50; and / or,

[0027] The LCDR2 comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 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 to one of SEQ ID NOs: 37, 40, 43, 46, 49 and 52, or consists of one of SEQ ID NOs: 37, 40, 43, 46, 49 and 52.

[0029] Furthermore, the above-mentioned isolated antibody or antigen-binding fragment thereof, wherein:

[0030] The HCDR1 comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 56, 59, 62, 65, 68 and 71; and / or,

[0031] the HCDR2 comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 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 to one of SEQ ID NOs: 58, 61, 64, 67, 70 and 73, or consists of one of SEQ ID NOs: 58, 61, 64, 67, 70 and 73.

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

[0034] (a) HCDR1 set forth in SEQ ID NO:56, HCDR2 set forth in SEQ ID NO:57, and HCDR3 set forth in SEQ ID NO:58, LCDR1 set forth in SEQ ID NO:35, LCDR2 set forth in SEQ ID NO:36, and LCDR3 set forth 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 set forth in SEQ ID NO:62, HCDR2 set forth in SEQ ID NO:63, and HCDR3 set forth in SEQ ID NO:64, LCDR1 set forth in SEQ ID NO:41, LCDR2 set forth in SEQ ID NO:42, and LCDR3 set forth in SEQ ID NO:43; or

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

[0038] (e) HCDR1 set forth in SEQ ID NO:68, HCDR2 set forth in SEQ ID NO:69, and HCDR3 set forth in SEQ ID NO:70, LCDR1 set forth in SEQ ID NO:47, LCDR2 set forth in SEQ ID NO:48, and LCDR3 set forth 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] Further, the above-mentioned isolated antibody or antigen-binding fragment thereof, 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 NOs: 20, 22, 24, 26, 28 and 30, or consists of one of SEQ ID NOs: 20, 22, 24, 26, 28 and 30.

[0041] Further, the above-mentioned isolated antibody or antigen-binding fragment thereof, 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 consists 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 or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20;

[0044] wherein the light chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19;

[0045] (b) the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22;

[0046] wherein the light chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21;

[0047] (c) the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24;

[0048] wherein the light chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23;

[0049] (d) the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26;

[0050] wherein the light chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25;

[0051] (e) the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28;

[0052] wherein the light chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27;

[0053] (f) the heavy chain variable region comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30;

[0054] 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 to SEQ ID NO: 29, or consists of SEQ ID NO: 29.

[0055] Furthermore, the above-mentioned isolated antibody or antigen-binding fragment thereof also comprises a heavy chain constant region, wherein the heavy chain constant 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: 34 or consists of SEQ ID NO: 34.

[0056] Furthermore, the above-mentioned isolated antibody or antigen-binding fragment thereof also comprises a light chain constant region, which light chain constant 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: 33 or consists of SEQ ID NO: 33.

[0057] Furthermore, the above-mentioned isolated antibody or antigen-binding fragment thereof comprises:

[0058] (a) a light chain comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 5, 7, 9, 11, 13, and 15;

[0059] (b) a heavy chain comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14 and 16.

[0060] Furthermore, the above-mentioned isolated antibody or antigen-binding fragment thereof comprises:

[0061] (a) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO:5; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of one of SEQ ID NO:6, 8, 10, 12, 14, and 16;

[0062] (b) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO:7; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of one of SEQ ID NO:6, 8, 10, 12, 14, and 16;

[0063] (c) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO:9; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO:6, 8, 10, 12, 14, and 16;

[0064] (d) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO: 11; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0065] (e) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO: 13; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16, or a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to or consisting of SEQ ID NO: 6, 8, 10, 12, 14, and 16;

[0066] (f) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 6, 8, 10, 12, 14 and 16, or consisting of one of SEQ ID NOs: 6, 8, 10, 12, 14 and 16.

[0067] Furthermore, the above-mentioned isolated antibody or antigen-binding fragment thereof, wherein:

[0068] (a) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:5; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:6;

[0069] (b) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:7; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:8;

[0070] (c) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:9; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:10;

[0071] (d) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO: 11; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, one of SEQ ID NO: 12;

[0072] (e) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:13; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, or consisting of, SEQ ID NO:14;

[0073] (f) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:15; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:16 or consisting of SEQ ID NO:16.

[0074] Further, the antibody or antigen-binding fragment thereof according to any one of the above claims, wherein the antibody is a monoclonal antibody.

[0075] Further, the antibody or antigen-binding fragment thereof according to any one of the above claims, wherein the antibody is a chimeric antibody or a humanized antibody or an improved antibody such as an improved chimeric antibody.

[0076] Furthermore, an isolated nucleic acid molecule comprises a nucleic acid sequence encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0077] Furthermore, a vector comprises the above-mentioned nucleic acid molecule.

[0078] Furthermore, a host cell comprises the above-mentioned nucleic acid molecule or vector.

[0079] Furthermore, a conjugate comprises the above-mentioned antibody or antigen-binding fragment thereof coupled to at least one detectable label.

[0080] Furthermore, an antibody-drug conjugate comprising an antibody includes one or more drug moieties, wherein the drug moieties are covalently linked to the above-mentioned antibody or antigen-binding fragment thereof directly or via a linker.

[0081] Further, a multispecific molecule comprises the above-mentioned antibody or antigen-binding fragment thereof; preferably, the multispecific molecule specifically binds to TREM2, and additionally specifically binds to one or more other targets; further preferably, the multispecific molecule also comprises at least one molecule having a second binding specificity for a second target.

[0082] Furthermore, a pharmaceutical composition or kit comprises the above-mentioned antibody or antigen-binding fragment thereof, or nucleic acid molecule, or vector, or host cell, or conjugate, or antibody-drug conjugate, or multispecific molecule, or pharmaceutically acceptable carrier.

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

[0084] (i) expressing the above-mentioned antibody or antigen-binding fragment thereof in the above-mentioned host cell; and optionally

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

[0086] Furthermore, the above-mentioned antibodies or antigen-binding fragments thereof, or nucleic acid molecules, or vectors, or host cells, or conjugates, or antibody-drug conjugates, or multispecific molecules, or pharmaceutical compositions, or kits are used in the preparation of kits for diagnosing, detecting or monitoring diseases related to TREM2 expression.

[0087] Furthermore, the above-mentioned antibodies or antigen-binding fragments thereof, or nucleic acid molecules, or vectors, or host cells, or conjugates, or antibody-drug conjugates, or multispecific molecules, or pharmaceutical compositions, or kits are used in the preparation of drugs for treating diseases related to TREM2 expression or determining the prognosis thereof.

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

[0089] Furthermore, in the use as described above, the disease associated with TREM2 expression is cancer, and the cancer is selected from the following group: gastric cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, brain glioma, and ovarian cancer.

[0090] Furthermore, in the above-mentioned use, the disease associated with TREM2 expression is a neurodegenerative disease, and the neurodegenerative disease is selected from the following group: Alzheimer's disease (AD), Parkinson's disease (PD), autism syndrome (ASDs), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS).

[0091] Further, as described above, the disease associated with TREM2 expression is metabolic syndrome, and the metabolic syndrome is selected from the following group: adipocyte hypertrophy, systemic hypercholesterolemia, inflammation and glucose intolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the embodiments of the present disclosure, they are used to explain the present disclosure and do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0093] Figure 1A Results of the binding activity test between anti-TREM2 antibodies C1 and G3 and cell surface huTREM2;

[0094] Figure 1B Results of the binding activity test between anti-TREM2 antibodies 2A6 and 2A10 and cell surface huTREM2;

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

[0096] Figure 2A Binding activity test results of anti-TREM2 antibodies C1 and G3 with monkey TREM2;

[0097] Figure 2B Results of the binding activity test of anti-TREM2 antibodies 2A6 and 2A10 with monkey TREM2;

[0098] Figure 2C Binding activity test results of anti-TREM2 antibodies mAb006c and mAb008c to monkey TREM2;

[0099] Figure 3A Binding activity test results of anti-TREM2 antibodies C1 and G3 with mouse TREM2;

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

[0101] Figure 3C Binding activity test results of anti-TREM2 antibodies mAb006c and mAb008c with mouse TREM2;

[0102] Figure 4 TREM2 reporter gene activity detection results, where the horizontal axis is the antibody concentration and the vertical axis RLU represents the relative light unit;

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

[0104] Figure 6 Survival curves of mice in each group, where Group represents group, ip represents intraperitoneal injection, and BIW represents administration twice a week. DETAILED DESCRIPTION

[0105] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present disclosure. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0106] The preferred embodiments of the present disclosure are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0107] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly 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". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0108] definition

[0109] The term "antibody" is used in its broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0110] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. The variable regions in the two chains typically comprise 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 antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are between flanks called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

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

[0112] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions 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 M. P. et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J.Mol.Biol., 309:657-670 (2001), wherein the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of an antibody or a grafted antibody or a variant thereof is intended to be within the scope of the terms defined and used herein. The amino acid residues covering the CDRs defined by each reference cited above are listed in Table 1 below as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol.Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in the present disclosure and may be included in one or more claims herein.In some embodiments, the CDR sequences provided herein are based on the IMGT definition.For example, CDR sequences can be determined by the VBASE2 tool (http: / / www.vbase2.org / vbase2.php, see also Retter I, Althaus HH, 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). 5 Residue numbering follows the nomenclature of Honegger and Plückthun above. The expression "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and its variants refers to the numbering system for the heavy chain variable domain or light chain variable domain of the antibody compilation in Kabat et al. above. Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids, which corresponds to a shortening or insertion of a FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after the residue of H2 (residue 52a according to Kabat) and an inserted residue after the heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of the residues of a given antibody can be determined by alignment with the "standard" Kabat numbering sequence in the homology region of the antibody sequence. Unless otherwise specified herein, the numbering of the residues in the immunoglobulin heavy chain is the numbering of the EU index as in Kabat et al. above. "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

[0113] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0114] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after alignment of the sequences, taking into account any conservative substitutions as part of sequence identity. For example, alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in various ways within the technical scope of the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequence being compared. However, for the purposes of this article, % amino acid sequence identity values ​​are 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] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if the respective positions in the two protein molecules are occupied by lysine, or if the respective positions in the two DNA molecules are occupied by adenine, the molecules are homologous at this position. The homology percentage between the 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 6 out of 10 positions in the two sequences are matched or homologous, the two sequences have 60% homology. For example, the protein sequences SGTSTD and TGTSDA have 50% homology. Typically, the comparison is performed when the two sequences are aligned to provide maximum homology. The term "constant domain" refers to the part of the immunoglobulin molecule that has a more conservative amino acid sequence relative to the other parts (variable domains) of the immunoglobulin that contain the antigen binding site. The constant domains comprise the CH1, CH2 and CH3 domains (collectively referred to as CH) of the heavy chain and the CHL (or CL) domain of the light chain.

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

[0117] The "CH1 domain" (also called "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 as the region from Glu216 to Pro230 in IgG corresponding to human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position, the hinge region of other IgG isotypes can be aligned with the IgG1 sequence. The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region generally extends from about amino acid 231 to about amino acid 340. The uniqueness of the CH2 domain is that it is not tightly paired with another domain. Instead, two N-connected branched carbohydrate chains are inserted between the two CH2 domains of the complete native IgG molecule. It is speculated that carbohydrates can provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22: 161-206 (1985).

[0119] The "CH3 domain" (also called the "C2" domain) comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (ie, from about amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 in IgG).

[0120] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from position Pro230 to its carboxyl terminus. For example, during the production or purification of the antibody, or by recombinant engineering 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 can be removed. Therefore, the composition of the complete antibody can include 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. The native sequence Fc region suitable for use with the antibodies described herein includes human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0121] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR that binds to an IgG antibody (gamma receptor) and includes receptors of FcγRI, FcγRII, FcRN and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, the main difference being their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M.Annu.Rev.Immunol.15:203-234 (1997)). FcRN is essential for antibody circulation to the blood and can prolong the serum half-life of antibodies. FcRs are reviewed by Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immuno methods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0122] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0123] As used herein, a first antibody or fragment thereof "competes" for binding to a target antigen with a second antibody or fragment thereof when the first antibody or fragment thereof inhibits target antigen binding of a second antibody or fragment thereof by at least about 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of equimolar concentrations of the first antibody or fragment thereof, and vice versa. A high throughput process for "binning" 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 for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to the target with greater affinity, avidity, greater ease and / or greater duration than binding to other targets. In some embodiments, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, for example as measured by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target (or an antibody that is specific for an antigen) refers to an antibody that binds to the target with less than about 10% affinity, avidity, greater ease and / or greater duration. -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 In some embodiments, the antibody specifically binds to an epitope on a conserved protein in proteins from different species. In some embodiments, specific binding may include but does not require exclusive binding. The binding specificity of an antibody or antigen binding domain may be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, BLI, RIA-, ECL-, IRMA-, EIA-, BIACORE™-tests, and peptide scanning.

[0125] An "isolated" or "purified" antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from all other components of its production environment.

[0126] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof as described herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule that is normally associated with it in the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with all components associated with the production environment. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from the form or environment in which it is found in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or in a chromosomal position different from its natural chromosomal position.

[0127] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes, for example, include promoters, optional operator 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 linked". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked to a coding sequence; or if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and continuous in the reading frame. However, enhancers do not have to be continuous. The connection is accomplished by connecting at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used as a matter of routine practice.

[0129] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0130] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0131] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cell are included herein.

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

[0133] As used herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result (including a clinical result). For the purposes of this disclosure, a beneficial or desired clinical result includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing body weight, and / or prolonging survival. "Treatment" also includes reducing the pathological consequences of cancer (e.g., tumor volume). The method of this disclosure contemplates any one or more of these treatment aspects.

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

[0135] The term "inhibition" refers to the reduction or cessation of any phenotypic characteristic or the reduction or cessation of the incidence, degree or possibility of the characteristic. "Reduction" or "inhibition" refers to reducing, reducing or preventing activity, function and / or amount compared to a reference. In certain embodiments, "reduction" or "inhibition" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reduction" or "inhibition" refers to the ability to cause an overall reduction of 50% or more. In yet another embodiment, "reduction" or "inhibition" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. As used herein, "reference" refers to any sample, standard or level for comparison purposes. Reference can be obtained from healthy and / or non-diseased samples. In some instances, reference can be obtained from untreated samples. In some instances, reference is obtained from non-diseased or untreated samples of an individual. In some instances, reference is obtained from one or more healthy individuals who are not the individual or patient.

[0136] As used herein, "delaying the progression of a disease" means delaying, hindering, slowing, arresting, stabilizing, inhibiting and / or delaying the progression of a disease (e.g., cancer). Such delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one skilled in the art, sufficient or significant delay can actually include prevention, i.e., the individual does not develop the disease. For example, the progression of advanced cancer, such as metastasis, can be delayed.

[0137] As used herein, "prevention" includes providing protection against the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0138] As used herein, "inhibiting" a function or activity means reducing a function or activity when compared to the same conditions other than the target condition or parameter, or optionally, when compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of a tumor compared to the growth rate of the tumor 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, the individual is a human. An "effective amount" of an agent refers to an amount effective to achieve the desired therapeutic or preventive result at the necessary dosage and time period. The specific dosage may vary depending on one or more of the following: the specific agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the time of administration, the tissue to be imaged, and the physical delivery system carrying the agent.

[0140] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist of the present disclosure may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the substance / molecule, agonist or antagonist are offset by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations.

[0141] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in subjects prior to disease or at an earlier stage of disease.

[0142] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to preparations that are in such form as to permit the biological activity of the active ingredient to be effective and that contain no additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such preparations 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 conventional in the art for use with a therapeutic agent, which together constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0144] A "sterile" preparation is sterile or substantially free of viable microorganisms and their spores.

[0145] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and serial or sequential administration in any order.

[0146] The term "simultaneous" is used herein to refer to the administration of two or more therapeutic agents, wherein at least a portion of the administration overlaps in time, or wherein 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 no more than about any of 30, 15, 10, 5, or 1 minute.

[0147] The term "sequentially" is used herein 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 stopped. For example, two or more therapeutic agents are administered at a time interval of more than about 15 minutes, such as about any of 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 herein, "in combination" refers to administering one treatment modality in addition to another treatment modality. Thus, "in combination" refers to administering one treatment modality before, during, or after another treatment modality is administered to an individual.

[0149] An "article of manufacture" is any article of manufacture (e.g., packaging or container) or kit comprising at least one agent (e.g., a drug for treating a disease or disorder (e.g., cancer)), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0150] In the present disclosure, the amino acid at a certain position may be allowed to vary, for example, [K / R] indicates that the amino acid at the position may be either K or R; another example is [S / *], where the * indicates a deletion, indicating that the amino acid at the position may be either S or deleted.

[0151] It should be understood that embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0152] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field (e.g., Molecular Cloning Experiment Guide, 3rd edition, Science Press, by J. Sambrook et al., translated by Huang Peitang et al.) or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0153] Anti-TREM2 antibodies

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

[0155] In the context of the present application, "antibodies" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including mutant proteins and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, as long as they show preferential association or binding with TREM2 protein. In addition, unless the context otherwise dictates, 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 an improved chimeric monoclonal antibody.

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

[0157] In some embodiments, the isolated antibody or antigen-binding fragment thereof may comprise conservative substitutions or modifications or replacements (e.g., conservative replacements), deletions or additions (e.g., replacements, deletions or additions of 1, 2, 3, 4 or 5 amino acids; or conservative replacements of up to 20, up to 15, up to 10, or up to 5 amino acids) of amino acids in the variable region of the heavy chain and / or light chain, or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity with the antibody or antigenic fragment thereof from which it is derived. Amino acid deletion variants comprising deletions at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncation variants. It is understood in the art that certain modifications of conservative sequences that do not eliminate antigen binding may be performed. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0158] As described above, the term "conservative substitution / conservative replacement" as used herein refers to an amino acid substitution / replacement that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitution / conservative replacement can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue with a similar side chain, such as a physical or functionally similar residue (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.) to a 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, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0159] According to the present invention, if reference is made to comprising a specific antibody heavy chain and / or a specific antibody light chain (e.g., chains comprising specific CDR sequences), the two heavy chains and / or the two light chains of the antibody are preferably constituted by 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 blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competition assays).

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

[0162] In some embodiments, the antibody or fragment may have a binding property of: -7 M or lower KD binds to human, mouse or monkey TREM2; 5×10 -8 M or lower KD binds to human, mouse or monkey TREM2; 1×10 -8 M or lower KD binds to human, mouse or monkey TREM2; 5×10 -9 M or lower KD binds to human, mouse or monkey TREM2; 1×10 -9 M or lower KD binds to human, mouse or monkey TREM2; or -9 Binds to human, mouse, or monkey TREM2 with a KD of M or lower.

[0163] In some embodiments, the antibody or fragment may have an EC50 binding property of: 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; or binding to human TREM2 or monkey TREM2 with an EC50 of 2 μg / mL or less; or binding to human TREM2 or monkey TREM2 with an EC50 of 1 μg / mL or less; or 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 the present invention refers to the ability to bind to one or more TREM2 epitopes, especially TREM2 epitopes in natural conformation, in particular human TREM2 specificity.

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

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

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

[0168] In the art, various methods are used to modify the desired properties of antibodies without changing them, such as the method used in the present disclosure of recombining the light chain and heavy chain of the antibody, replacing amino acids, etc. For example, the sequences of the present invention, including chimeric antibody sequences or humanized antibody sequences, can be subjected to conservative amino acid substitutions.

[0169] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid sequences of CDRs are more diverse than other sequences between antibodies. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that contain CDR sequences from the specific naturally occurring antibodies, which are grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germ lines are sequences that are different from mature antibody gene sequences because they do not contain fully assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also have sequences that are different from the high-affinity second antibody library (secondary repertoireantibody) at individual locations evenly across the variable region.

[0170] Mouse antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when used repeatedly, with the main immunogenicity mediated by the heavy chain constant region. If the individual antibodies are chimerized or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided.

[0171] Chimeric antibodies refer to antibodies in which different parts are from different animal species, for example, antibodies with variable regions from mouse antibodies and human immunoglobulin constant regions. The variable regions of mouse antibody heavy and light chains are linked together with the constant regions of human heavy and light chains to obtain the chimeric nature of the antibody (for example, as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by connecting human kappa light chain constant regions to mouse light chain variable regions. In another preferred embodiment, chimeric antibodies can be produced by connecting human lambda light chain constant regions to mouse light chain variable regions.

[0172] Humanized antibodies are antibodies in which CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0173] In order to reduce the immunogenicity of the antibody to humans, the sequence of the TREM2 antibody of the present disclosure is used to produce a humanized anti-TREM2 antibody, and the CDR region of the murine anti-TREM2 antibody is combined with the human framework region (e.g., human immunoglobulin) to form the humanized anti-TREM2 antibody of the present 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] Preparation or generation of antibodies

[0175] The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although hybridoma technology is preferred, in principle, other techniques for producing monoclonal antibodies can be used, such as virus or oncogene transformation of B lymphocytes or phage display technology using antibody gene libraries, somatic cell hybridization methods, and for example, obtaining through genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into expression vectors, and then transfected into host cells, and then, the transfected host cells are cultured under specific conditions, and the antibodies of the present invention are expressed.

[0176] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (described, for example, 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 very 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 technology, recombinant technology, phage display technology, transgenic animals or some combination thereof. For example, hybridomas and biochemical and genetic engineering techniques recognized in the art can be used to produce monoclonal antibodies, such 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 altered, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies comprising altered target binding sequences are also antibodies of the present invention.

[0179] In some embodiments, the method of producing the antibodies or fragments described herein comprises the following steps:

[0180] (i) expressing the antibody or fragment in a host cell; and optionally

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

[0182] In a preferred embodiment, anti-TREM2 monoclonal antibodies are prepared by using hybridomas.

[0183] In order to obtain hybridomas that produce antibodies of the invention, such as human monoclonal antibodies of the invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The production 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 produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In some embodiments, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors so that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating transcription and translation of the antibody gene.

[0185] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors.In some embodiments, the variable region is used to produce the full-length antibody gene of any antibody isotype by inserting it into the expression vector of the heavy chain constant region and the light chain constant region encoding the desired isotype, so that the VH segment is operably connected to the CH segment and the VL segment in the carrier are operably connected to the CL segment in the carrier.In addition or alternatively, the recombinant expression vector can encode the signal peptide that promotes the secretion of the antibody chain from the host cell.The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminal of the antibody chain gene.The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0186] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran 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 appropriately folded and immunologically active antibodies.

[0187] Mammalian host cells for expressing the recombinant antibodies of the 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 a DHFR selection marker (e.g., as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0188] In another preferred embodiment, transgenic or transchromosomal mice with parts of the human immune system (rather than the mouse system) can be used to generate human monoclonal antibodies against TREM2.

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

[0190] To prepare chimeric antibodies, murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Pat. No. 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 linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, US 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. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably connected" as used herein is intended to indicate that two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0191] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of the above transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for 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 the present invention

[0193] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the above-mentioned isolated antibody or fragment thereof as disclosed herein.

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

[0195] In order to prepare chimeric antibodies, methods known in the art can be used to connect the mouse immunoglobulin variable region to the human immunoglobulin constant region (see, for example, U.S. Pat. No. 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 connecting the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3), and DNA fragments containing these regions can be obtained by standard PCR amplification. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably connecting the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0196] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof as described in the present disclosure.

[0197] In one aspect, the present disclosure provides a vector comprising the above-mentioned nucleic acid molecule.

[0198] In one aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or the vector as described above.

[0199] Conjugate

[0200] In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as described above coupled to at least one detectable marker. Detectable markers include, but are 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) affecting mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape or other physical parameters, or (iv) providing a capture moiety, such as affinity, antibody / antigen or ion complexation.

[0201] Suitable structures as labels include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particle labels (especially metal particle labels, magnetic particle labels, polymer particle labels), organic small molecules (such as biotin, receptor ligands or binding molecules (such as cell adhesion proteins or phosphatidylcholine), which can be used to bind The marker sequence containing nucleic acid and / or amino acid residues can be detected by the marker. The marker includes, but is not limited to, barium sulfate, iodine, iopanoic acid, amiopromide calcium, diatrizoate sodium, diatrizoate meglumine, meglumine, sodium tyropanate, and radiodiagnostic agents (including positron emitters, (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131 and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridinium esters), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0202] Detectable labels as 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 labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be connected to antibodies or antigen-binding fragments thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0203] Antibody Drug Conjugates / Immunoconjugates

[0204] On the one hand, the present disclosure provides an antibody-drug conjugate comprising an antibody, which includes one or more drug moieties / therapeutic agents, which are directly or via a linker (e.g., covalently linked) to an antibody or fragment thereof as described above. In the antibody-drug conjugate of the present application, the linker structure for conjugating the anti-TREM2 antibody to the drug is not particularly limited, as long as the resulting antibody-drug conjugate can be used.

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

[0206] Multispecific molecules

[0207] The antibodies or antigen-binding fragments thereof of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present invention can be part of a multispecific molecule (e.g., a bispecific molecule), wherein the bispecific or multispecific molecule comprises 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 thereof of the present invention, thereby being able to bind to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be connected to a second antibody or antigen-binding fragment thereof that can specifically bind to any protein that can be used as a potential target for combined therapy. To produce the bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present invention can be connected (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

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

[0209] In certain preferred embodiments, the multispecific molecule specifically binds TREM2 (e.g., human TREM2 or monkey TREM2) and specifically binds one or more other targets.

[0210] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target (eg, a second antibody).

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

[0212] Pharmaceutical composition

[0213] In some aspects, the present invention relates to a pharmaceutical composition. The present disclosure provides a pharmaceutical composition or kit comprising the antibody or fragment as described above, the nucleic acid molecule as described above, the vector as described above, the host cell as described above, the conjugate as described above, the antibody-drug conjugate as described above, the 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 antibody or drug. The pharmaceutical composition 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. A pharmaceutically acceptable carrier may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous medium, a non-aqueous medium, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspending / dispersing agent, a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, a combination of various components known in the art, or more.

[0215] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, 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, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed in the present invention, in a solvent containing an antibody or its antigen-binding fragment of the composition disclosed in the present invention containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment, it may be oxidized. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides methods wherein the antibodies or antigen-binding fragments thereof are mixed with one or more antioxidants such as methionine, whereby the antibodies or antigen-binding fragments thereof can be protected from oxidation to extend their shelf life and / or increase their activity.

[0216] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, 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 carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), partitioning 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 in multiple dose containers comprising phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0217] Administration, formulation and dosage

[0218] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable preparations and routes of administration can be selected according to the intended application and treatment regimen.

[0219] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0220] Preparations 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 ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (including dosage, time, and repetition) will depend on specific individuals and individual medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0221] The requirements for effective pharmaceutical carriers for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, eds. Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0222] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing the proliferation of tumor cells or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0223] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, administration time, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, condition, general health and previous medical history of the patient. However, the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0224] Generally, the antibodies or antigen-binding fragments thereof of the present invention can be administered in various ranges. These include about 4 μg / kg body weight to about 100 mg / kg body weight per dose; about 20 μg / kg body weight to about 50 mg / kg body weight per dose; about 50 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 15 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0225] In certain preferred embodiments, the course of treatment involving the antibodies or antigen-binding fragments thereof of the present invention will include multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen-binding fragments thereof of the present invention may be administered every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it is understood that the dosage may be changed or the interval may be adjusted based on patient response and clinical practice.

[0226] Compatible formulations for parenteral administration (e.g., intravenous injection) will include an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the 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 / 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.

[0227] The antibodies of the invention can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, anti-tumor agents, anti-angiogenic agents, or immunosuppressive agents) to reduce the induction of an immune response against the antibodies of the invention. The antibody can be linked to the therapeutic agent (as an immune complex) or can be administered separately from the therapeutic agent.

[0228] In the context of administering a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which the treatment or therapeutic agent is administered to a subject. The treatment or therapeutic agent may be administered before, simultaneously, or after the second treatment or therapeutic agent is administered to the patient. Preferably, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that the treatment or therapeutic agent can work together. In a specific embodiment, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that they provide an increased benefit than if they were administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0229] Medical Uses

[0230] The antibodies, antibody compositions and methods of the invention have many in vitro and in vivo uses, including, for example, detection of TREM2 or enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo.

[0231] Preferred subjects include mammals, such as humans / patients. Mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals.

[0232] Treating conditions associated with TREM2 expression

[0233] In some aspects, the invention provides methods of treating a disorder in a mammal comprising administering to a subject (eg, a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

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

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

[0236] In some aspects, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of an agent (or drug) for treating a disease associated with TREM2 expression or determining the prognosis thereof.

[0237] In one embodiment, a disease associated with TREM2 includes a neoplastic disease, such as cancer.

[0238] In some embodiments, the disease associated with TREM2 is cancer, and the cancer is preferably selected from the following group:

[0239] Gastric cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, brain glioma, and ovarian cancer.

[0240] The antibodies or antigen-binding fragments thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiation therapy.

[0241] The antibodies or antigen-binding fragments thereof can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0242] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, anti-metastatic agents, and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such an engineered conjugate is explicitly contemplated within the scope of the present invention. In other embodiments, the disclosed anticancer agent will be administered in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0243] In some embodiments, the TREM2-related disease is a neurodegenerative disease, and the neurodegenerative disease is selected from the following group: Alzheimer's disease (AD), Parkinson's disease (PD), autism disorders (ASDs), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS).

[0244] In some embodiments, the TREM2-related disease is metabolic syndrome, and the metabolic syndrome is selected from the group consisting of adipocyte hypertrophy, systemic hypercholesterolemia, inflammation, and glucose intolerance.

[0245] diagnosis

[0246] The present invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods for screening cells from patients to identify tumor cells including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, including contacting 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 or binding level of the antibody bound to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

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

[0248] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for diagnosing, detecting or monitoring a disease associated with TREM2 in a subject.

[0249] In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent (or drug) for diagnosing, detecting or monitoring a disease associated with TREM2 expression.

[0250] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, etc., known to those skilled in the art.

[0251] The methods described in the present invention for in vitro detection or monitoring of TREM2 expression or TREM2-expressing cell levels can also be used for non-diagnostic purposes.

[0252] Preferred subjects include mammals, such as humans / patients in need thereof.

[0253] The sample from the subject is blood, excreta (urine or feces), oral or nasal secretions, or alveolar lavage fluid, tissue fluid, sweat, or extracts thereof from the subject.

[0254] Pharmaceutical packaging and kits

[0255] Also provided are pharmaceutical packaging and kits containing one or more containers of one or more doses of an antibody or an antigen-binding fragment thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or an antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may contain saline, sucrose or the like; a buffer such as phosphate, etc.; and / or be formulated in a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted after adding a suitable liquid (e.g., sterile water or saline). In certain 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 encapsulated conjugate composition is used to treat a selected tumor disease condition.

[0256] Such kits typically include a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally include one or more anticancer agents or other agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combined therapy.

[0257] More specifically, the kit can have a single container containing an antibody or antigen-binding fragment thereof of the present disclosure, with or without additional components, or they can have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or one component more than another. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers before being administered to a patient. The kit can also include a second / third container device for containing a sterile pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and glucose solution.

[0258] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as dry powders. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0259] Example

[0260] Example 1 Antigen preparation, mouse immunization and screening

[0261] A. DNA Antigen Preparation

[0262] 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) (the sequence is shown in SEQ ID NO: 1), and clone the coding sequence of the above gene into an expression vector by enzyme digestion.

[0263] SEQ ID NO:1 sequence:

[0264] ATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCCCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGCCCCTATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGAGAGAAGGGCCCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCAGTGCCAGAGCCTCCATGGCAGTGAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTGGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCA CAGCATCTCCAGGAGCCTCTTGGAAGGAGAAATCCCCTTCCCACCCACTTCCATCCTTCTCCTCCTGGCCTGCATCTTTCTCATCAAGATTCTAGCAGCCAGCGCCCTCTGGGCTGCAGCCTGGCATGGACAGAAGCCAGGGACACATCCACCCAGTGAACTGGACTGTGGCCATGACCCAGGGTATCAGCTCCAAACTCTGCCAGGGCTGAGAGACACGTGA

[0265] B. Preparation of protein antigen

[0266] The method for preparing the human TREM2 ECD-hFc protein for immunizing Balb / c mice is as follows: synthesize a nucleotide fragment encoding the extracellular region Fc fusion protein of human TREM2 (Uniprot ID Q9NZC2) (sequence as shown in SEQ ID NO: 2), and load it into the expression vector by DNA ligase. The plasmid containing Human TREM2 ECD-hFc was transferred into ExpiCHO (Gibco, cat: A29127) cells by PEI transfection. Nine days after transfection, the culture supernatant containing the target protein was harvested by centrifugation and filtration, and then the target protein was purified by affinity column (Cytiva, cat: 17549851). The purified protein will be identified by SDS-PAGE and SEC-HPLC.

[0267] SEQ ID NO:2 sequence:

[0268]

[0269] C. Preparation of reference antibody PY314

[0270] This study used the anti-TREM2 monoclonal antibody developed by Pionyr Immunotherapeutics as a reference, i.e., the reference antibody. The amino acid sequence was derived from the 37012 sequence in patent US2020 / 0140546A1. The DNA sequence of the variable region amino acids was synthesized (the sequence is shown in SEQ ID NO: 3). The heavy chain and light chain variable region sequences were loaded into the pTT5 vector (HonorGene, cat: HG-VPW0888) by DNA ligase, respectively. The heavy chain constant region subtype was hIgG1 and the light chain constant region subtype was kappa. The plasmids containing the heavy and light chains of the tool antibody were co-transfected into ExpiCHO cells using the PEI transfection method. 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 antibodies will be identified by SDS-PAGE and SEC-HPLC. The amino acid sequence of the reference antibody PY314, 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.

[0271] SEQ ID NO:3 sequence:

[0272] Heavy Chain:

[0273] GAGGTGCAGCTGCTGGAAAGCGGAGGCGGACTGGTGCAACCCGGCGGCAGCCTGAGACTGAGCTGCGCTGCTTCTGGCTTTACATTCAGCAACTACTACATGGCCTGGGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCAGCCTCACCAACAGCGGCGGATCTACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGATAATAGCAAGAACAC CCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTACAAGAGAGTGGGCCGGCAGCGGCTACTTCGACTACTGGGGCCAGGGCACCCTGGTCACAGTGTCCTCT

[0274] SEQ ID NO:4 sequence:

[0275] Light chain:

[0276] GACATCCAGATGACCCAGAGCCCATCCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATTACATGCAAGGCTTCTCAGAACGTGGGCAACAACCTGGCCTGGTACCAGCAGAAACCTGGCAAGGCCCCTAAGCTGCTGATCTACTATACATCTAATAGATTCACCGGCGTCCCCAGCAGATTCAGCGGATCTGGCAGCGGCACAGATTTTACCCTGACCATCAGCAGCCTGCAACCTGAGGACTTCGCCACCTACTACTGTCAGCGGATCTACAACAGCCCCTGGACCTTCGGCCAGGGAACAAAGCTGGAAATCAAG

[0277] D. Generation and screening of monoclonal antibodies

[0278] The human TREM2 expression vector prepared above and the human TREM2 ECD-hFc protein prepared above were used to immunize Balb / c mice (commercially purchased mice from Ruizhi Chemical). The above DNA antigen was mixed with gold powder and freeze-dried, and then the abdomen of the mouse was immunized multiple times using a gene gun. 4ug of the expression vector DNA was used for each immunization, with an interval of two weeks between each immunization. After two immunizations, blood was collected from the mice to measure the titer, and mice with high serological titers were selected for booster immunization. Each mouse was intraperitoneally injected with 25ug of human TREM2 ECD-hFc protein. After 3 days, the spleen, lymph nodes and bone marrow cells of the target animals were collected respectively. Plasma cells were separated and enriched for immune cells using a mouse CD138+ plasma cell separation kit (Miltenyi, cat: 130-092-530). Plasma cells are the cells that mainly secrete antibodies. After the plasma cells were adjusted to an appropriate concentration, they were irradiated with a Beacon light-guided system (Berkeley Lights, BLI Optofluidic System) into the independent pen on the chip, screened for positive B cells. Single cells can be lysed in the Beacon pen and directly exported to a 96-well plate. The RNA of the lysed single cells is collected by Agencourt RNAClean XPBeads (Beckman Coulter, cat: A63987) and then used cDNA synthesis was performed using RACE 5' / 3' kit (Clontech, cat: 634859). cDNA purification was performed using Agencourt AMPure XP Beads (Beckman Coulter, cat: A63881). Using cDNA as a template and mouse Ig-Primer Set (Novagen, 69831-3) as a gene-specific primer (GSP), PCR was performed to amplify the heavy and light chain variable regions.

[0279] Example 2 Detection of binding activity of anti-TREM2 antibodies to cell surface huTREM2

[0280] The plasmids containing the heavy and light chains of the TREM2 antibody were co-transfected into FUT8-KO ExpiCHO (Wisdom Chemical Construction) cells using the PEI transfection method. 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 will be identified by SDS-PAGE and SEC-HPLC. The antibody is C1.

[0281]

[0282] SEQ ID NO:5 Antibody C1 light chain full length sequence

[0283] DIVMTQAAPSVSVTPGESVSVSCRSSKSLLYSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0284] SEQ ID NO:6 Antibody C1 heavy chain full length sequence

[0285] EVQLQQSGPVLVKPGASVKMSCQASGYTFTDYYMNWVKQSHGKSLEWIGFINPYSGTTNYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARSLYYYG SEGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0286] The EC50 of antibody molecules binding to cell surface huTREM2 at different concentrations was measured by FACS to evaluate their binding activity.

[0287] As described above, the stable cell line CHOK1-human TREM2-DAP12 expressing the human TREM2 antigen was cultured and treated by the flow cytometry method. The cells harvested after digestion were centrifuged at 300g 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, the supernatant was discarded after centrifugation, and the cells were resuspended with a diluted antibody solution, 100uL / well, mixed, and incubated at 4°C for 1 hour. Centrifuge at 4°C, 300g for 5 minutes, discard the supernatant antibody solution, wash the cells twice with FACS buffer, add Goat anti human lgG-Alexafluro488 secondary antibody solution, 100uL / well, and incubate at 4 degrees for 1 hour. Centrifuge at 4°C, 300g for 5 minutes, discard the supernatant antibody solution, wash the cells twice with FACS buffer, and finally resuspend the cells with PBS, and detect the signal value on a BD flow cytometer.

[0288] The results are as follows Figure 1A , Figure 1B and Figure 1C As shown, C1, G3, 2A6, 2A10, mAb006c and mAb008c have strong binding activity with 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 that overexpress human TREM2, and have a certain dose dependence. The binding ability of C1, G3, 2A6, 2A10, mAb006c and mAb008c is significantly better than that of the reference antibody PY314.

[0289] Example 3 Species Cross-Activity Detection

[0290] The binding of C1, G3, 2A6, 2A10, mAb006c and mAb008c to cell surface CynoTREM2 at different concentrations was detected by FACS, and the EC50 was measured to evaluate their species cross-reactivity.

[0291] As described above, the stable cell line CHOK1-cyno TREM2-DAP12 expressing monkey TREM2 antigen was cultured and treated by flow cytometry. The cells harvested after digestion were centrifuged at 300g 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, the supernatant was discarded after centrifugation, and the cells were resuspended with a diluted antibody solution, 100uL / well, mixed, and incubated at 4°C for 1 hour. Centrifuge at 4°C, 300g for 5 minutes, discard the supernatant antibody solution, wash the cells twice with FACS buffer, add Goat anti human lgG-Alexafluro488 secondary antibody solution, 100uL / well, and incubate at 4 degrees for 1 hour. Centrifuge at 4°C, 300g for 5 minutes, discard the supernatant antibody solution, wash the cells twice with FACS buffer, and finally resuspend the cells with PBS, and detect the signal value on a BD flow cytometer.

[0292] Species cross-reactivity results with monkeys are as follows Figure 2A , Figure 2B and Figure 2C As shown, all six antibodies have good cynoTREM2 binding activity, among which C1, G3, 2A6, 2A10, mAb006c and mAb008 have significantly better binding activity to Cyno than the reference antibody.

[0293] The binding of C1, G3, 2A6, 2A10, mAb006c and mAb008c to cell surface mouseTREM2 at different concentrations was detected by FACS, and the EC50 was measured to evaluate their species cross-reactivity.

[0294] As described above, the stable cell line CHOK1-mouse TREM2-DAP12 expressing mouse TREM2 antigen was cultured and treated by flow cytometry. The cells harvested after digestion were centrifuged at 300g 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, the supernatant was discarded after centrifugation, and the cells were resuspended with a diluted antibody solution, 100uL / well, mixed, and incubated at 4°C for 1 hour. Centrifuge at 4°C, 300g for 5 minutes, discard the supernatant antibody solution, wash the cells twice with FACS buffer, add Goat anti human lgG-Alexafluro488 secondary antibody solution, 100uL / well, and incubate at 4 degrees for 1 hour. Centrifuge at 4°C, 300g for 5 minutes, discard the supernatant antibody solution, wash the cells twice with FACS buffer, and finally resuspend the cells with PBS, and detect the signal value on a BD flow cytometer.

[0295] Species cross-reactivity results with mice are as follows Figure 3A , Figure 3B and Figure 3C As shown, all six antibodies were able to bind to mouseTREM2, among which mAb008c, C1, and G3 antibodies had significantly better binding activity to mouse than the reference antibody.

[0296] Example 4 TREM2 reporter gene experiment

[0297] Genetically engineered Jurkat cells are used as effector cells, and the TREM2 receptor and DAP12 are stably expressed on Jurkat cells, as well as the Luciferase gene expressed by the NFAT response element. The activity of luciferase produced by the activation of the NFAT pathway can be used to quantitatively detect the activation or inhibition of the TREM2 signaling pathway by therapeutic antibodies.

[0298] The antibody is coated or mixed with Crosslinker (anti-Fc) for crosslinking, and then

[0299] Jurkat-NFAT-TREM2-DAP12 cells were incubated in a 37°C incubator for 6 hours, and an equal volume of ONE-Glo (Promega, E6120) was added. After mixing, the cells were incubated at room temperature in the dark for 10-15 minutes. The fluorescence signal was read with a microplate reader and the data was analyzed.

[0300] A crosslinker was used to increase the aggregation of antibodies (Abs:crosslinker = 1:0.5). The cloned NFAT signal was detected after incubation with Jurkat-TREM2-DAP12-NFAT cells at 37°C for 6 hours. The results are shown in Figure 4 As shown, the effects of C1, G3 and mAb008c antibodies on cells were slightly inhibitory, and the inhibitory effects were comparable to those of the reference antibody PY314, while 2A6, 2A10 and mAb006c had stronger inhibitory effects on cells, which were better than PY314.

[0301] Example 5 Affinity Detection

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

[0303] SPR technology was used for capture detection. A CM5 chip coupled with an Anti--mFc antibody was used to capture recombinant hTREM2, cyno-TREM2 or mTREM2 protein with a His tag as a ligand (Ligand). Different concentrations of TREM2 antibodies were set as analytes (Analysis). Kinetic detection was performed with the captured human recombinant hTREM2, cyno-TREM2 or mTREM2 protein. Biacore software was used to perform Kinetics fitting on the binding curve to obtain 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 in the EMT6 cell subcutaneous homologous transplant mouse tumor model was evaluated.

[0308] 2×10 6 Each mouse was subcutaneously inoculated with 0.1 mL of cell suspension (containing 2x10 5 On the 5th day after inoculation, tumors with a volume range of 40 to 67 mm 3 (mean tumor volume 55mm 3 ) tumor-bearing mice were divided into groups according to the tumor volume by stratified randomization, with 8 mice in each group. Drug administration began on the day of grouping (denoted as PG-D0) and was administered twice a week. The groups of this experiment are shown in Table 2 (ip means intraperitoneal injection, BIW means twice a week):

[0309] Table 2 Drug efficacy grouping experiment

[0310]

[0311]

[0312] Table 3 Tumor growth inhibition rate

[0313]

[0314] Tumor growth and inhibition rate results are as follows Figure 5As shown in Table 3, compared with the solvent control group, statistics showed that there were significant differences in tumor volume in treatment group 2, treatment group 3, treatment group 4, treatment group 5, treatment group 6, treatment group 7, treatment group 8 and treatment group 9. Compared with the efficacy of the reference molecule PY314 combined with PD-1 drug, the efficacy of 2A6+RMP1-14 and mAb006c+RMP1-14 was significantly better than the reference molecule.

[0315] When the tumor volume reaches 2000mm 3 As the survival endpoint, the time from the start of drug administration to the euthanasia of the mice was considered the survival time of the mice. 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 ​​corresponding to treatment group 2, treatment group 3, treatment group 4, treatment group 5, treatment group 6, treatment group 7, treatment group 8 and treatment group 9 were 18 days, 23 days, 19.5 days, 22 days, 29 days, 22 days, 24 days and 18 days, respectively. Compared with the solvent control group, treatment group 3, treatment group 5, treatment group 6, treatment group 7 and treatment group 8 can significantly prolong the survival of mice (ILS>25%). Statistical Kaplan-Meier survival analysis showed that compared with the solvent control group, treatment group 5, treatment group 6, treatment group 7 and treatment group 8 had significant differences.

[0316] Table 4. Survival analysis of mice in each group

[0317]

[0318]

[0319] Finally, it should be noted that the above is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Although the present disclosure is 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 replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

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

37.

2. The antibody or fragment according to claim 1, wherein The heavy chain variable region is shown in SEQ ID NO: 20; The light chain variable region is shown in SEQ ID NO:

19.

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 shown in SEQ ID NO: 34; It also comprises a light chain constant region, which is shown in SEQ ID NO:

33.

4. The isolated antibody or antigen-binding fragment thereof according to claim 3, wherein: Wherein the antibody comprises: the light chain as set forth in SEQ ID NO:5; and the heavy chain as set forth in SEQ ID NO:

6. The antibody or antigen-binding fragment thereof according to claim 4 , wherein the antibody is a monoclonal antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a chimeric antibody or a humanized antibody or an improved antibody such as an improved chimeric antibody.

7. The antibody or antigen-binding fragment thereof as claimed in claim 6, wherein the improved antibody is an improved chimeric antibody.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule according to claim 8.

10. A host cell comprising the nucleic acid molecule according to claim 8 or the vector according to claim 9.

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