Methods of using Anti-TREM2 antibodies

Abstract

To provide a pharmaceutical composition for treating ovarian cancer.SOLUTION: Provided is a pharmaceutical composition for treating ovarian cancer in a subject in need thereof, the pharmaceutical composition comprising an isolated antibody that specifically binds to TREM2, the antibody comprising a variable heavy chain domain (VH) sequence including CDR-H1, CDR-H2, and CDR-H3, and a variable light chain domain (VL) sequence including CDR-L1, CDR-L2, and CDR-L3.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of International Application No. PCT / US2018 / 065026, filed December 11, 2018, and U.S. Provisional Application No. 62 / 889,990, filed August 21, 2019, both of which are incorporated by reference in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on XX / XX / 2019, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] background Immunity plays a role in preventing tumor growth. Complex microenvironments can develop within the lesion, and despite T cell recruitment, there is often no effective control of the emerging tumor mass. Understanding the balance between tumor elimination and tumor escape may depend on understanding the different roles that myeloid cells play in the tumor microenvironment.

[0004] Myeloid populations in the tumor microenvironment prominently include monocytes and neutrophils (sometimes loosely grouped as myeloid-derived suppressor cells), macrophages, and dendritic cells. Although intratumoral myeloid populations have long been thought to be, overall, non-stimulatory or suppressive, it has more recently been recognized that not all tumor-infiltrating myeloid cells are equal.

[0005] In normal tissues, many of these myeloid cells are essential for the proper functioning of both innate and adaptive immunity, particularly wound repair. However, in the setting of cancer, a significant excess of macrophages and dysfunctional or distorted populations of these and other cell types are commonly described. Macrophage infiltration, when considered as an aggregate population defined by a single marker such as CD68 or CD163, correlates with worse subject outcomes for multiple tumor types ((de Visser, Cancer Immunol Immunother, 2008;57:1531-9); (Hanada et al., Int J Urol 2000;7:263-9); (Yao et al., Clin Cancer Res, 520, 2001;7:4021-6); (Ruffell et al., PNAS, 523, 2012;109:2796-801) (Non-Patent Documents 1-4)). However, the phenotypic and functional subsetting of macrophages in the tumor microenvironment is a complex issue in tumor biology, complicated by the similarities between macrophages and dendritic cells. Morphological criteria have often been applied to the problem, with one approach attempting to distinguish dendritic cells from macrophages based on a more processive or dendritic morphology in the former and a more veiled or bulbous morphology in the latter (Bell et al., J Exp Med 555, 1999;190:1417-26). Other groups attempt to distinguish based on genetic and cell surface markers.

[0006] There is diversity in antigen-presenting compartments within tumors, and T cells can distinguish between the characteristics of antigen-presenting cells (APCs). Because T cells are the primary drivers of tumor immunity, it will be important to understand the precise characteristics of allogeneic APCs. Myeloid cells are prominent among cells capable of presenting tumor-derived antigens to T cells, thereby maintaining the latter in an activated state. Antigen presentation occurs within the tumor itself and can affect the function of tumor cytotoxic T lymphocytes (CTLs). T cell activation by antigen-presenting cells (APCs) is a critical component in antigen-specific immune responses and tumor cell killing. Because these myeloid populations represent the primary T cell interaction partners and antigen-presenting cells for subsequent tumor-reactive cytotoxic T lymphocytes, understanding their identity can guide therapeutic approaches.

[0007] Related patent applications include PCT / US2015 / 052682 (Patent Document 1), filed September 28, 2015, PCT / US2016 / 054104 (Patent Document 2), filed September 28, 2016, and PCT / US2018 / 065026 (Patent Document 3), filed December 11, 2018, each of which is incorporated by reference herein in its entirety for all purposes.

[0008] All patents, patent applications, publications, documents, and articles cited herein are hereby incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] PCT / US2015 / 052682 [Patent Document 2] PCT / US2016 / 054104 [Patent Document 3] PCT / US2018 / 065026 [Non-patent literature]

[0010] [Non-Patent Document 1] de Visser,Cancer Immunol Immunother,2008;57:1531-9 [Non-patent document 2] Hanada et al.,Int J Urol 2000;7:263-9 [Non-patent document 3] Yao et al.Clin Cancer Res,520,2001;7:4021-6 [Non-patent document 4] Ruffell et al.,PNAS,523 2012;109:2796-801 [Non-patent document 5] Bell et al., J Exp Med 555, 1999;190:1417-26 Summary of the Invention

[0011] overview In one aspect, provided herein is a method of treating ovarian cancer in a subject in need thereof, the method comprising administering to the subject an isolated humanized antibody that binds to human TREM2 (SEQ ID NO: 15) and competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17). In one aspect, provided herein is a method of treating solid tumors in a subject in need thereof, the method comprising administering to the subject an isolated humanized antibody that binds to human TREM2 (SEQ ID NO: 15) and competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17).

[0012] In some embodiments, the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO:9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO:10, a CDR-H3 comprising the sequence set forth in SEQ ID NO:11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO:12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO:13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:14 or 44. In some embodiments, the antibody is afucosylated and comprises a VH sequence set forth in SEQ ID NO:1; a VL sequence set forth in SEQ ID NO:2; and an active human IgG1 Fc region. In some embodiments, the antibody comprises a sequence set forth in at least one of SEQ ID NO:9 or 39, SEQ ID NO:10 or 40, SEQ ID NO:11 or 41, SEQ ID NO:12 or 42, SEQ ID NO:13 or 43, and SEQ ID NO:14 or 44. In some embodiments, the antibody comprises a sequence set forth in each of SEQ ID NO:9 or 39, SEQ ID NO:10 or 40, SEQ ID NO:11 or 41, SEQ ID NO:12 or 42, SEQ ID NO:13 or 43, and SEQ ID NO:14 or 44. In some embodiments, the antibody comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the antibody comprises the sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody comprises the sequence set forth in SEQ ID NO: 1 and the sequence set forth in SEQ ID NO: 2.

[0013] In some embodiments, the antibody comprises a VH sequence comprising an A to T substitution at position 97 of the sequence set forth in SEQ ID NO: 7; and a K to R substitution at position 98 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 1, 3, or 5. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 1, 3, or 5 and a VL sequence set forth in SEQ ID NO: 2, 4, or 6. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 1.

[0014] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody is antibody 37012. In some embodiments, the antibody comprises the heavy chain sequence set forth in SEQ ID NO: 25 and the light chain sequence set forth in SEQ ID NO: 26.

[0015] In another aspect, provided herein is a method of treating a solid cancer in a subject in need thereof, the method comprising administering to the subject an isolated antibody that binds to human TREM2 (SEQ ID NO: 15), wherein the antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); ii) comprises a human Fc region. In another aspect, provided herein is a method of treating a solid cancer in a subject in need thereof, the method comprising administering to the subject an isolated antibody that binds to human TREM2 (SEQ ID NO: 15), wherein the antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); ii) comprises a human Fc region. In another aspect, provided herein is a method of treating ovarian cancer in a subject in need thereof, the method comprising administering to the subject an isolated antibody that binds to human TREM2 (SEQ ID NO: 15), wherein the antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); ii) comprises a human Fc region.

[0016] In another aspect, provided herein is a method of treating a solid cancer in a subject in need thereof, the method comprising administering to the subject an isolated humanized antibody, wherein the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, a CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44. In another aspect, provided herein is a method of treating ovarian cancer in a subject in need thereof, the method comprising administering to the subject an isolated humanized antibody, wherein the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, a CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44.

[0017] In some embodiments, the antibody comprises a VH sequence comprising an A to T substitution at position 97 of the sequence set forth in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence set forth in SEQ ID NO:7. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:1, 3, or 5. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:1, 3, or 5 and a VL sequence set forth in SEQ ID NO:2, 4, or 6. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:1. The method of claim 16, wherein the antibody comprises a VH sequence set forth in SEQ ID NO:1 and a VL sequence set forth in SEQ ID NO:2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises a heavy chain sequence set forth in SEQ ID NO:25 and a light chain sequence set forth in SEQ ID NO:26.

[0018] In some embodiments, the antibody has a cytoplasmic affinity of about 1, 2, 3, 4, or 5 x 10 as measured by surface plasmon resonance (SPR) assay. -9 K below M D It binds to human TREM2 at

[0019] In some embodiments, the antibody is capable of specifically killing, depleting, or neutralizing TREM2+ myeloid cells, optionally unstimulated myeloid cells, optionally intratumoral myeloid cells.

[0020] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody has antibody-mediated cellular phagocytosis (ADCP) activity. In some embodiments, the antibody has complement-dependent cytotoxicity (CDC) activity.

[0021] In some embodiments, the antibody is at least one of a monoclonal antibody, a neutralizing antibody, an antagonistic antibody, an agonist antibody, a polyclonal antibody, an IgG1 antibody, an IgG3 antibody, an afucosylated antibody, a bispecific antibody, a human antibody, a chimeric antibody, a full-length antibody, and an antigen-binding fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is afucosylated.

[0022] In some embodiments, the antibody is an antigen-binding fragment thereof, a Fab, a Fab', a F(ab')2, an Fv, a scFv, an (scFv)2, a single-chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V-domain antibody.

[0023] In some embodiments, the antibody comprises a scaffold, optionally the scaffold is Fc, optionally a human Fc. In some embodiments, the antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibody comprises a heavy chain constant region of the class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody comprises a heavy chain constant region of IgG1.

[0024] In some embodiments, the Fc comprises one or more modifications that result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more modifications. In some embodiments, the Fc binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

[0025] In some embodiments, the antibody binds to the extracellular domain of TREM2 on a TREM2+ myeloid cell, optionally the myeloid cell is in a tumor.

[0026] In some embodiments, the antibody binds to the extracellular domain of TREM2 on myeloid cells, the myeloid cells are unstimulated myeloid cells that are CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3-, and the antibody kills, neutralizes, or depletes the unstimulated myeloid cells via ADCC, CDC, and / or ADCP to a level that is less than the level of unstimulated myeloid cells present in the cancer before contacting the unstimulated myeloid cells with the antibody, and the unstimulated myeloid cells are present in a population of immune cells that includes stimulated myeloid cells and unstimulated myeloid cells that are CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+, and the killing, neutralization, or depletion of the unstimulated myeloid cells treats the cancer.

[0027] In some embodiments, the contacting enhances the subject's immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the subject has previously received, is concurrently receiving, or will subsequently receive immunotherapy.

[0028] In some embodiments, the immunotherapy is at least one of a checkpoint inhibitor, a T cell checkpoint inhibitor, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, an adoptive T cell therapy, a CAR-T cell therapy, a dendritic cell vaccine, a monocyte vaccine, an antigen binding protein that binds to both T cells and antigen-presenting cells, a BiTE dual antigen binding protein, a Toll-like receptor ligand, a cytokine, a cytotoxic therapy, chemotherapy, radiation therapy, a small molecule inhibitor, a small molecule agonist, an immunomodulatory agent, and an epigenetic modulator. In some embodiments, the immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0029] In another aspect, provided herein is a method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject an isolated humanized antibody that binds to human TREM2 (SEQ ID NO: 15) and competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17).

[0030] In some embodiments, the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, a CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44. In some embodiments, the antibody is afucosylated and comprises the VH sequence set forth in SEQ ID NO: 1; the VL sequence set forth in SEQ ID NO: 2; and an active human IgG1 Fc region.

[0031] In some embodiments, the antibody comprises a VH sequence comprising an A to T substitution at position 97 of the sequence set forth in SEQ ID NO: 7; and a K to R substitution at position 98 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 1, 3, or 5. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 1, 3, or 5 and a VL sequence set forth in SEQ ID NO: 2, 4, or 6. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 1.

[0032] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody is antibody 37012. In some embodiments, the antibody comprises the heavy chain sequence set forth in SEQ ID NO: 25 and the light chain sequence set forth in SEQ ID NO: 26.

[0033] In another aspect, provided herein is a method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject an isolated antibody that binds to human TREM2 (SEQ ID NO: 15), wherein the antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); and ii) comprises a human Fc region.

[0034] In another aspect, provided herein is a method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject an isolated humanized antibody, wherein the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, a CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44.

[0035] In some embodiments, the antibody comprises a VH sequence comprising an A to T substitution at position 97 of the sequence set forth in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence set forth in SEQ ID NO:7. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:1, 3, or 5. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:1, 3, or 5 and a VL sequence set forth in SEQ ID NO:2, 4, or 6. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:1. The method of claim 16, wherein the antibody comprises a VH sequence set forth in SEQ ID NO:1 and a VL sequence set forth in SEQ ID NO:2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises a heavy chain sequence set forth in SEQ ID NO:25 and a light chain sequence set forth in SEQ ID NO:26.

[0036] In some embodiments, the antibody has a cytoplasmic affinity of about 1, 2, 3, 4, or 5 x 10 as measured by surface plasmon resonance (SPR) assay. -9 K below M D It binds to human TREM2 at

[0037] In some embodiments, the antibody is capable of specifically killing, depleting, or neutralizing TREM2+ myeloid cells, optionally unstimulated myeloid cells, optionally intratumoral myeloid cells.

[0038] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody has antibody-mediated cellular phagocytosis (ADCP) activity. In some embodiments, the antibody has complement-dependent cytotoxicity (CDC) activity.

[0039] In some embodiments, the antibody is at least one of a monoclonal antibody, a neutralizing antibody, an antagonistic antibody, an agonist antibody, a polyclonal antibody, an IgG1 antibody, an IgG3 antibody, an afucosylated antibody, a bispecific antibody, a human antibody, a chimeric antibody, a full-length antibody, and an antigen-binding fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is afucosylated.

[0040] In some embodiments, the antibody is an antigen-binding fragment thereof, a Fab, a Fab', a F(ab')2, an Fv, a scFv, an (scFv)2, a single-chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V-domain antibody.

[0041] In some embodiments, the antibody comprises a scaffold, optionally the scaffold is Fc, optionally a human Fc. In some embodiments, the antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibody comprises a heavy chain constant region of the class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody comprises a heavy chain constant region of IgG1.

[0042] In some embodiments, the Fc comprises one or more modifications that result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more modifications. In some embodiments, the Fc binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

[0043] In some embodiments, the antibody binds to the extracellular domain of TREM2 on a TREM2+ myeloid cell, optionally the myeloid cell is in a tumor.

[0044] In some embodiments, the antibody binds to the extracellular domain of TREM2 on myeloid cells, the myeloid cells are unstimulated myeloid cells that are CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3-, and the antibody kills, neutralizes, or depletes the unstimulated myeloid cells via ADCC, CDC, and / or ADCP to a level that is less than the level of unstimulated myeloid cells present in the cancer before contacting the unstimulated myeloid cells with the antibody, and the unstimulated myeloid cells are present in a population of immune cells that includes stimulated myeloid cells and unstimulated myeloid cells that are CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+, and the killing, neutralization, or depletion of the unstimulated myeloid cells treats the cancer.

[0045] In some embodiments, the contacting enhances the subject's immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the subject has previously received, is concurrently receiving, or will subsequently receive immunotherapy.

[0046] In some embodiments, the immunotherapy is at least one of a checkpoint inhibitor, a T cell checkpoint inhibitor, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, an adoptive T cell therapy, a CAR-T cell therapy, a dendritic cell vaccine, a monocyte vaccine, an antigen binding protein that binds to both T cells and antigen-presenting cells, a BiTE dual antigen binding protein, a Toll-like receptor ligand, a cytokine, a cytotoxic therapy, chemotherapy, radiation therapy, a small molecule inhibitor, a small molecule agonist, an immunomodulatory agent, and an epigenetic modulator. In some embodiments, the immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0047] In another aspect, provided herein are methods of enhancing an immune response, the methods comprising administering to a subject an isolated humanized antibody that binds to human TREM2 (SEQ ID NO: 15) and competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17). In some aspects, the subject has a solid tumor. In some aspects, the solid tumor is an ovarian tumor. In some aspects, the solid tumor is a gastric tumor. In some aspects, the immune response comprises ADCC, CDC, and / or ADCP.

[0048] In some embodiments, the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, a CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44.

[0049] In some embodiments, the antibody is afucosylated and comprises the VH sequence set forth in SEQ ID NO: 1; the VL sequence set forth in SEQ ID NO: 2; and an active human IgG1 Fc region. In some embodiments, the antibody comprises a VH sequence comprising an A to T substitution at position 97 of the sequence set forth in SEQ ID NO: 7; and a K to R substitution at position 98 of the sequence set forth in SEQ ID NO: 7.

[0050] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1, 3, or 5. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1, 3, or 5 and the VL sequence set forth in SEQ ID NO: 2, 4, or 6. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises the heavy chain sequence set forth in SEQ ID NO: 25 and the light chain sequence set forth in SEQ ID NO: 26.

[0051] In another aspect, provided herein is a method of enhancing an immune response, the method comprising administering to a subject an isolated antibody that binds human TREM2 (SEQ ID NO: 15), wherein the antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); and ii) comprises a human Fc region. In some aspects, the subject has a solid tumor. In some aspects, the solid tumor is an ovarian tumor. In some aspects, the solid tumor is a gastric tumor. In some aspects, the immune response comprises ADCC, CDC, and / or ADCP.

[0052] In another aspect, provided herein is a method of enhancing an immune response, the method comprising administering an isolated humanized antibody to a subject, wherein the antibody comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, a CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, a CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44. In some aspects, the subject has a solid tumor. In some aspects, the solid tumor is an ovarian tumor. In some aspects, the solid tumor is a gastric tumor. In some aspects, the immune response comprises ADCC, CDC, and / or ADCP.

[0053] In some embodiments, the antibody comprises a VH sequence comprising an A to T substitution at position 97 of the sequence set forth in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence set forth in SEQ ID NO:7.

[0054] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1, 3, or 5. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1, 3, or 5 and the VL sequence set forth in SEQ ID NO: 2, 4, or 6. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 2.

[0055] In some embodiments, the antibody is antibody 37012. In some embodiments, the antibody comprises a heavy chain sequence set forth in SEQ ID NO: 25 and a light chain sequence set forth in SEQ ID NO: 26. In some embodiments, the antibody has a molecular weight of about 1, 2, 3, 4, or 5 x 10 as measured by surface plasmon resonance (SPR) assay. -9 K below M D It binds to human TREM2 at

[0056] In some embodiments, the subject has ovarian cancer. In some embodiments, the subject has gastric cancer.

[0057] In some embodiments, the subject has previously received, is concurrently receiving, or will subsequently receive immunotherapy. In some embodiments, the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds to both T cells and antigen-presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator. In some embodiments, the immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0058] In some embodiments, the antibody is capable of specifically killing, depleting, or neutralizing TREM2+ myeloid cells, optionally unstimulated myeloid cells, optionally intratumoral myeloid cells.

[0059] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody has antibody-mediated cellular phagocytosis (ADCP) activity. In some embodiments, the antibody has complement-dependent cytotoxicity (CDC) activity.

[0060] In some embodiments, the antibody has receptor-ligand blocking, agonist, or antagonist activity, hi some embodiments, the antibody has agonist activity.

[0061] In some embodiments, the antibody induces increased expression of at least one cytokine or chemokine in cells compared to an isotype control antibody. In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, TNF-α, CXCL1, or CXCL10. In some embodiments, the cytokine or chemokine is CXCL10.

[0062] In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the antibody induces a memory immune response.

[0063] In some embodiments, the cell is a TREM2+ cell. In some embodiments, the TREM2+ cell is selected from the group consisting of a dendritic cell, a tumor-associated macrophage (TAM), and a neutrophil.

[0064] In some embodiments, the subject is a human. [The present invention 1001] A method for treating ovarian cancer in a subject in need thereof, comprising administering to the subject an isolated humanized antibody that binds to human TREM2 (SEQ ID NO: 15) and competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17). [The present invention 1002] The antibody a. CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39; b. CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40; c. CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41; d. CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42; e. CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and f. CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44 The method of the present invention 1001, comprising: [The present invention 1003] 1003. The method of claim 1002, wherein said antibody is afucosylated and comprises a VH sequence shown in SEQ ID NO: 1; a VL sequence shown in SEQ ID NO: 2; and an active human IgG1 Fc region. [The present invention 1004] The antibody A VH sequence comprising an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence shown in SEQ ID NO:7. The method of the present invention 1002, comprising: [The present invention 1005] 1004. The method of claim 1003, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO: 1, 3, or 5. [The present invention 1006] 1005. The method of claim 10, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6. [The present invention 1007] 1004. The method of claim 1003, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO:1. [The present invention 1008] 1007. The method of claim 10, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. [The present invention 1009] 1008. The method of the present invention, wherein the antibody is 37012 antibody. [The present invention 1010] 1001. The method of claim 1001, wherein said antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. [The present invention 1011] 1. A method of treating ovarian cancer in a subject in need thereof, comprising administering to the subject an isolated antibody that binds to human TREM2 (SEQ ID NO: 15); The antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); ii) comprises a human Fc region; The method. [The present invention 1012] 1. A method of treating ovarian cancer in a subject in need thereof, comprising: a. CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39; b. CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40; c. CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41; d. CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42; e. CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and f. CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44 administering to said subject an isolated humanized antibody comprising: [The present invention 1013] The antibody A VH sequence comprising an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence shown in SEQ ID NO:7. The method of the present invention 1012, comprising: [The present invention 1014] 1013. The method of claim 1013, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO: 1, 3, or 5. [The present invention 1015] 1014. The method of claim 1014, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6. [The present invention 1016] 1015. The method of claim 10, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO:1. [The present invention 1017] 1016. The method of claim 1016, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. [The present invention 1018] The method of the present invention 1017, wherein the antibody is 37012 antibody. [The present invention 1019] 1012. The method of claim 1012, wherein said antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. [The present invention 1020] The antibody has a cytoplasmic affinity of about 1, 2, 3, 4, or 5×10 as measured by a surface plasmon resonance (SPR) assay. -9 K below M D Any of the preceding methods of the invention, wherein the antibody binds to human TREM2 at [The present invention 1021] Any of the prior methods of the invention, wherein said antibody is capable of specifically killing, depleting or neutralizing TREM2+ myeloid cells, optionally unstimulated myeloid cells, optionally intratumoral myeloid cells. [The present invention 1022] Any of the prior methods of the invention, wherein said antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [The present invention 1023] Any of the prior methods of the invention, wherein said antibody has antibody-mediated cellular phagocytosis (ADCP) activity. [The present invention 1024] Any of the prior methods of the invention, wherein said antibody has complement dependent cytotoxicity (CDC) activity. [The present invention 1025] Any of the preceding methods of the invention, wherein the antibody is at least one of a monoclonal antibody, a neutralizing antibody, an antagonistic antibody, an agonist antibody, a polyclonal antibody, an IgG1 antibody, an IgG3 antibody, an afucosylated antibody, a bispecific antibody, a human antibody, a chimeric antibody, a full-length antibody, and an antigen-binding fragment thereof. [The present invention 1026] The method of any of the prior inventions, wherein said antibody is a monoclonal antibody. [The present invention 1027] The method of any one of the preceding inventions, wherein said antibody is multispecific. [The present invention 1028] Any of the preceding methods of the invention, wherein said antibody is afucosylated. [The present invention 1029] Any of the preceding methods of the invention, wherein the antibody is an antigen-binding fragment thereof, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V-domain antibody. [The present invention 1030] Any of the prior methods of the invention, wherein said antibody comprises a scaffold, and optionally said scaffold is an Fc, optionally a human Fc. [The present invention 1031] Any of the preceding methods of the invention, wherein said antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. [The present invention 1032] Any of the preceding methods of the invention, wherein said antibody comprises a heavy chain constant region of said IgG class and subclass selected from IgG1, IgG2, IgG3, and IgG4. [The present invention 1033] Any of the prior methods of the invention, wherein said antibody comprises an IgG1 heavy chain constant region. [The present invention 1034] Any of the preceding methods of the invention, wherein the Fc comprises one or more modifications that result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to the Fc without said one or more modifications. [This invention 1035] Any of the preceding methods of the invention, wherein the Fc binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. [The present invention 1036] Any of the prior methods of the invention, wherein said antibody binds to the extracellular domain of TREM2 on a TREM2+ myeloid cell, and optionally said myeloid cell is intratumor. [This invention 1037] the antibody binds to the extracellular domain of TREM2 on myeloid cells; the bone marrow cells are unstimulated bone marrow cells that are CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3-; the antibody kills, disables, or depletes the unstimulated bone marrow cells via ADCC, CDC, and / or ADCP to a level that is less than the level of unstimulated bone marrow cells present in the cancer prior to contacting the unstimulated bone marrow cells with the antibody; the unstimulated bone marrow cells are present in a population of immune cells that includes stimulator bone marrow cells that are CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+, and the unstimulated bone marrow cells; said killing, disabling, or depleting said unstimulated bone marrow cells treats said cancer; Any method of the preceding invention. [The present invention 1038] Any of the preceding methods of the present invention, wherein said contacting enhances an immune response in said subject. [This invention 1039] The method of claim 1038, wherein said enhanced immune response is an adaptive immune response. [The present invention 1040] The method of claim 1038, wherein said enhanced immune response is an innate immune response. [The present invention 1041] The method of any of claims 1001 to 1040, wherein said subject has previously undergone, is concurrently undergoing, or will subsequently undergo immunotherapy. [The present invention 1042] The method of claim 1041, wherein the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds to both T cells and antigen presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator. [This invention 1043] 1043. The method of claim 1042, wherein said immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody. [This invention 1044] A method for treating gastric cancer in a subject in need thereof, comprising administering to the subject an isolated humanized antibody that binds to human TREM2 (sequence number 15) and competes with the 37017 antibody (sequence numbers 31 and 32) for binding to mouse TREM2 (sequence number 17). [This invention 1045] The antibody a. CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39; b. CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40; c. CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41; d. CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42; e. CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and f. CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44 The method of the present invention 1044, comprising: [The present invention 1046] 1045. The method of claim 1045, wherein said antibody is afucosylated and comprises a VH sequence shown in SEQ ID NO: 1; a VL sequence shown in SEQ ID NO: 2; and an active human IgG1 Fc region. [This invention 1047] The antibody A VH sequence comprising an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence shown in SEQ ID NO:7. The method of the present invention 1045, comprising: [This invention 1048] 1046. The method of claim 1046, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO: 1, 3, or 5. [This invention 1049] 1048. The method of claim 1048, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6. [The present invention 1050] 1046. The method of claim 1046, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO:1. [This invention 1051] 1050. The method of claim 1050, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. [This invention 1052] 1051. The method of claim 1051, wherein the antibody is antibody 37012. [This invention 1053] 104. The method of claim 1044, wherein said antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. [This invention 1054] 1. A method of treating gastric cancer in a subject in need thereof, comprising administering to the subject an isolated antibody that binds to human TREM2 (SEQ ID NO: 15); The antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); ii) comprises a human Fc region; The method. [This invention 1055] 1. A method of treating gastric cancer in a subject in need thereof, comprising: a. CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39; b. CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40; c. CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41; d. CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42; e. CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and f. CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44 administering to said subject an isolated humanized antibody comprising: [The present invention 1056] The antibody A VH sequence comprising an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence shown in SEQ ID NO:7. The method of the present invention 1055, comprising: [This invention 1057] 1056. The method of claim 1056, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO: 1, 3, or 5. [This invention 1058] 1057. The method of claim 1057, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6. [This invention 1059] 1058. The method of claim 1058, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO:1. [The present invention 1060] 1059. The method of claim 1059, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. [The present invention 1061] The method of claim 1060, wherein the antibody is antibody 37012. [The present invention 1062] 1055. The method of claim 1055, wherein said antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. [This invention 1063] The antibody has a cytoplasmic affinity of about 1, 2, 3, 4, or 5×10 as measured by a surface plasmon resonance (SPR) assay. -9 K below M D The method of any one of claims 1044 to 1062, wherein the antibody binds to human TREM2. [The present invention 1064] 4. The method of any of claims 1044 to 1063, wherein said antibody is capable of specifically killing, depleting or neutralizing TREM2+ bone marrow cells, optionally unstimulated bone marrow cells, optionally intratumoral bone marrow cells. [This invention 1065] The method according to any one of claims 1044 to 1064, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [The present invention 1066] The method of any one of claims 1044 to 1065, wherein the antibody has antibody-mediated cellular phagocytosis (ADCP) activity. [This invention 1067] The method according to any one of claims 1044 to 1066, wherein the antibody has complement-dependent cytotoxicity (CDC) activity. [The present invention 1068]

[0033] Any of the methods of claims 1044 to 1067, wherein the antibody is at least one of a monoclonal antibody, a neutralizing antibody, an antagonistic antibody, an agonist antibody, a polyclonal antibody, an IgG1 antibody, an IgG3 antibody, an afucosylated antibody, a bispecific antibody, a human antibody, a chimeric antibody, a full-length antibody, and an antigen-binding fragment thereof. [The present invention 1069] The method of any one of claims 1044 to 1068, wherein the antibody is a monoclonal antibody. [The present invention 1070] 1069. The method of any of claims 1044 to 1069, wherein said antibody is multispecific. [This invention 1071] The method of any of claims 1044 to 1070, wherein the antibody is afucosylated. [This invention 1072] Any of the methods of claims 1044 to 1071, wherein the antibody is an antigen-binding fragment thereof, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V-domain antibody. [This invention 1073] The method of any of claims 1044 to 1072, wherein said antibody comprises a scaffold, and optionally said scaffold is Fc, optionally human Fc. [This invention 1074] The method of any of claims 1044 to 1073, wherein the antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. [This invention 1075] The method of any of claims 1044 to 1074, wherein the antibody comprises a heavy chain constant region of the IgG class and a subclass selected from IgG1, IgG2, IgG3, and IgG4. [This invention 1076] The method of any one of claims 1044 to 1075, wherein the antibody comprises a heavy chain constant region of IgG1. [This invention 1077] Any of the methods of claims 1044 to 1076, wherein the Fc comprises one or more modifications, and said one or more modifications result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to said Fc without said one or more modifications. [This invention 1078] The method of any of claims 1044 to 1077, wherein the Fc binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. [This invention 1079] 9. The method of any of claims 1044 to 1078, wherein said antibody binds to the extracellular domain of TREM2 on TREM2+ myeloid cells, and optionally said myeloid cells are intratumoral. [The present invention 1080] the antibody binds to the extracellular domain of TREM2 on myeloid cells; the bone marrow cells are unstimulated bone marrow cells that are CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3-; the antibody kills, disables, or depletes the unstimulated bone marrow cells via ADCC, CDC, and / or ADCP to a level that is less than the level of unstimulated bone marrow cells present in the cancer prior to contacting the unstimulated bone marrow cells with the antibody; the unstimulated bone marrow cells are present in a population of immune cells that includes stimulator bone marrow cells that are CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+, and the unstimulated bone marrow cells; said killing, disabling, or depleting said unstimulated bone marrow cells treats said cancer; Any of methods 1044 to 1079 of the present invention. [This invention 1081] The method of any of claims 1044 to 1080, wherein said contacting enhances an immune response in said subject. [This invention 1082] 1081. The method of claim 1081, wherein said enhanced immune response is an adaptive immune response. [This invention 1083] 1081. The method of claim 1081, wherein said enhanced immune response is an innate immune response. [This invention 1084] 4. The method of any of claims 1044 to 1083, wherein said subject has previously undergone, is concurrently undergoing, or will subsequently undergo, immunotherapy. [This invention 1085] The method of claim 1084, wherein the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds to both T cells and antigen presenting cells; a BiTE dual antigen binding protein; a toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator. [The present invention 1086] 1085. The method of claim 1085, wherein said immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody. [This invention 1087] A method for enhancing an immune response, comprising administering to a subject an isolated humanized antibody that binds to human TREM2 (sequence number 15) and competes with the 37017 antibody (sequence numbers 31 and 32) for binding to mouse TREM2 (sequence number 17). [This invention 1088] The antibody a. CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39; b. CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40; c. CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41; d. CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42; e. CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and f. CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44 The method of the present invention 1087, comprising: [This invention 1089] 108. The method of claim 1088, wherein said antibody is afucosylated and comprises a VH sequence shown in SEQ ID NO: 1; a VL sequence shown in SEQ ID NO: 2; and an active human IgG1 Fc region. [The present invention 1090] The antibody A VH sequence comprising an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence shown in SEQ ID NO:7. The method of the present invention 1088, comprising: [This invention 1091] 1090. The method of claim 1090, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO: 1, 3, or 5. [This invention 1092] 1090. The method of claim 1090, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6. [This invention 1093] 1090. The method of claim 1090, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO:1. [This invention 1094] 1093. The method of claim 1093, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. [This invention 1095] The method of claim 1094, wherein the antibody is antibody 37012. [This invention 1096] 1087. The method of claim 1087, wherein said antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. [This invention 1097] 1. A method of enhancing an immune response, comprising administering to a subject an isolated antibody that binds human TREM2 (SEQ ID NO: 15); The antibody i) competes with the 37017 antibody (SEQ ID NOs: 31 and 32) for binding to mouse TREM2 (SEQ ID NO: 17); ii) comprises a human Fc region; The method. [This invention 1098] 1. A method of enhancing an immune response, comprising: a. CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39; b. CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40; c. CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41; d. CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42; e. CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and f. CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44 The method comprises administering to a subject an isolated humanized antibody comprising: [This invention 1099] The antibody A VH sequence comprising an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a K to R substitution at position 98 of the sequence shown in SEQ ID NO:7. The method of the present invention 1098, comprising: [The present invention 1100] 1099. The method of claim 1099, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO: 1, 3, or 5. [The present invention 1101] 110. The method of claim 1100, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6. [The present invention 1102] 1102. The method of claim 1101, wherein said antibody comprises a VH sequence as set forth in SEQ ID NO:1. [The present invention 1103] 1103. The method of claim 1102, wherein said antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. [The present invention 1104] 1103. The method of claim 1103, wherein the antibody is antibody 37012. [This invention 1105] 1098. The method of claim 1098, wherein said antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. [The present invention 1106] The antibody has a cytoplasmic affinity of about 1, 2, 3, 4, or 5×10 as measured by a surface plasmon resonance (SPR) assay. -9 K below M D The method of any one of claims 1087 to 1105, wherein the antibody binds to human TREM2. [This invention 1107] The method of any one of claims 1087 to 1106, wherein the subject has ovarian cancer. [This invention 1108] The method of any one of claims 1087 to 1106, wherein the subject has gastric cancer. [This invention 1109] The method of any of claims 1087 to 1108, wherein said subject has previously undergone, is concurrently undergoing, or will subsequently undergo immunotherapy. [The present invention 1110] The method of the present invention 1109, wherein the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds to both T cells and antigen presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator. [The present invention 1111] 1110. The method of claim 1110, wherein said immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody. [The present invention 1112] 2. The method of any of claims 1087 to 1111, wherein said antibody is capable of specifically killing, depleting, or neutralizing TREM2+ bone marrow cells, optionally unstimulated bone marrow cells, optionally intratumoral bone marrow cells. [The present invention 1113] The method according to any one of claims 1087 to 1111, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [This invention 1114] The method of any one of claims 1087 to 1111, wherein the antibody has antibody-mediated cellular phagocytosis (ADCP) activity. [This invention 1115] The method according to any one of claims 1087 to 1111, wherein the antibody has complement-dependent cytotoxicity (CDC) activity. [The present invention 1116] 6. The method of any one of claims 1087 to 1115, wherein said antibody has receptor-ligand blocking, agonist, or antagonist activity. [This invention 1117] 1116. The method of claim 1116, wherein said antibody has agonist activity. [This invention 1118] The method of any of claims 1087 to 1117, wherein said antibody induces increased expression of at least one cytokine or chemokine in the cells compared to an isotype control antibody. [This invention 1119] 1118. The method of claim 1118, wherein said at least one cytokine or chemokine is selected from the group consisting of IFN-γ, TNF-α, CXCL1, or CXCL10. [The present invention 1120] 1119. The method of claim 1119, wherein said cytokine or chemokine is CXCL10. [This invention 1121] The method of any of claims 1087 to 1120, wherein said enhanced immune response is an adaptive immune response. [This invention 1122] The method of any of claims 1087 to 1120, wherein the enhanced immune response is an innate immune response. [This invention 1123] The method of any one of claims 1087 to 1120, wherein the antibody induces a memory immune response. [This invention 1124] The method of any one of claims 1087 to 1123, wherein the cells are TREM2+ cells. [Invention 1125] 1125. The method of claim 1124, wherein said TREM2+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), and neutrophils. [The present invention 1126] The method of any one of claims 1087 to 1125, wherein the subject is a human. [Brief explanation of the drawings]

[0065] [Figure 1A] Figure 1 shows anti-TREM2 PI-7012-mediated anti-tumor activity in combination with anti-PD-1 in the CT-26 syngeneic mouse tumor model. Afucosylation of PI-7012 improves anti-tumor activity in combination with anti-PD-1. Mean tumor volumes (10 mice / group) are shown. [Figure 1B] Individual tumor volumes for PI-7012 are shown. [Figure 1C] Individual tumor volumes of afucosylated-PI-7012 (afuc-PI-7012) are shown. [Figure 2]There was no significant weight loss due to the combined treatment. Ten mice in each group were treated with the indicated antibodies, and body weights were recorded frequently. The mean body weight for each group was plotted against the number of days of the study. [Figure 3] In addition to H&E staining, tissues were also stained for macrophages using anti-CD68. The intracellular marker CD68 is widely used in the literature as a reliable cytochemical marker for immunostaining monocytes / macrophages in inflamed tissues and tumors. In the lung (Panel E) and other tissues analyzed, no discernible changes in the number of CD68+ macrophages were observed in any of the treatment groups compared to controls, indicating that anti-TREM2-mediated depletion occurred specifically in the TME. [Figure 4] Figure 4A shows anti-CD68 staining of FFPE lung tissue from the indicated treatment groups. Figure 4B shows quantification by light microscopy of 8–9 fields of view in each section. [Figure 5A] We show that TREM2 expression is absent or very low in cells of selected tissues. [Figure 5B] TREM2 expression is absent or very low in cells of selected tissues. Gray histograms are from TREM2KO mice, and open histograms are from wild-type mice. The antibody used for anti-TREM2 staining was clone 237920 from R&D Systems. [Figure 6] Cell surface expression of TREM2 (open histograms) was significantly higher in TAMs compared to granulocytic or monocytic MDSCs in both MC38 and CT26 tumors. Lymphocytes do not express TREM2. Isotype control staining is shown in gray filled histograms. [Figure 7]Cell surface expression of TREM2 (open histogram) was significantly higher in CD14-derived macrophages compared to PBMC subsets. Human PBMCs or macrophages were surface stained for either TREM2 (open histogram) or an isotype control (gray histogram). PBMC subsets were identified as neutrophils, monocytes, or T cells using a pre-validated multicolor FACS panel. [Figure 8] Cell surface expression of TREM2 (open histogram) was significantly higher in TAMs compared with other infiltrating cells or non-CD45-positive cells. Single-cell suspensions derived from human tumor tissue were surface stained for either TREM2 (open histogram) or an isotype control (gray histogram). Immune and non-immune subsets were identified using a pre-validated multicolor FACS panel. [Figure 9-1] Figure 9A shows that the anti-TREM2 mAb afuc-PI7012 in combination with an anti-PD-1 mAb resulted in significant anti-tumor activity in the Panc-02 pancreatic tumor model. Tumor volume was tracked over time in female C57BL / 6J mice implanted with Panc-02 tumor cells and treated with the indicated mAbs. The Y-axis represents the mean + / - standard deviation of the average tumor volume of 10 mice in each group. [Figure 9-2] Figure 9B shows tumor volumes from individual animals treated with an isotype control mAb. Figure 9C shows tumor volumes from individual animals treated with anti-TREM2 mAb afuc-PI7012. Figure 9D shows tumor volumes from individual animals treated with anti-PD-1. Figure 9E shows tumor volumes from individual animals treated with anti-TREM2 mAb afuc-PI7012 and anti-PD-1. [Figure 9-3] FIG. 9F shows the statistical analysis of group mean tumor volumes at day 32 post-implantation for each treatment group. [Figure 10]Tumor-free BALB / c mice treated with anti-TREM2 mAb and anti-PD-1 mAb were re-challenged with CT26 tumor cells (square symbols) 3 months later. Age-matched, treatment-naive mice (circle symbols) were administered equivalent numbers of CT26 cells and followed for tumor growth during the study period. Mice received no additional treatment during the study period. [Figure 11] Figure 1 shows that the anti-TREM2 mAb afuc-PI7012 in combination with an anti-PD-1 mAb resulted in significant antitumor activity in the ID8 ovarian tumor model. Tumor cell luminescence was tracked over time in female C57BL / 6J mice implanted with ID8 tumor cells and treated with the indicated mAbs. The Y-axis represents the mean + / - standard deviation of the average tumor luminescence of 10 mice in each group. [Figure 12-1] Figure 12A shows TREM2 expression in ovarian cancer, Figure 12B shows TREM2 expression in ovarian cancer, Figure 12C shows TREM2 expression in gastric cancer, and Figure 12D shows TREM2 expression in liver cancer. [Figure 12-2] Figure 12E shows TREM2 expression in prostate cancer, Figure 12F shows TREM2 expression in pancreatic cancer, Figure 12G shows TREM2 expression in bladder cancer, and Figure 12H shows TREM2 expression in lung cancer. [Figure 12-3] Figure 12I shows TREM2 expression in colon cancer, Figure 12J shows TREM2 expression in kidney cancer, Figure 12K shows TREM2 expression in breast cancer, and Figure 12L shows TREM2 expression in TNBC cancer. [Figure 12-4] Figure 12M shows TREM2 expression in melanoma, Figure 12N shows TREM2 expression in endometrial carcinoma, and Figure 12O shows TREM2 expression in lung adenocarcinoma. [Figure 13]Figure 13A shows that anti-TREM2 antibody treatment resulted in a reduction in tumor size in an in vivo EMT6 model. Figure 13B shows that anti-TREM2 antibody therapy reduced the number of MHCII-low TAMs and increased the number of MHCII-high TAMs compared to isotype antibody alone. Figure 13C shows that anti-TREM2 antibody therapy increased the number of CD8+ T cells and NKp46+ NK cells compared to isotype antibody alone. [Figure 14] Figure 14A shows the production of the pro-inflammatory cytokines IFN-γ, TNF-α, and CXCL1 after treatment with anti-TREM2 and PD-1 antibodies, and Figure 14B shows the increase in the number of CD8+ T cells expressing granzyme B (GrzB), TNF-α, or IFN-γ after treatment with both anti-TREM2 and anti-PD-1 antibodies. [Figure 15] 1 shows a dose-dependent increase in CXCL10 secretion by BMDMs after incubation with anti-TREM2 antibody. [Figure 16] We show that TREM2 expression levels in gastric cancer are inversely correlated with patient survival chances. [Figure 17] Figure 17A shows that TREM2 expression levels in ovarian cancer are inversely correlated with the likelihood of patient survival. Figure 17B shows that TREM2 expression levels in ovarian cancer are inversely correlated with the likelihood of patient survival. Figure 17C shows that TREM2 expression levels in ovarian cancer are inversely correlated with the likelihood of patient survival. [Figure 18] We show that TREM2 expression levels in ovarian cancer are inversely correlated with tumor malignancy. [Figure 19] A comparison of the amount of soluble TREM2 (ng / ml) in ovarian plasma samples and normal non-cancer plasma samples is shown. [Figure 20] Figure 1 shows that TREM2 is primarily expressed in tumor-associated macrophages (TAMs). Bars indicate the % of CD45+ immune infiltrating cell types isolated from ovarian tumors. Inset dark gray bars indicate the percentage of TREM2+ cells in each cell subset. TREM2-positive cells were primarily found in the TAM and macrophage populations. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description definition For the purposes of interpreting this specification, the following definitions will apply, and wherever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition below conflicts with any document incorporated herein by reference, the stated definition shall control.

[0067] Aspects and embodiments of the invention described herein are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0068] For all compositions and methods of using the compositions described herein, the composition may include any recited components or steps or may "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" recited components, the composition contains the recited components and may contain other components other than the explicitly recited components that do not substantially affect the condition being treated, but does not contain any other components that substantially affect the condition being treated, or if the composition contains additional components other than the recited components that substantially affect the condition being treated, the composition does not contain the additional components in concentrations or amounts sufficient to substantially affect the condition being treated. When a method is described as "consisting essentially of" recited steps, the method contains the recited steps and may contain other steps that do not substantially affect the condition being treated, but the method does not contain any other steps other than those explicitly recited that substantially affect the condition being treated. As a specific, non-limiting example, when a composition is described as "consisting essentially of" a component, the composition may further contain any amount of a pharmaceutically acceptable carrier, vehicle, or diluent, and such other components which do not substantially affect the condition being treated.

[0069] The term "optionally," when used sequentially, means to include from one to all of the listed combinations, and contemplates all subcombinations.

[0070] As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic compound, such as an anti-TREM2 antigen binding agent or anti-TREM2 antibody, administered to an individual, either as a single dose or as part of a series, that is effective to produce or is responsible for the desired therapeutic effect, either alone or in combination with another therapy. Examples of desired therapeutic effects are an enhanced immune response; slowing or delaying tumor progression; stabilizing disease; or amelioration of one or more symptoms. An effective amount may be given in one or more administrations.

[0071] As used herein, the term "treating" refers to slowing or reversing the progression of a condition, such as cancer. As used herein, the term "treatment" refers to the act of treating a condition, such as cancer.

[0072] As used herein, "individual" or "subject" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual is a human. In some embodiments, the individual is a mouse.

[0073] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0074] The terms "increase" and "activate" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater increase in the recited variable.

[0075] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater decrease in the recited variable.

[0076] The term "stimulate" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and stimulates a receptor.

[0077] The term "antagonize" refers to the inhibition of receptor signaling to inhibit the biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0078] The term "about" as used herein refers to the normal error range for the respective value that would be readily apparent to those skilled in the art. An exemplary error range is plus or minus 5%. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0079] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0080] For any of the structural and functional properties described herein, methods for determining these properties are known in the art.

[0081] antibody structure The present application provides antibodies and compositions, including antibodies that bind to TREM2 protein, including antibodies that neutralize unstimulated bone marrow cells.

[0082] The term "antibody" is used herein in the broadest sense to include a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0083] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0084] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminal domain of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. An IgG1 heavy chain is composed of, from N- to C-terminus, VH, CH1, CH2, and CH3 domains, respectively. The light chain is composed of, from N- to C-terminus, VL and CL domains. An IgG1 heavy chain contains a hinge between the CH1 and CH2 domains. In certain embodiments, an immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are derived from or are derived from immunoglobulin-based constructs, such as diabodies or nanobodies. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain derived from a heavy chain antibody, such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain derived from a mammalian antibody, such as a bovine antibody, a human antibody, a camelid antibody, a murine antibody, or any chimeric antibody.

[0085] In some embodiments, an antibody provided herein comprises a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0086] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, a naturally occurring four-chain antibody comprises six HVRs: three VH (H1, H2, H3) and three VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or complementarity-determining regions (CDRs), the latter of which are of the highest sequence variability and / or are involved in antigen recognition. With the exception of CDR1 of VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen-binding region. This particular region is described by Kabat et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), and the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody.

[0087] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of a number of known numbering schemes, including those described in Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0088] Table A provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0089] CDRs may be assigned using antibody numbering software such as Abnum, for example, as described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, available at www.bioinf.org.uk / abs / abnum / and incorporated by reference in its entirety.

[0090] Table A: CDR residues according to the Kabat and Chothia numbering scheme TIFF2025128164000002.tif42146*The C-terminus of CDR-H1 varies between H32 and H34 depending on the length of the CDR when numbered using the Kabat numbering convention.

[0091] The "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0092] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain such embodiments, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain of the single-chain Fab molecule. As described in more detail herein, an scFv has a variable domain of the light chain (VL) linked by a polypeptide chain from the C-terminus to the N-terminus of the variable domain of the heavy chain (VH). Alternatively, an scFv is composed of a polypeptide chain in which the C-terminus of the VH is linked by a polypeptide chain to the N-terminus of the VL.

[0093] A "Fab fragment" (also called fragment antigen binding) contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), along with the variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain the complementarity-determining loops (CDRs, also called hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0094] An "F(ab')2" fragment contains two Fab' fragments linked near the hinge region by a disulfide bond. F(ab')2 fragments may be produced, for example, by recombinant methods or by pepsin digestion of intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0095] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0096] A "single-chain Fv" or "scFv" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0097] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. The Fc domain may be linked to the C-terminus of the scFv (i.e., V H -V L or V L -V H Depending on the orientation of the variable domain in H or V L Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0098] The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. sdAbs are fairly stable and amenable to expression as fusion partners with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Appl. Microbiol. Biotechnol. 77(1):13-22).

[0099] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain that includes an Fc region. For example, when used to refer to an IgG molecule, a "full length antibody" is an antibody that includes two heavy chains and two light chains.

[0100] The term "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for determining epitopes, such as, for example, testing antibody binding to TREM2 variants with different point mutations or chimeric TREM2 variants.

[0101] A "multispecific antibody" is an antibody that comprises two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (e.g., a single TREM2 molecule expressed by a cell) or different antigens (e.g., different TREM2 molecules, or a TREM2 molecule and a non-TREM2 molecule expressed by the same cell). In some embodiments, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some embodiments, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody").

[0102] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is a naturally occurring IgG molecule that is bivalent (i.e., has two antigen-binding domains), but recognizes the same epitope in each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.

[0103] The term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope(s), excluding variations that may normally arise during the production of monoclonal antibodies. Such variations are generally present only in small amounts. Monoclonal antibodies are typically obtained by a process that includes selecting a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve production in cell culture, and / or to reduce immunogenicity in a subject.

[0104] "Effector function" refers to a biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP), receptor-ligand blockade, agonism, or antagonism.

[0105] Anti-TREM2 antibodies can include those described herein, such as the clones listed in the Tables. In some embodiments, the antibody comprises an alternative scaffold. In some embodiments, the antibody consists of an alternative scaffold. In some embodiments, the antibody consists essentially of an alternative scaffold. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the antibody consists of an antibody fragment. In some embodiments, the antibody consists essentially of an antibody fragment. A "TREM2 antibody," "anti-TREM2 antibody," or "TREM2-specific antibody" is an antibody as provided herein that specifically binds to the antigen TREM2. In some embodiments, the antibody binds to the extracellular domain of TREM2. In certain embodiments, the TREM2 antibodies provided herein bind to an epitope of TREM2 that is conserved between or among TREM2 proteins from different species.

[0106] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0107] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, e.g., a mouse, rat, rabbit, chicken, or non-human primate antibody with the desired specificity, affinity, or biological effect. When administered to a human subject, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response than antibodies from non-human species. In some cases, selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. Examples of methods for making humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293, each of which is incorporated by reference in its entirety. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0108] In one embodiment, constant domain(s) from a human antibody are fused to variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when it is administered to a human subject, either because none of the altered amino acid residues are important for immunospecific binding of the antibody to the antigen, or because the changes made to the amino acid sequence are conservative changes such that binding of the humanized antibody to the antigen is significantly lower than binding of the non-human antibody to the antigen.

[0109] A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies may be prepared in a variety of ways, including immunization with the antigen of interest of mice that have been genetically modified to express antibodies derived from genes encoding human heavy and / or light chains.

[0110] In some embodiments, the antibodies provided herein comprise an antibody fragment. In some embodiments, the antibodies provided herein consist of an antibody fragment. In some embodiments, the antibodies provided herein consist essentially of an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment. In some embodiments, the antibody fragment is a fragment of a single domain antibody.

[0111] TREM2 antibody sequence V H domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 1, 3, 5, and 7. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 1. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:3. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:5.H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:7. H Contains arrays.

[0112] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 1, 3, 5, and 7. H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H In some embodiments, the antibodies provided herein comprise a V sequence as provided in SEQ ID NOs: 1, 3, 5, and 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. H In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0113] V L domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 2, 4, 6, and 8. L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO:2. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:4. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:6. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 8. L Contains arrays.

[0114] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 2, 4, 6, and 8. L V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence L In some embodiments, the antibodies provided herein comprise a VL1 sequence as provided in SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. L In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0115] V H -V L combination of In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 1, 3, 5, and 7. H V selected from the sequence SEQ ID NOs: 2, 4, 6, and 8 L Contains arrays.

[0116] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 2 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:3. H Sequence and V of SEQ ID NO: 4 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:5. H Sequence and V of SEQ ID NO: 6 LIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:7. H Sequence and V of SEQ ID NO: 8 L In certain embodiments, any of SEQ ID NOs: 1, 3, 5, and 7 can be combined with any of SEQ ID NOs: 2, 4, 6, and 8. For example, SEQ ID NO: 1 can be combined with any of SEQ ID NOs: 2, 4, 6, or 8. As another example, SEQ ID NO: 2 can be combined with any of SEQ ID NOs: 1, 3, 5, or 7.

[0117] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 1, 3, 5, and 7. H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H Sequences and exemplary V provided in SEQ ID NOs: 2, 4, 6, and 8 L In some embodiments, the antibodies provided herein comprise VH sequences as provided in SEQ ID NOs: 1, 3, 5, and 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions, and VL sequences as provided in SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0118] CDR In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 1, 3, 5, and 7. H In some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 1, 3, 5, and 7. H In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7. In some aspects, the CDRs are exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0119] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 1, 3, 5, and 7. In some embodiments, the CDR-H1 is the CDR-H1 of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H2 is the CDR-H2 of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 is the CDR-H3 of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0120] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, the antibodies provided herein comprise three CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some aspects, the CDRs are exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0121] The CDRs of VH and VL defined using Kabat, Chothia, AbM, Contact, and IMGT are shown in Table A1 below. (Table A1) TIFF2025128164000003.tif180128

[0122] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, CDR-L1 is CDR-L1 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR-L2 is CDR-L2 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR-L3 is CDR-L3 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0123] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7, and one to three CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7, and two to three CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7, and three CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some aspects, the CDRs are exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0124] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NO: 11 or 41. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 11 or 41. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NO: 11 or 41 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0125] In some embodiments, the antibodies provided herein comprise a CDR-H2 of SEQ ID NO: 10 or 40. In some aspects, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 10 or 40. In some embodiments, the CDR-H2 is a CDR-H2 of SEQ ID NO: 10 or 40 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0126] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 9 or 39. In some aspects, the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 9 or 39. In some embodiments, the CDR-H1 is a CDR-H1 of SEQ ID NO: 9 or 39 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0127] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 11 or 41 and a CDR-H2 of SEQ ID NO: 10. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 11 or 41, a CDR-H2 of SEQ ID NO: 10 or 40, and a CDR-H1 of SEQ ID NO: 9 or 39. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 11 or 41; the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 10 or 40; and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 9 or 39. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 11 or 41 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 10 or 40 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 9 or 39 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0128] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 14 or 44. In some aspects, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L3 of SEQ ID NO: 14 or 44. In some embodiments, the CDR-L3 is a CDR-L3 of SEQ ID NO: 14 or 44 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0129] In some embodiments, the antibodies provided herein comprise a CDR-L2 of SEQ ID NO: 13 or 43. In some aspects, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L2 of SEQ ID NO: 13 or 43. In some embodiments, the CDR-L2 is a CDR-L2 of SEQ ID NO: 13 or 43 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0130] In some embodiments, the antibodies provided herein comprise a CDR-L1 of SEQ ID NO: 12 or 42. In some aspects, the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-L1 is a CDR-L1 of SEQ ID NO: 12 or 42 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0131] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 14 or 44 and a CDR-L2 of SEQ ID NO: 13 or 43. In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 14 or 44, a CDR-L2 of SEQ ID NO: 13 or 43, and a CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L3 of SEQ ID NO: 14 or 44, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L2 of SEQ ID NO: 13 or 43, and the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-L3 is CDR-L3 of SEQ ID NO: 14 or 44 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 13 or 43 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 12 or 42 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0132] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 11 or 41, a CDR-H2 of SEQ ID NO: 10 or 40, a CDR-H1 of SEQ ID NO: 9 or 39, a CDR-L3 of SEQ ID NO: 14 or 44, a CDR-L2 of SEQ ID NO: 13 or 43, and a CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 11 or 41, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 10 or 40, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 9 or 39. wherein CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 14 or 44; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 13 or 43; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 11 or 41 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 10 or 40 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 9 or 39 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 14 or 44 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 13 or 43 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 12 or 42 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants."In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0133] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 9, a CDR-H2 of SEQ ID NO: 10, a CDR-H3 of SEQ ID NO: 11, a CDR-L1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13, and a CDR-L1 of SEQ ID NO: 14. In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 39, a CDR-H2 of SEQ ID NO: 40, a CDR-H3 of SEQ ID NO: 41, a CDR-L1 of SEQ ID NO: 42, a CDR-L2 of SEQ ID NO: 43, and a CDR-L1 of SEQ ID NO: 44.

[0134] Fc area The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc polypeptide of a dimeric IgGFc comprises IgG CH2 and IgG CH3 constant domain sequences. The Fc may be of the classes IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0135] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, an FcR may be a native-sequence human FcR. Generally, FcRs bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes can also bind to specific FcRs (see, e.g., Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994), and de Haas 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. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0136] In some embodiments, the antibody is an IgG1 antibody.

[0137] In some embodiments, the antibody is an IgG3 antibody.

[0138] In some embodiments, the antibody is an IgG2 antibody.

[0139] In some embodiments, the antibody is an IgG4 antibody.

[0140] Modifications of the CH2 domain can affect FcR binding to Fc. Numerous amino acid modifications of the Fc region to selectively alter the affinity of Fc for different Fc-gamma (Fcγ) receptors are known in the art. In one embodiment, the Fc contains one or more modifications to promote selective binding to Fc-gamma receptors.

[0141] Exemplary mutations that alter binding of FcR to Fc are listed below: S298A / E333A / K334A, S298A / E333A / K334A / K326A(Lu Y, Vernes JM, Chiang N, et al.J Immunol Methods.2011 Feb 28;365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L(Stavenhagen JB,Gorlatov S,Tuaillon N,et al.Cancer Res.2007 Sep 15;67(18):8882-90;Nordstrom JL, Gorlatov S, Zhang W, et al.Breast Cancer Res.2011 Nov 30;13(6):R123); F243L(Stewart R,Thom G,Levens M,et al.Protein Eng Des Sel.2011 Sep;24(9):671-8.),S298A / E333A / K334A(Shields RL,Namenuk AK,Hong K,et al.J Biol Chem.2001 Mar 2;276(9):6591-604); S239D / I332E / A330L, S239D / I332E(Lazar GA,Dang W,Karki S,et al.Proc Natl Acad Sci US A.2006 Mar 14;103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S, et al.Mol Immunol.2008 Sep;45(15):3926-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D and other mutations listed in WO2011 / 120134 and WO2011 / 120135 (incorporated herein by reference). Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012) lists the mutations on page 283.

[0142] In some embodiments, the antibodies described herein comprise modifications that improve their ability to mediate effector function. Such modifications are known in the art and include afucosylation or engineering the affinity of Fc for activating receptors, primarily FCGR3a for ADCC and C1q for CDC. Table B below summarizes various designs reported in the literature for effector function engineering.

[0143] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at one or more of the Fc region positions 298, 333, and 334. In some embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions at positions 239, 332, and 330 as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103:4005-4010, which is incorporated by reference in its entirety.

[0144] In some embodiments, the antibodies provided herein contain one or more modifications that improve or decrease C1q binding and / or CDC. See U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 2000, 164:4178-4184 (each of which is incorporated by reference in its entirety).

[0145] Thus, in one embodiment, the antibodies described herein may comprise a dimeric Fc containing one or more amino acid modifications that confer improved effector function, as described in Table B. In another embodiment, the antibodies may be afucosylated to improve effector function. (Table B) CH2 domain and effector function engineering TIFF2025128164000004.tif127148

[0146] Fc modifications that reduce FcγR and / or complement binding and / or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curr Opin Biotech 20:685-691, and Strohl, WR and Strohl LM, "Antibody Fc engineering for optimal antibody performance," In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp. 225-249). These strategies include reducing effector function by modifying glycosylation, using an IgG2 / IgG4 scaffold, or introducing mutations into the hinge or CH2 region of the Fc. For example, U.S. Patent Publication No. 2011 / 0212087 (Strohl), International Patent Publication No. WO2006 / 105338 (Xencor), U.S. Patent Publication No. 2012 / 0225058 (Xencor), U.S. Patent Publication No. 2012 / 0251531 (Genentech), and Strop et al ((2012) J. Mol. Biol. 420:204-219) describe certain modifications that reduce FcγR or complement binding to Fc.

[0147] Specific non-limiting examples of known amino acid modifications that reduce FcγR or complement binding to Fc include those identified in Table C below. Table C: Modifications that reduce FcγR or complement binding to Fc TIFF2025128164000005.tif109128

[0148] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn297EU numbering) without modifying the amino acid sequence are well known in the art. GlymaxX® technology (ProBioGen AG) is based on the introduction of a gene encoding an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the addition of the sugar "fucose" to N-linked antibody carbohydrate moieties by antibody-producing cells (von Horsten et al. (2010) Glycobiology. 2010 Dec;20(12):1607-18). Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Publication No. 2003 / 0157108; WO2004 / 056312, each of which is incorporated by reference in its entirety), which are deficient in protein fucosylation and have stable overexpression of the bacterial GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), as well as knockout cell lines such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al., J. Immunol. 2004, 20:1607-1618). al., Biotech. Bioeng., 2004, 87:614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107 (each of which is incorporated by reference in its entirety). Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Patent No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to produce reduced levels of antibody fucosylation.

[0149] Antibodies can be fully afucosylated, meaning they contain no detectable fucose, or they can be partially afucosylated, meaning that the isolated antibody contains less than 95%, 85%, 75%, 65%, 55%, 45%, 35%, 25%, 15%, or 5% of the amount of fucose typically found in a similar antibody produced in a mammalian expression system.

[0150] In some embodiments, the antibodies provided herein comprise an IgG1 domain with a reduced fucose content at Asn297 compared to naturally occurring IgG1 domains. Such Fc domains are known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, which is incorporated by reference in its entirety. In some embodiments, such antibodies do not comprise any fucose at Asn297. The amount of fucose may be determined using any suitable method, such as, for example, that described in WO2008 / 077546, which is incorporated by reference in its entirety.

[0151] In some embodiments, the antibodies provided herein comprise bisected oligosaccharides, such as biantennary oligosaccharides attached to the Fc region of the antibody, which are bisected at GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Patent No. 6,602,684; and U.S. Patent Publication No. 2005 / 0123546, each of which is incorporated by reference in its entirety.

[0152] Other exemplary glycosylation variants that can be incorporated into the antibodies provided herein are described in, e.g., U.S. Patent Publication Nos. 2003 / 0157108, 2004 / 0093621, 2003 / 0157108, 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0132140, 2004 / 011 0704, 2004 / 0110282, 2004 / 0109865; International Patent Publication Nos. 2000 / 61739, 2001 / 29246, 2003 / 085119, 2003 / 084570, 2005 / 035586, 2005 / 035778, 2005 / 053742, 2002 / 031140, Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249, and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87:614-622, each of which is incorporated by reference in its entirety.

[0153] In some embodiments, the antibodies provided herein comprise an Fc region having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764, each of which is incorporated by reference in its entirety.

[0154] Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Publication No. 2003 / 0157108; WO2004 / 056312, each of which is incorporated by reference in its entirety), which are deficient in protein fucosylation and have stable overexpression of the bacterial GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), as well as knockout cell lines such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al., J. Immunol. 2004, 20:1607-1618). al., Biotech. Bioeng., 2004, 87:614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107 (each of which is incorporated by reference in its entirety).

[0155] In some embodiments, the antibody has antibody-dependent cellular phagocytosis (ADCP) activity. ADCP can occur when an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell. Phagocytes, including monocytes and macrophages, which have Fc receptors on their cell surface, recognize and bind the Fc region of antibodies bound to target cells. Binding of the Fc receptor to the antibody-bound target cell can initiate phagocytosis of the target cell. ADCP can be considered a form of ADCC.

[0156] In some embodiments, the antibody is capable of forming an immune complex, for example, an immune complex may be a tumor cell coated with the antibody.

[0157] In some embodiments, the anti-TREM2 antibody does not substantially bind to myeloid cells present outside the cancer tissue, hi some embodiments, the anti-TREM2 antibody does not substantially bind to stimulatory myeloid cells present in the cancer tissue.

[0158] In some embodiments, the antibody is a monoclonal antibody.

[0159] In some embodiments, the antibody is a polyclonal antibody.

[0160] In some embodiments, the antibody is produced by a hybridoma, hi other embodiments, the antibody is produced by a recombinant cell that has been engineered to express the desired variable and constant domains.

[0161] In some embodiments, the antibody may be a single chain antibody or other antibody derivative that retains the antigen specificity and a lower hinge region or variant thereof.

[0162] In some embodiments, the antibody may be a polyfunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment, or a variant thereof. In certain embodiments, the antibody fragment or derivative thereof is selected from a Fab fragment, a Fab'2 fragment, a CDR, and an ScFv.

[0163] In some embodiments, the antibody is specific for a surface antigen such as a TREM2 protein. In some embodiments, the therapeutic antibody is specific for a tumor antigen (e.g., a molecule specifically expressed by tumor cells). In certain embodiments, the therapeutic antibody may have a human or non-human primate IgG1 or IgG3 Fc portion.

[0164] join With respect to antibody binding to a target molecule, the terms "binds with," "specifically binding to," "specifically binds to," "specific for," "selectively binds to," and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is somehow distinct from nonspecific or nonselective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a molecule of interest that mimics the epitope recognized on the target molecule. Specific binding is then indicated if binding of the antibody to the target molecule is competitively inhibited by the molecule of interest.

[0165] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise specified, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of molecule X for partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0166] As used herein, "k" d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is off Also called value.

[0167] As used herein, "k" a " (M -1 ×sec -1 The term k ) refers to the association rate constant for a particular antibody-antigen interaction. This value ison Also called value.

[0168] As used herein, "K" D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. D =k d / k a In some embodiments, the affinity of an antibody is determined by the K D For clarity, as known in the art, the smaller K D values ​​indicate a high affinity interaction, but a large K D Values ​​indicate low affinity interactions.

[0169] As used herein, "K" A " (M -1 The term K ) refers to the association equilibrium constant of a particular antibody-antigen interaction. A =k a / k d .

[0170] As used herein in the context of two or more antibodies, the terms "compete with" or "cross-compete with" indicate that the two or more antibodies compete for binding to an antigen (e.g., TREM2). In one exemplary assay, TREM2 is coated on a surface and contacted with a first TREM2 antibody, after which a second TREM2 antibody is added. In another exemplary assay, a first TREM2 antibody is coated on a surface and contacted with TREM2, after which a second TREM2 antibody is added. In either assay, antibodies compete with each other if the presence of the first TREM2 antibody reduces binding of the second TREM2 antibody. The term "compete with" also includes antibody combinations in which one antibody reduces binding of another antibody, but no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit each other's binding regardless of the order in which they are added. In some embodiments, one antibody reduces the binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. One skilled in the art can select the concentration of antibody to use in a competition assay based on the affinity of the antibody for TREM2 and the valency of the antibody. The assays described in this definition are exemplary, and one skilled in the art can utilize any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358, each of which is incorporated by reference in its entirety.

[0171] In some embodiments, the antibodies provided herein have a mAb content of about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 3, 4, 5, 6, 7, 8, 9, or 10 x 10 as measured by a Biacore assay. -9 K below M D In some embodiments, the K of the antibodies provided herein binds to human TREM2. D is approximately 0.001–0.01, 0.01–0.1, 0.01–0.05, 0.05–0.1, 0.1–0.5, 0.5–1, 0.25–0.75, 0.25–0.5, 0.5–0.75, 0.75–1, 0.75–2, 1.1–1.2, 1.2–1.3, 1.3–1.4, 1.4–1.5, 1.5–1.6, 1.6–1.7, 1.7–1.8, 1.8–1.9, 1.9–2, 1–2, 1–5, 2–7, 3–8, 3–5, 4–6, 5–7, 6–8, 7–9, 7–10, or 5–10 × 10, as measured by Biacore assay. -9 I am M.

[0172] In some embodiments, the antibodies provided herein have a cytotoxicity of about 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, or 1.4 x 10 as measured by a Biacore assay. -9 K below M D In some embodiments, the antibody binds to human TREM2 at 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, or 1.9-1.5 x 10 as measured by a Biacore assay. -9 K of M D Provided herein are antibodies that bind to human TREM2 at about 10, 9.56, 9.5, 9.0, 8.88, 8.84, 8.5, 8, 7.5, 7.32, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 x 10 as measured by a Biacore assay. -4(1 / s) or less, or K d In some embodiments, the antibodies provided herein bind to human TREM2 at a concentration of 7-10, 7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, or 9.5-10 x 10 as measured by a Biacore assay. -4 (1 / s)K d In some embodiments, the antibodies provided herein bind to human TREM2 at about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 45, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 7, 8, 9, or 10 x 10 as measured by a Biacore assay. 5 (1 / Ms) or greater a In some embodiments, the antibodies provided herein bind to human TREM2 at a ribonucleotide concentration of 4-7, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10 x 10 as measured by a Biacore assay. 5 (1 / Ms)K a It binds to human TREM2 at

[0173] In some embodiments, the antibodies provided herein bind to human TREM2 with an EC50 of 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM or less as measured by flow cytometry. In some embodiments, the antibodies bind to human TREM2 with an EC50 of 0.6-1.4 nM as measured by flow cytometry. In some embodiments, the antibodies bind to human TREM2 with an EC50 of about 0.5, 0.6, 0.9, 1.1, 1.2, 1.3, 1.4, or 1.5 nM as measured by flow cytometry.

[0174] In some embodiments, the antibodies provided herein bind to mouse TREM2 with an EC50 of 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM or less as measured by flow cytometry. In some embodiments, the antibodies bind to mouse TREM2 with an EC50 of 0.6-1.4 nM as measured by flow cytometry. In some embodiments, the antibodies bind to mouse TREM2 with an EC50 of about 0.5, 0.6, 0.9, 1.1, 1.2, 1.3, 1.4, or 1.5 nM as measured by flow cytometry.

[0175] In some embodiments, the antibodies provided herein do not bind to human TREM2 with an EC50 of 20 nM or greater, as measured by flow cytometry. In some embodiments, the antibodies provided herein do not bind to mouse TREM2 with an EC50 of 3 nM or greater, as measured by flow cytometry.

[0176] To screen for antibodies that bind to the epitope on the target antigen to which the antibody of interest (e.g., TREM2) binds, routine cross-blocking assays can be performed, such as those described in *Antibodies, A Laboratory Manual*, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, or additionally, epitope mapping can be performed by methods known in the art.

[0177] Competition between antibodies can be determined in an assay in which the antibody under test inhibits or blocks the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990; Fendly et al. Cancer Research 50:1550-1558; US 6,949,245). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits or blocks the binding of the reference antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (as measured in a competitive binding assay). Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to allow steric hindrance. For example, a second, competing antibody can be identified that competes with a first antibody described herein for binding to TREM2. In certain cases, the second antibody can block or inhibit binding of the first antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (as measured in a competitive binding assay). In certain cases, the second antibody can displace the first antibody by more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0178] function In some embodiments, the antibody has antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC can occur when an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell. Effector cells bearing Fc gamma receptors (FcγR or FCGR) on their cell surface, including cytotoxic T cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, dendritic cells, or monocytes, recognize and bind the Fc region of an antibody bound to a target cell. Such binding can cause activation of intracellular signaling pathways leading to cell death. In certain embodiments, the immunoglobulin Fc region subtype (isotype) of the antibody comprises human IgG1 and IgG3. As used herein, ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize antibody bound to a target cell and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0179] In some embodiments, the antibody has complement-dependent cytotoxicity (CDC) activity. Antibody-induced CDC is mediated by proteins of the typical complement cascade and is triggered by the binding of the complement protein C1q to the antibody. The antibody Fc region that binds to C1q can induce activation of the complement cascade. In certain embodiments, the immunoglobulin Fc region subtype (isotype) of the antibody includes human IgG1 and IgG3. As used herein, CDC refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide (e.g., an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0180] In some embodiments, the antibody is an agonist antibody. An agonist antibody can induce (e.g., increase) one or more activities or functions of NSM after the antibody binds to the TREM2 protein expressed on a cell. An agonist antibody can bind to and activate NSM, causing changes in cell proliferation or modifying antigen-presenting ability. An agonist antibody can bind to and activate NSM, triggering intracellular signaling pathways that lead to the modification of cell growth or apoptosis.

[0181] In some embodiments, the antibody is an antagonist antibody. An antagonist antibody can block (e.g., reduce) one or more activities or functions of NSM after the antibody binds to the TREM2 protein expressed on a cell. For example, an antagonist antibody can bind to and block ligands that bind to one or more NSM proteins, preventing cell differentiation and proliferation or modifying antigen-presenting ability. An antagonist antibody can bind to and prevent activation of the TREM2 protein by ligands, preventing binding and activation and modifying intracellular signaling pathways that contribute to cell growth and survival.

[0182] In some embodiments, the antibody is a depleting antibody. A depleting antibody kills unstimulated bone marrow cells upon contact via antibody interaction with other immune cells. For example, when the antibody binds to cells bearing the TREM2 protein, it can capture complement proteins and induce complement-dependent cell lysis. When the antibody binds to cells bearing the TREM2 protein, it can also trigger nearby cells with Fc receptors to kill them by antibody-dependent cellular cytotoxicity (ADCC).

[0183] In some embodiments, the antibody is a neutralizing antibody, and the antibody neutralizes one or more biological activities of the NSM. In some embodiments, the TREM2 protein is expressed on the surface of unstimulated bone marrow cells, and the antibody recognizes the extracellular domain of the TREM2 protein.

[0184] In some embodiments, the antibody is selective for NSM (binds preferentially to TREM2). In certain embodiments, an antibody that selectively binds to NSM has a dissociation constant (Kd) in the range of 0.0001 nM to 1 μM. In certain embodiments, the antibody specifically binds to an epitope on the TREM2 protein that is conserved among proteins from different species. In other embodiments, selective binding includes, but does not require, exclusive binding.

[0185] In one embodiment, an anti-TREM2 antibody bound to a target causes in vivo depletion of unstimulated bone marrow cells to which it binds. In some embodiments, effector proteins induced by clustered antibodies can elicit various responses, including the release of inflammatory cytokines, control of antigen production, endocytosis, or cell killing. In one embodiment, the antibody can recruit and activate complement, mediate antibody-dependent cellular cytotoxicity (ADCC) in vivo, or mediate phagocytosis by binding Fc receptors in vivo. The antibody can also deplete unstimulated bone marrow cells by inducing apoptosis or necrosis of the unstimulated bone marrow cells upon binding.

[0186] In some embodiments, deactivation of unstimulated bone marrow cells is in vitro and is achieved by a) killing the unstimulated bone marrow cells, b) magnetic bead depletion of the unstimulated bone marrow cells; or c) fluorescence-activated cell sorting (FACS) sorting of the unstimulated bone marrow cells.

[0187] In some embodiments, the antibody is linked or conjugated to an effector molecule, hi certain embodiments, the antibody is conjugated to at least one therapeutic agent selected from the group consisting of a radionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a prodrug, a toxin, an enzyme, an immunomodulatory agent, an antiangiogenic agent, a proapoptotic agent, a cytokine, a hormone, an oligonucleotide, an antisense molecule, an siRNA, a second antibody, and a second antibody fragment.

[0188] In certain embodiments, the antibody is conjugated to a drug, such as a toxin, a chemotherapeutic agent, an immunomodulator, or a radioisotope. Several methods for preparing ADCs (antibody-drug conjugates) are known in the art, and are described, for example, in U.S. Patent Nos. 8,624,003 (pot method), 8,163,888 (one-step method), and 5,208,020 (two-step method). The antibody or its antigen-binding fragment can be conjugated to at least one agent, including an antigen-binding radionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a prodrug, a toxin, an enzyme, an immunomodulator, an antiangiogenic agent, a proapoptotic agent, a cytokine, a hormone, an oligonucleotide, an antisense molecule, an siRNA, a second antibody, and a second antibody fragment.

[0189] Non-stimulated bone marrow cells (NSM) Described herein are methods and compositions for neutralizing and / or detecting non-stimulated myeloid cells (NSM), including the use of anti-TREM2 antibodies. Also provided herein are methods and compositions for targeting and / or detecting non-stimulated myeloid cells that express NSM proteins.

[0190] Also provided herein are methods and compositions for neutralizing and / or detecting unstimulated bone marrow cells in the non-human individual, comprising the use of antibodies directed against non-human homologs of human NSM proteins.

[0191] As used herein, unstimulated bone marrow cells are bone marrow cells that are not sufficiently effective at stimulating an immune response (e.g., not as effective at stimulating an anti-tumor response in the tumor microenvironment as stimulated bone marrow cells). In some embodiments, unstimulated bone marrow cells are not as effective at presenting antigens (e.g., tumor antigens) to T cells or at stimulating tumor-specific T cell responses as stimulated bone marrow cells. In some embodiments, unstimulated bone marrow cells may exhibit a reduced ability to uptake, process, and / or present tumor-associated antigens to T cells as stimulated bone marrow cells. Unstimulated bone marrow cells may have reduced or no ability to reprime cytotoxic T lymphocytes, or in some cases, may not stimulate effective tumor cell killing. Unstimulated bone marrow cells may exhibit reduced expression of genes and cell surface markers involved in antigen processing, antigen presentation, and / or antigen costimulation, including, but not limited to, CD80, CD86, MHC1, and MHCII, as stimulated bone marrow cells.

[0192] Unstimulated bone marrow cells may exhibit decreased expression of genes associated with cross-presentation, costimulation, and / or stimulatory cytokines, including, but not limited to, any one or more of TAP1, TAP2, PSMB8, PSMB9, TAPBP, PSME2, ​​CD24a, CD274, BTLA, CD40, CD244, ICOSL, ICAM1, TIM3, PDL2, RANK, FLT3, CSF2RB, CSF2RB2, CSF2RA, IL12b, XCR1, CCR7, CCR2, CCL22, CXCL9, and CCL5, and increased expression of the anti-inflammatory cytokine IL-10, when compared to stimulated bone marrow cells. In some embodiments, unstimulated bone marrow cells depend on the transcription factor IRF4 and the cytokines GM-CSF or CSF-1 for differentiation and survival. In some embodiments, unstimulated bone marrow cells can contribute to tumor angiogenesis by secreting vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS), and support tumor growth by secreting epidermal growth factor (EGF).

[0193] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages (TAMs), neutrophils, monocytes, or dendritic cells (DCs). In some embodiments, the unstimulated bone marrow cells are not dendritic cells (DCs). In some embodiments, the unstimulated bone marrow cells are neutrophils.

[0194] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages (TAMs). TAMs are macrophages that reside near or within cancerous tumors and are derived from circulating monocytes or resident tissue macrophages.

[0195] In some embodiments, unstimulated and stimulated bone marrow cells are distinguished based on the markers they express or preferentially express. Expression of a cell surface marker can be described as "+" or "positive." Absence of a cell surface marker can be described as "-" or "negative." Expression of a cell surface marker can be further described as "high" (cells expressing high levels of the marker) or "low" (cells expressing low levels of the marker), indicating the relative expression of each marker on the cell surface. Marker levels may be determined by various methods known in the art, such as immunostaining and FACS analysis, or gel electrophoresis and Western blotting.

[0196] In some embodiments, the non-stimulatory bone marrow cells are dendritic cells (DCs). In some embodiments, dendritic cells can be distinguished by the morphology of their processes or dendrites. In one embodiment, the non-stimulatory dendritic cells are at least CD45+, HLA-DR+, CD14-, CD11c+, and BDCA1+ (also referred to as DC1 cells). In one embodiment, the non-stimulatory dendritic cells are not CD45+, HLA-DR+, CD14-, CD11c+, and BDCA3+ (also referred to as DC2 cells). In one embodiment, dendritic cells that are CD45+, HLA-DR+, CD14-, CD11c+, and BDCA3+ are stimulatory bone marrow cells.

[0197] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages. In some embodiments, for example in humans, the unstimulated tumor-associated macrophages are at least CD45+, HLA-DR+, CD14+. In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , CD11c+ In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11c + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + is.

[0198] In some embodiments, the methods and compositions of the present invention are useful for targeting TAMs and DCs in other mammals, e.g., mice. In such embodiments, mouse TAMs and DCs are contacted with a TREM2 antibody. In one embodiment, for example, in mice, tumor-associated macrophages are at least CD45+, HLA-DR+, CD14+, CD11b high , and CD11c low In one embodiment, for example in a mouse, tumor-associated macrophages are at least CD45+, HLA-DR+, CD14+, CD11b low , and CD11chigh (also known as TAM2). high The term "macrophage" refers to a macrophage that expresses high levels of CD11b. low The term "macrophage" refers to CD11b high As used herein, "CD11c" refers to macrophages that express CD11b on their surface at levels substantially lower than those of macrophages. high The term "CD11c" refers to macrophages that express high levels of CD11c. low The term "macrophage" refers to Cd11c high It concerns macrophages that express CD11c on their surface at levels substantially lower than those of macrophages.

[0199] In some embodiments, the unstimulated bone marrow cells of the invention comprise one or more of TAM and DC1 cells.

[0200] In some embodiments, for example in a mouse, the unstimulated bone marrow cells of the invention comprise one or more of TAM1, TAM2, and DC1 cells. In such embodiments, the unstimulated bone marrow cells of the invention are contacted with a TREM2 antibody.

[0201] In some embodiments, the unstimulated bone marrow cells are bone marrow cells that are tumor cells.

[0202] In some embodiments, unstimulated bone marrow cells localize within the margins of tumor lesions or within transformed tumor ducts, where they are in contact with allogeneic T cells. In one embodiment, the localization of unstimulated bone marrow cells is modified, such that the cells no longer localize to the tumor margin or are no longer in contact with T cells.

[0203] In some embodiments, the unstimulated bone marrow cells are in a population of immune cells that includes stimulated and unstimulated bone marrow cells. In some embodiments, the unstimulated bone marrow cells are in a population of immune cells that includes only unstimulated bone marrow cells. The immune cell populations of the present invention may be pure, homogeneous, heterogeneous, derived from a variety of sources (e.g., diseased tissue, tumor tissue, healthy tissue, cell banks), maintained in primary cell culture, and / or maintained in ex vivo culture.

[0204] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages.

[0205] In some embodiments, the unstimulated bone marrow cells are dendritic cells.

[0206] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + The cell of the present invention essentially consists of cells that are

[0207] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3- In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - The cell of the present invention essentially consists of cells that are

[0208] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + The cell of the present invention essentially consists of cells that are

[0209] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11c+ In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11c + The cell of the present invention essentially consists of cells that are

[0210] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + The cell of the present invention essentially consists of cells that are

[0211] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 +, BDCA3 - , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + The cell of the present invention essentially consists of cells that are

[0212] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + The cell of the present invention essentially consists of cells that are

[0213] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c+ In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + The cell of the present invention essentially consists of cells that are

[0214] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA3 + In some embodiments, the unstimulated bone marrow cells are not CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA3 + Includes cells that are not

[0215] In some embodiments, for example in mice, unstimulated bone marrow cells express CD45 + , HLA-DR + , CD14 + , CD11b high , and CD11c low In some embodiments, for example in mice, unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11bhigh , and CD11c low In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b high , and CD11c low In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b high , and CD11c low In such embodiments, unstimulated mouse bone marrow cells are contacted with the TREM2 antibody.

[0216] In some embodiments, for example in mice, unstimulated bone marrow cells express CD45 + , HLA-DR + , CD14 + , CD11b low , and CD11c high In some embodiments, for example in mice, unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b low , and CD11c high In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b low , and CD11c high In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b low , and CD11c high In such embodiments, unstimulated mouse bone marrow cells are contacted with the TREM2 antibody.

[0217] In some embodiments, the unstimulated bone marrow cells are in cancer tissue.

[0218] In some embodiments, the population of immune cells is in cancer tissue.

[0219] In some embodiments, the unstimulated cells and the stimulated bone marrow cells are in cancer tissue.

[0220] In some embodiments, the biological sample comprises a population of immune cells comprising unstimulated bone marrow cells and stimulated bone marrow cells.

[0221] NSM cells may collectively refer to DC1, TAM1, and TAM2 cells, which are present in tumor tissues and can be distinguished from other cell types by the expression of NSM cell markers. For example, genes and associated proteins that are more abundantly expressed or translated in NSM cells than in SDCs can serve as NSM markers. An exemplary NSM marker is CD11b. Additional exemplary NSM markers are listed in Table A. NSM cells may express TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and TMEM119 on the cell surface. In some embodiments, NSM cells do not express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1.

[0222] In one embodiment, the NSM cells express one or more of the NSM marker genes listed in Table A. In another embodiment, the NSM cells express 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more of the NSM markers listed in Table D. In another embodiment, the NSM cells express most or all of the NSM markers listed in Table D. In another embodiment, the NSM cells are identified as expressing MRC1, MS4A7, C1QC, APOE, C1QB, C1QA, and C5AR1.

[0223] (Table D) TIFF2025128164000006.tif78128

[0224] Stimulating bone marrow cells As used herein, stimulated bone marrow cells (also referred to in certain aspects as SDCs) are bone marrow cells that are effective at stimulating an immune response (e.g., more effective at stimulating an anti-tumor response in a tumor microenvironment compared to unstimulated bone marrow cells). In some embodiments, stimulated bone marrow cells are more effective at presenting antigens (e.g., tumor antigens) to T cells or more effective at stimulating tumor-specific T cell responses compared to unstimulated bone marrow cells. In some embodiments, stimulated bone marrow cells may exhibit increased ability to uptake, process, and / or present tumor-associated antigens to T cells compared to unstimulated bone marrow cells. Stimulated bone marrow cells may have increased ability to reprime cytotoxic T lymphocytes or, in some cases, stimulate effective tumor cell killing compared to unstimulated bone marrow cells. Stimulated bone marrow cells may exhibit higher expression of genes and cell surface markers involved in antigen processing, antigen presentation, and / or antigen costimulation, including, but not limited to, CD80, CD86, MHC1, and MHCII, compared to unstimulated bone marrow cells.

[0225] Exemplary stimulatory myeloid cell markers are listed in Table A. For example, in human SDC, expression of Xcr1, Clec9a, and BDCA3 (CD141) are markers of SDC identity. In mice, CD103 can also be used as a strong marker of SDC identity, although it will be noted that it is not expressed in human SDC.

[0226] In one embodiment, SDCs are tumor-infiltrating myeloid cells that have a dendritic cell identity and also express one or more of the SDC markers listed in Table A. In another embodiment, SDCs are tumor-infiltrating myeloid cells that have a dendritic cell identity and also express two, three, four, five, six, seven, eight, nine, or all of the SDC markers listed in Table A. In another embodiment, SDCs are identified as tumor-infiltrating myeloid dendritic cells that express BDCA3, KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, XCR1, and CLEC9A. SDC cells may express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1. In some embodiments, the SDCs do not substantially express TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and / or TMEM119 on the cell surface. In some embodiments, the SDCs do not substantially express C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, MERTK, C1QC, TMEM119, MS4A7, APOE, CYP4F18, TREM2, TLR7, and / or LILRB4. Flow cytometry and PCR, among other art-recognized assays, can be used to assess expression of the markers disclosed herein.

[0227] Stimulated bone marrow cells express CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA3 + The stimulated bone marrow cells may be CD45 + , HLA-DR + , and BDCA3 + The stimulated bone marrow cells may be CD45 + , HLA-DR + , CD14 - , and BDCA3 + The stimulated bone marrow cells may be CD45+ , HLA-DR + , CD11c + , and BDCA3 + It could be.

[0228] Proteins, nucleotides, and homologs Provided herein are methods and compositions for neutralizing and / or detecting unstimulated human bone marrow cells that express an NSM protein. In some embodiments, the invention relates to neutralizing and / or detecting unstimulated bone marrow cells from non-human mammalian cells that express an NSM protein homolog. For example, mouse NSM proteins may express a restricted expression pattern equivalent to that of the human homolog. Thus, in one embodiment, provided herein are methods and compositions for neutralizing and / or detecting unstimulated mouse bone marrow cells that express an NSM protein. Also provided herein are analogous methods and compositions for neutralizing and / or detecting unstimulated cells from any individual that expresses a homolog of an NSM protein with a similar expression pattern, where the cells exhibit an expression pattern equivalent to that of the NSM protein.

[0229] The NSM protein or nucleotide may include at least one of C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, MERTK, C1QC, TMEM119, MS4A7, APOE, CYP4F18, TREM2, TLR7, and LILRB4, and homologs thereof. The SDC protein or nucleotide may include at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1, and homologs thereof. The cell surface NSM protein may include at least one of TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and TMEM119. Cell surface NSM proteins can be targeted by one or more anti-TREM2 antibodies, either alone or in combination. Generally, NSMs are positive for NSM proteins or nucleotides and negative for SDC proteins or nucleotides; conversely, SDCs are generally positive for SDC proteins or nucleotides and negative for NSM proteins or nucleotides.

[0230] The antibodies described herein comprise at least one polypeptide, but typically comprise a HC / LC dimer, i.e., four polypeptides. Polynucleotides encoding the polypeptides described herein are also described. The antibodies are typically isolated.

[0231] As used herein, "isolated" refers to an agent (e.g., a polypeptide or polynucleotide) that has been identified and separated and / or recovered from components of its natural cell culture environment. Contaminant components of the natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated also refers to an agent that has been produced synthetically, for example, by human intervention.

[0232] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0233] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Reference to amino acids includes, for example, naturally occurring proteinogenic L-amino acids; chemically modified amino acids, such as D-amino acids, amino acid variants, and derivatives; naturally occurring non-proteinogenic amino acids, such as β-alanine and ornithine; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methylamino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine), amino acids with an additional methylene in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid). The incorporation of synthetic non-natural amino acids, substituted amino acids, or non-natural amino acids, including one or more D-amino acids, into proteins of the invention can be advantageous in a number of different ways. D-amino acid-containing peptides and the like exhibit increased stability in vitro or in vivo compared to their L-amino acid-containing counterparts. Thus, constructs such as peptides incorporating D-amino acids may be particularly useful when greater intracellular stability is desired or required.More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule and extended in vivo longevity when such properties are required. Furthermore, D-peptides and the like cannot be efficiently processed for major histocompatibility complex class II-restricted presentation to helper T cells and are therefore less likely to induce a humoral immune response in the whole organism.

[0234] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0235] The present invention also includes polynucleotides encoding antibody polypeptides. The terms "polynucleotide" or "nucleotide sequence" are intended to refer to a continuous stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combination thereof.

[0236] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof in either single- or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), and DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0237] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified change. All nucleic acid sequences herein that encode a polypeptide also describe all possible silent variations of the nucleic acid. Those of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0238] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants," where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles described herein.

[0239] Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman & Co.; 2nd edition (December 1993)).

[0240] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. When compared and aligned for maximum correspondence over a comparison window, or a designated region as measured using one of the sequence comparison algorithms below (or other algorithms available to those skilled in the art), or by manual alignment and visual inspection, sequences are "substantially identical" if they have the same percentage of amino acid residues or nucleotides (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over the designated region). Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. This definition also refers to the complement of the test sequence. Identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where specified, over the entire sequence of the polynucleotide or polypeptide. Polynucleotides encoding the polypeptides of the invention, including homologs from species other than human, may be obtained by a process comprising screening a library under stringent hybridization conditions with a labeled probe having a polynucleotide sequence described herein, or a fragment thereof, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art.

[0241] For sequence comparison, one sequence to which a test sequence is compared usually serves as a reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as needed, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence compared to the reference sequence based on the program parameters.

[0242] As used herein, a "comparison window" includes reference to any one of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, over which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are known to those of skill in the art. Optimal alignment of sequences for comparison can be performed, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0243] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, available on the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, or M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix uses an alignment (B) of 50 (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915), an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically run with the "low complexity" filter turned off.

[0244] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the smallest sum probability in the comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001, the nucleic acid is considered to be similar to the reference sequence.

[0245] The phrase "selectively (or specifically) hybridizes" refers to the binding, duplexing, or hybridization of a molecule to only a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (including, but not limited to, total cellular or library DNA or RNA).

[0246] The phrase "stringent hybridization conditions," as known in the art, refers to hybridization of sequences of DNA, RNA, or other nucleic acids, or combinations thereof, under conditions of low ionic strength and high temperature. Typically, under stringent conditions, a probe will hybridize to a target subsequence of a complex mixture of nucleic acids (including, but not limited to, whole cellular or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize particularly at higher temperatures. An extensive guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[0247] As used herein, the terms "engineered, engineered, manipulating" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Manipulation includes altering the amino acid sequence, altering the glycosylation pattern, or altering the side groups of individual amino acids, as well as combinations of these approaches. Designed proteins are expressed and produced by standard molecular biology techniques.

[0248] An "isolated nucleic acid molecule or polynucleotide" refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or polynucleotides that have been purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or in a chromosomal location that differs from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive- and negative-stranded forms, and double-stranded forms. Isolated polynucleotides or nucleic acids described herein further include such molecules that are synthetically produced, for example, via PCR or chemical synthesis. In addition, polynucleotides or nucleic acids, in certain embodiments, contain regulatory elements such as a promoter, ribosome binding site, or transcription terminator.

[0249] The term "polymerase chain reaction" or "PCR" generally refers to a method for amplifying a desired nucleotide sequence in vitro, as described, for example, in U.S. Patent No. 4,683,195. In general, the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of preferentially hybridizing to a template nucleic acid.

[0250] By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to the reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.These changes in the reference sequence may occur between the residues of the reference sequence, or at the 5' or 3' end of the reference nucleotide sequence, or between the end positions of the reference nucleotide sequence, interspersed with one or more adjacent groups within the reference sequence. In practice, whether a particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be routinely determined using known computer programs such as those discussed above for polypeptides (e.g., ALIGN-2).

[0251] A derivative or variant of a polypeptide shares "homology" with, or is said to be "homologous" to, a peptide if the amino acid sequence of the derivative or variant has at least 50% identity with the 100 amino acid sequence derived from the original peptide. In certain embodiments, a derivative or variant is at least 75% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, a derivative or variant is at least 85% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of a derivative is at least 90% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of a derivative is at least 95% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, a derivative or variant is at least 99% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative.

[0252] The term "modified" as used herein refers to any alteration made to a given polypeptide, such as altering the length, amino acid sequence, chemical structure, co-translational modification, or post-translational modification of the polypeptide. The term "(modified)" in the form means that the polypeptide under consideration is optionally modified, i.e., the polypeptide under consideration can be modified or unmodified.

[0253] In some aspects, a polypeptide comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the relevant (e.g., polypeptide and / or antibody) amino acid sequence, or a fragment thereof, set forth in the table(s) or accession number(s) disclosed herein. In some aspects, an isolated antibody or protein disclosed herein comprises an amino acid sequence encoded by a polynucleotide that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the relevant nucleotide sequence, or a fragment thereof, set forth in the table(s) or accession number(s) disclosed herein. In some embodiments, the nucleotide sequence comprises a nucleotide sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleotide sequence disclosed herein as set forth in the table(s) or accession number(s) disclosed herein.

[0254] Pharmaceutical Composition The present application provides compositions comprising antibodies, including pharmaceutical compositions comprising any one or more of the antibodies described herein, together with one or more pharmaceutically acceptable excipients. In some embodiments, the compositions are sterile. Pharmaceutical compositions generally comprise an effective amount of an antibody.

[0255] These compositions may contain, in addition to one or more of the antibodies disclosed herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other substance may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0256] The pharmaceutical composition for oral administration can be in tablet, capsule, powder, or liquid form.Tablets can contain solid carriers such as gelatin or adjuvants.Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil.Saline, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0257] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0258] Whether it is a polypeptide, antibody (e.g., an anti-TREM2 antibody), nucleic acid, small molecule, or other pharmaceutically useful compound to be given to an individual, the administration is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (in some cases, prophylaxis can be considered treatment, although prophylaxis can be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Treatment formulation, such as determining dosage, is within the responsibility of general practitioners and other physicians and will usually take into account the disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0259] The compositions may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition to be treated.

[0260] method Preparation method The antibodies described herein can be produced using recombinant methods and compositions, such as those described in US Pat. No. 4,816,567.

[0261] In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or VH of the antibody (e.g., the light and / or heavy chain of the antibody) or an amino acid sequence comprising the VHH of a single-domain antibody. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In one embodiment, the nucleic acid is provided in a multicistronic vector. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antigen-binding polypeptide construct. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a YO, NS, or Sp20 cell). In one embodiment, a method of producing an antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0262] For recombinant production of antibodies, nucleic acids encoding the antibodies, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0263] The term "substantially purified" refers to constructs described herein, or variants thereof, which may be substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., naturally occurring cells or, in certain embodiments, host cells in the case of recombinantly produced heteromultimers, substantially free of extracellular material, includes preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein in certain embodiments is present at no more than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% of the dry weight of the cell. When the heteromultimer or a variant thereof is recombinantly produced by a host cell, the protein is present in the culture medium, in certain embodiments, at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of dry weight of cells. In certain embodiments, the "substantially purified" heteromultimers produced by the methods described herein have a level of purification of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically at least about 75%, 80%, 85%, more specifically at least about 90%, at least about 95%, at least about 99%, or more, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0264] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0265] "Recombinant host cell" or "host cell" refers to a cell containing an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells. The exogenous polynucleotide may be maintained as a non-integrated vector, e.g., a plasmid, or alternatively, may be integrated into the host genome. Host cells may include CHO, CHO derivatives, NS0, Sp20, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0266] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0267] As used herein, the term "prokaryote" refers to prokaryotic organisms. For example, non-eukaryotic organisms may belong to the Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the Archaea (including, but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, e.g., Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

[0268] For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0269] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast lineages whose glycosylation pathways have been "humanized," resulting in the production of antibodies that contain partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0270] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly Spodoptera frugiperda cells for transfection.

[0271] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0272] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); e.g., Mather et al., Annals Other useful mammalian host cell lines include TRI cells, as described in NYAcad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. - Examples of suitable host cell lines for antibody production include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and SP2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0273] In one embodiment, an antibody described herein is produced in a stable mammalian cell by a method comprising transfecting at least one stable mammalian cell with nucleic acids encoding the antibody in a predetermined ratio and expressing the nucleic acids in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that results in the highest proportion of antibody in the expression product.

[0274] In some embodiments, there are methods of producing antibodies in stable mammalian cells as described herein, wherein the expression product of at least one stable mammalian cell comprises a greater proportion of the desired glycosylated antibody compared to monomeric heavy or light chain polypeptides or other antibodies.

[0275] In some embodiments, there are methods for producing glycosylated antibodies in stable mammalian cells as described herein, the methods comprising identifying and purifying a desired glycosylated antibody, in some embodiments, by one or both of liquid chromatography and mass spectrometry.

[0276] If necessary, antibodies can be purified or isolated after expression. Proteins may be isolated or purified by a variety of methods known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, size, or gel filtration, and reversed phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins may find use in the present invention for antibody purification. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can often be enabled by specific fusion partners. For example, antibodies can be purified by binding to glutathione resins when GST fusions are used, Ni when His tags are used, or immobilized. +2 If a Flag tag is used, it may be purified using affinity chromatography, or an anti-Flag antibody. For general guidance in suitable purification techniques, see, e.g., Protein Purification: Principles and Practice, 3, incorporated by reference in its entirety. rd Ed., Scopes, Springer-Verlag, NY, 1994. The degree of purification required will vary depending on the use of the antibody. In some cases, no purification is necessary.

[0277] In certain embodiments, antibodies are purified using anion exchange chromatography, including but not limited to, chromatography on Q-Sepharose, DEAE Sepharose, Poros HQ, Poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0278] In certain embodiments, the proteins described herein are purified using cation exchange chromatography, including but not limited to SP-Sepharose, CM Sepharose, Poros HS, Poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and derivatives.

[0279] Additionally, the antibodies described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NY, and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids generally include, but are not limited to, the D isomers of the common amino acids 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, designer amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs. Additionally, amino acids can be D (dextrorotatory) or L (levorotatory).

[0280] How to use In one aspect, the present application provides a method of contacting unstimulated bone marrow cells with an anti-TREM2 antibody, such as a human antibody, which results in the neutralization of the unstimulated bone marrow cells.

[0281] In another aspect, the present application provides a method of contacting unstimulated bone marrow cells with an anti-TREM2 mouse antibody, which results in the neutralization of the unstimulated bone marrow cells.

[0282] In some embodiments, the non-stimulatory cells are one or more of DC1 cells and TAM cells.

[0283] In some embodiments, the present application provides a method for neutralizing unstimulated bone marrow cells, comprising contacting the unstimulated bone marrow cells with a TREM2 antibody, thereby killing the unstimulated bone marrow cells. Neutralization refers to rendering the cells partially or completely non-functional. In some embodiments, neutralization of the unstimulated bone marrow cells results in inducing growth arrest in the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in apoptosis of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in lysis of the cells, e.g., by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, neutralization of the unstimulated bone marrow cells results in necrosis of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in inducing growth arrest in the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in inactivation of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in neutralization of the activity of the TREM2 protein in the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in reduced proliferation of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in differentiation of the cells. In some embodiments, nullifying the non-stimulated bone marrow cells results in a decrease in the ability of the cells to act as inhibitory antigen-presenting cells or an increase in the ability of the cells to act as activating antigen-presenting cells. In some embodiments, nullifying the non-stimulated bone marrow cells results in a mallocalization of the cells within the tumor tissue or tumor microenvironment (TME). In some embodiments, nullifying the non-stimulated bone marrow cells results in an alteration of the spatial organization of cells within the tumor tissue or tumor microenvironment. In some embodiments, nullifying the non-stimulated bone marrow cells results in an alteration of the temporal expression of cells within the tumor tissue or TME. In some embodiments, the method further comprises removing the non-stimulated bone marrow cells.

[0284] In any and all aspects of neutralizing unstimulated bone marrow cells described herein, any increase or decrease or change in aspect of property(ies) or function(ies) is as compared to cells not contacted with the anti-TREM2 antibody.

[0285] In another aspect, the application provides a method of contacting unstimulated bone marrow cells with an anti-TREM2 antibody, resulting in modulation of the function of the unstimulated bone marrow cells. The modulation can be any one or more of the following: In some embodiments, the unstimulated cells are one or more of DC1 cells, TAM1 cells, and TAM2 cells. In some embodiments, the modulation of function results in neutralization of the unstimulated bone marrow cells. In some embodiments, the modulation of function of the unstimulated bone marrow cells results in an increased ability of the cells to stimulate both naive and activated CD8+ T cells, e.g., by increasing the ability of the unstimulated cells to cross-present tumor antigens on MHCI molecules to naive CD8+ T cells. In some embodiments, the modulation increases the T cell stimulatory function of the unstimulated bone marrow cells, including, e.g., the ability of the cells to induce T cell receptor (TCR) signaling, T cell proliferation, or T cell cytokine production. In one embodiment, the survival of the unstimulated cells is decreased or the proliferation of the unstimulated cells is decreased. In one embodiment, the ratio of stimulated to unstimulated bone marrow cells is increased.

[0286] In any and all aspects of decreasing the function of unstimulated bone marrow cells described herein, any increase or decrease or change in property(ies) or aspect(s) of function(ies) is as compared to cells not contacted with the TREM2 antibody.

[0287] In some embodiments, the present application provides a method for killing unstimulated bone marrow cells (also referred to as inducing cell death), comprising contacting unstimulated bone marrow cells with an anti-TREM2 antibody, thereby killing the unstimulated bone marrow cells. In some embodiments, killing is increased compared to unstimulated bone marrow cells not contacted with the anti-TREM2 antibody. In some embodiments, the contacting induces apoptosis of the unstimulated bone marrow cells. In some embodiments, the contacting induces apoptosis of the unstimulated bone marrow cells. In some embodiments, the unstimulated bone marrow cells are in a population of immune cells that includes unstimulated bone marrow cells and stimulated bone marrow cells. In some embodiments, the method further comprises removing the unstimulated bone marrow cells. In some embodiments, 10% to 80% of the cells are killed. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the cells are killed.

[0288] In some embodiments, the present application provides a method for increasing the ratio of stimulated to unstimulated bone marrow cells in a population of immune cells, the ratio including stimulated and unstimulated bone marrow cells, comprising contacting the population of immune cells with an anti-TREM2 antibody. In some embodiments, the ratio is increased compared to a population of cells not contacted with the anti-TREM2 antibody. In some embodiments, the ratio of DC2 cells to DC1 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1 cells is increased. In some embodiments, the ratio of DC2 cells to TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1+TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1+DC1 cells is increased. In some embodiments, the ratio of DC2 cells to DC1+TAM2 cells is increased. In some embodiments, the ratio is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0289] In some embodiments, the ratio of stimulated bone marrow cells to unstimulated bone marrow cells before contact ranges from 0.001:1 to 0.1:1. In some embodiments, the ratio of stimulated bone marrow cells to unstimulated bone marrow cells after contact ranges from 0.1:1 to 100:1.

[0290] In some embodiments, the number of unstimulated bone marrow cells is reduced. In some embodiments, the stimulated bone marrow cells are DC2 cells. In some embodiments, the unstimulated bone marrow cells are killed, for example, by necrosis or apoptosis. In some embodiments, the unstimulated bone marrow cells are induced to undergo growth arrest. In some embodiments, the unstimulated bone marrow cells no longer proliferate. In some embodiments, the spatial localization of unstimulated bone marrow cells is altered, and the ratio is increased in specific regions of the TME. In some embodiments, the temporal expression of unstimulated bone marrow cells is altered, and the ratio is increased during specific times during tumor development.

[0291] In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In certain embodiments, the contacting is in vivo in a human. In some embodiments, the contacting occurs by administering an anti-TREM2 antibody. In some embodiments, the individual to whom the antibody (e.g., a human) is administered has cancer.

[0292] In another aspect, the invention provides methods for treating an immune-related condition (e.g., cancer) in an individual, comprising administering to the individual an effective amount of a composition comprising an anti-TREM2 antibody. In another aspect, the invention provides methods for enhancing an immune response in an individual, comprising administering to the individual an effective amount of a composition comprising an anti-TREM2 antibody. In some embodiments, these methods are further provided in combination with other combination therapies, such as PDL blockade therapy, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, CTLA4 blockade therapy, anti-CTLA4 antibodies, systemic checkpoint blockade therapy in which inhibitory molecules on T cells are blocked, adoptive T cell therapy, CAR T cell therapy, dendritic cell or other cell therapy, and conventional chemotherapy.

[0293] In some embodiments, the method further comprises determining the expression level of TREM2 protein in a biological sample from the individual. In some embodiments, the biological sample includes, but is not limited to, body fluids, tissue samples, organ samples, urine, feces, blood, saliva, CSF, and any combination thereof. In some embodiments, the biological sample is derived from tumor tissue. In some embodiments, the expression level comprises the mRNA expression level of mRNA encoding the TREM2 protein. In some embodiments, the expression level of the TREM2 protein comprises the protein expression level of NSM. In some embodiments, the expression level of the TREM2 protein is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof.

[0294] In another aspect, the present application provides a method for determining the presence or absence of unstimulated bone marrow cells in general, or a method for determining the presence or absence of specific unstimulated bone marrow cells (e.g., DC1 cells, TAM1 cells, and / or TAM2 cells), comprising contacting a population of cells comprising unstimulated bone marrow cells with an anti-TREM2 antibody and quantifying the number of unstimulated bone marrow cells. In another aspect, the present application provides a method for determining the presence or absence of unstimulated bone marrow cells, comprising contacting a population of immune cells comprising unstimulated bone marrow cells and stimulated bone marrow cells with an anti-TREM2 antibody, detecting complexes or moieties indicative of binding of the antibody to the cells, and optionally quantifying the number of unstimulated bone marrow cells in the population. In another aspect, the present application provides a method for determining the relative ratio of unstimulated bone marrow cells to stimulated bone marrow cells, comprising contacting a population of immune cells comprising unstimulated bone marrow cells and stimulated bone marrow cells with an anti-TREM2 antibody; quantifying the number of stimulated bone marrow cells and unstimulated bone marrow cells; and determining the relative ratio of unstimulated bone marrow cells to stimulated bone marrow cells.

[0295] In the embodiments described herein for detection and / or quantification, the anti-TREM2 antibody binds to the TREM2 protein but does not necessarily affect a biological response such as ADCC, although it may have an effect on a biological response.

[0296] In another aspect, the present invention provides methods for identifying an individual who may respond to immunotherapy (e.g., using an anti-TREM2 antibody) for the treatment of an immune-related condition (e.g., cancer), comprising detecting the expression level of TREM2 protein in a biological sample from the individual and determining whether the individual may respond to immunotherapy based on the expression level of TREM2 protein, wherein an elevated level of TREM2 protein in the individual compared to that of a healthy individual indicates that the individual may respond to immunotherapy. In some embodiments, these methods may also be used to diagnose an immune-related condition (e.g., cancer) in an individual, wherein an elevated level of TREM2 protein in the individual compared to that of a healthy individual based on the expression level of TREM2 protein indicates that the individual has cancer. In some embodiments, the expression level comprises the mRNA expression level of mRNA encoding TREM2 protein. In other embodiments, the expression level of TREM2 protein comprises the protein expression level of TREM2 protein. In some embodiments, the expression level of TREM2 protein is detected in a sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof. In these embodiments, the anti-TREM2 antibody binds to the TREM2 protein but does not necessarily affect a biological response such as ADCC. In some embodiments, the biological sample is derived from tumor tissue. In some embodiments, the biological sample includes, but is not limited to, body fluids, tissue samples, organ samples, urine, feces, blood, saliva, CSF, and any combination thereof.

[0297] Also disclosed herein is a method for enhancing the immune response of a subject against tumor or for enhancing the effectiveness of immunotherapy treatment.Generally, treatment that increases the abundance of SDC will improve subject outcomes such as recurrence-free survival time, and will enhance the effectiveness of cancer immunotherapy treatment.Treatment can increase the relative or absolute abundance of SDC cells in the tumor of a subject.Treatment can decrease the relative or absolute abundance of NSM cells in the tumor of a subject.

[0298] An exemplary general treatment strategy involves increasing the number of SDCs by systemic introduction of Flt3L. Another method is treatment of a subject's autologous bone marrow or blood cells with Flt3L while simultaneously blocking CSF1. For example, retroviral expression of SDC transcription factors such as IRF8, Mycl1, BATF3, or ZBTB46 in bone marrow or blood progenitor cell populations may also be used to promote the emergence of SDCs. Another treatment strategy involves systematically eliminating NSM cells while selectively sparing SDCs. This can result in an overall favorable change in the ratio of these populations. Elimination of NSM cells can be achieved by any means, including administration (systemically or locally to the tumor) of antibodies against the TREM2 surface protein.

[0299] In some embodiments, the SDC-enhancing treatment is applied as a therapeutic treatment to make the subject's natural immune system more capable of controlling or eradicating cancer. In another embodiment, the SDC-enhancing treatment of the present invention is applied in combination with a therapeutic treatment, such as an immunotherapy treatment (such application occurs before, simultaneously with, or after the immunotherapy treatment), and the SDC-enhancing treatment acts as a supplementary or adjuvant treatment to increase the effectiveness of the therapeutic treatment.

[0300] An enhanced immune response includes increasing, maintaining, initiating, or inducing an immune response following administration of an isolated antibody that binds TREM2 compared to the immune response following administration of an isotype control antibody. In some embodiments, an enhanced immune response comprises increasing the immune response compared to an isotype control antibody. In some embodiments, an enhanced immune response comprises maintaining immunity compared to an isotype control antibody. In some embodiments, an enhanced immune response comprises initiating an immune response compared to an isotype control antibody. In some embodiments, an enhanced immune response comprises inducing an immune response compared to an isotype control antibody.

[0301] In some embodiments, the treatment enhances an immune response in the subject. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the antibody induces a memory immune response.

[0302] In some embodiments, the antibody induces increased expression of at least one cytokine or chemokine in the cells compared to an isotype control antibody.

[0303] In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, TNF-α, CXCL1, or CXCL10.

[0304] In some embodiments, the cytokine or chemokine is CXCL10.

[0305] Administration method In some embodiments, the methods provided herein are useful for treating an immune-related condition in an individual. In one embodiment, the individual is a human and the antibody is a TREM2 antibody. In another embodiment, the individual is a mouse and the antibody is a TREM2 antibody.

[0306] In some embodiments, for in vivo administration of the anti-TREM2 antibodies described herein, typical dosages may range from about 10 ng / kg up to about 100 mg / kg of an individual's body weight or more per day, preferably about 1 mg / kg / day to 10 mg / kg / day, depending on the route of administration. For repeated administration over several days or longer, depending on the severity of the disease or disorder being treated, treatment is sustained until the desired suppression of symptoms is achieved. An exemplary dosing regimen involves administering an initial dose of about 2 mg / kg of anti-TREM2 antibody, followed by weekly maintenance doses of about 1 mg / kg every other week. Other dosing regimens may be useful depending on the pattern of pharmacokinetic decline the physician desires to achieve. For example, administering to an individual 1 to 21 times per week is contemplated herein. In certain embodiments, doses ranging from about 3 μg / kg to about 2 mg / kg (e.g., about 3 μg / kg, about 10 μg / kg, about 30 μg / kg, about 100 μg / kg, about 300 μg / kg, about 1 mg / kg, and about 2 mg / kg) may be used. In certain embodiments, the dosing frequency is three times a day, twice a day, once a day, every other day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once a month, once every two months, once every three months, or more. The progress of therapy is easily monitored by conventional techniques and assays. The dosing regimen, including the anti-TREM2 antibody administered, can vary over time, independently of the dose used.

[0307] In some embodiments, the methods provided herein (e.g., methods of enhancing an immune response or methods of resulting in the neutralization of unstimulated bone marrow cells) are useful for treating cancer, and the anti-TREM2 antibody or the individual to whom the anti-TREM2 antibody is administered has cancer.

[0308] Any suitable cancer can be treated with the antibodies provided herein. The cancer can be any carcinoma, adenocarcinoma, soft tissue cancer, sarcoma, teratoma, melanoma, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or brain cancer known in the medical field. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is immunoevasive. In some embodiments, the cancer is immunoresponsive. In some embodiments, the cancer is melanoma, kidney, hepatobiliary, head and neck squamous cell carcinoma (HNSC), pancreas, colon, bladder, glioblastoma, prostate, lung, breast (mammary), ovary, stomach, kidney, bladder, esophagus, kidney, melanoma, leukemia, lymphoma, or mesothelioma. In some embodiments, the cancer is colon cancer, pancreatic cancer, or breast cancer.

[0309] In some embodiments, the immune-related condition is an immune-related condition associated with expression of TREM2 protein on unstimulated (human) bone marrow cells or expression of a homologue of TREM2 protein in a non-human species. In some embodiments, the immune-related condition is an immune-related condition associated with overexpression of TREM2 protein in unstimulated bone marrow cells compared to stimulated bone marrow cells. In some embodiments, overexpression of TREM2 mRNA or TREM2 protein is at least about 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, or 100-fold higher compared to stimulated bone marrow cells.

[0310] In some embodiments, the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. An effective amount of an anti-TREM2 antibody may be administered to treat cancer. The appropriate dosage of the anti-TREM2 antibody may be determined based on the type of cancer to be treated, the type of anti-TREM2 antibody, the severity and course of the cancer, the individual's clinical condition, the individual's clinical history, and response to treatment, as well as the judgment of the attending physician.

[0311] Combination therapy In some embodiments, the antibodies provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with the antibodies provided herein. In some embodiments, the immunotherapy is selected from checkpoint inhibitors; T cell checkpoint inhibitors; anti-PD1 antibodies; anti-PDL1 antibodies; anti-CTLA4 antibodies; adoptive T cell therapy; CAR-T cell therapy; dendritic cell vaccines; monocyte vaccines; antigen binding proteins that bind to both T cells and antigen-presenting cells; BiTE dual antigen binding proteins; Toll-like receptor ligands; cytokines; cytotoxic therapy; chemotherapy; cytostatic agents; radiation therapy; small molecule inhibitors; small molecule agonists; immunomodulators; and epigenetic modulators, and combinations thereof.

[0312] In some embodiments, the additional therapeutic agent is an antibody. In some embodiments, the additional therapeutic agent is an antibody that binds to a protein(s) on the surface of a tumor cell.

[0313] For the treatment of cancer, anti-TREM2 antibodies may be combined with one or more antibodies that inhibit immune checkpoint proteins. Immune checkpoint proteins displayed on the surface of tumor cells are of particular interest. The most actively investigated immune checkpoint receptors in the context of clinical cancer immunotherapy, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4, also known as CD152) and programmed cell death protein 1 (PD1, also known as CD279), are both inhibitory receptors. The clinical activity of antibodies that block either of these receptors means that antitumor immunity can be enhanced at multiple levels and that combination strategies can be rationally designed, guided by mechanistic considerations and preclinical models.

[0314] The two ligands for PD-1 are PD-1 ligand 1 (PD-L1, also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). PD-L1 is expressed on cancer cells, and through binding to its receptor PD-1 on T cells, it inhibits T cell activation / function. Inhibitors that block the interaction of PD-1 with its cognate ligands PD-L1 and PD-L2 on cancer cells can result in increased T cell activation and function, preventing cancer cells from evading the immune system.

[0315] In some embodiments, the immunotherapy is an agent that interferes with the binding of PD-1 and PD-L1 or PD-L2. In some embodiments, the immunotherapy is an anti-PD1 antibody. In some embodiments, the immunotherapy is an anti-PD-L1 antibody. In some embodiments, the immunotherapy is an anti-PD-L2 antibody.

[0316] Various PD-1, PD-L1, and PD-L2 antibodies are known in the art. In some embodiments, the additional therapeutic agent is at least one of atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1), nivolumab (PD-1), pembrolizumab (PD-1), cemiplimab (PD-1), ipilimumab (CTLA4), tremelimumab (CTLA4), or any combination thereof.

[0317] The additional therapeutic agent can be administered by any suitable means. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in the same pharmaceutical composition. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in different pharmaceutical compositions.

[0318] In embodiments in which an antibody provided herein and an additional therapeutic agent are contained in different pharmaceutical compositions, administration of the antibody may occur prior to, simultaneously with, and / or after administration of the additional therapeutic agent. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one month of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one week of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one day of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about 12 hours of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one hour of each other.

[0319] Kits and Articles of Manufacture The present application provides kits comprising any one or more of the antibody compositions described herein. In some embodiments, the kit further contains a component selected from a secondary antibody, an immunohistochemistry reagent, a pharmaceutically acceptable excipient, and instructions, and any combination thereof. In a specific embodiment, the kit includes a pharmaceutical composition comprising any one or more of the antibody compositions described herein together with one or more pharmaceutically acceptable excipients.

[0320] The present application also provides an article of manufacture comprising any one of the antibody compositions or kits described herein. Examples of articles of manufacture include vials (including sealed vials). [Example]

[0321] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0322] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature, see, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0323] Example 1: Humanization of anti-TREM2 antibodies. Humanization of clone #237920 Monoclonal rat IgG specific for mouse and human TREM2 2B Clone #237920 (R&D Systems catalog number MAB17291) was used for sequencing and humanization. Briefly, the disulfide bonds of the antibody were reduced with dithiothreitol (DTT), and the free sulfhydryl groups were alkylated with iodoacetamide. The alkylated antibody was digested with sequencing-grade endoproteinase, purified using a spin column, and the sequence was determined by LC-MS / MS analysis. The sequence is shown below. TIFF2025128164000007.tif69166

[0324] The VH and VL sequences were compared to a library of known human germline sequences on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ; Ye, J. et al., Nucleic Acids Research 41:W34-W40 (2013)). The databases used were the IMGT human VH gene (F+ ORF, 273 germline sequences) and the IMGT human VL kappa gene (F+ ORF, 74 germline sequences).

[0325] For 237920VH, human germline IGHV3-23 (allele 1) was selected as the acceptor sequence, and the human heavy chain IGHJ4 (allele 1) joining region (J gene) was selected from human joining region sequences compiled by IMGT®, the international ImMunoGeneTics information system®, www.imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France).

[0326] For 237920VL, human germline IGKV1-39 (allele 1) was selected as the acceptor sequence, and the human light chain IGKJ2 (allele 1) joining region (J gene) was selected from human joining region sequences compiled by IMGT®, the international ImMunoGeneTics information system®, www.imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France).

[0327] Exemplary definitions or CDRs were defined according to the AbM definitions (see the website of Dr. Andrew C. Martin at www.bioinf.org.uk / abs / for a table comparing CDR definitions). For example, to optimize binding of the humanized antibodies, changes were made to human germline framework positions (i.e., non-CDR residues of VH and VL) relative to the corresponding parental murine sequences.

[0328] Table 1A shows the VL, VH, and complete heavy and light chain sequences of the humanized versions of mAb 237920 that were generated. 37017 is the parent humanized clone from which other humanized versions were generated by additional mutations. Table 1B shows the CDR sequences.

[0329] (Table 1A) TIFF2025128164000008.tif110170TIFF2025128164000009.tif161170

[0330] Table 1B. CDRs of humanized antibodies according to an exemplary system TIFF2025128164000010.tif52128

[0331] Table 1C: CDRs of humanized antibodies according to the AbM system TIFF2025128164000011.tif52128

[0332] Alignment of humanized antibody frameworks (SEQ ID NOS: 7, 3, 5, 21, 6, 23, and 24, respectively, in order of appearance) TIFF2025128164000012.tif117158

[0333] In the CDRs within the VL domain, Asn28, Asn31, Asn32, and Asn53 have low potential for deamidation based on sequence and conformation. Asn93 has low to moderate potential for deamidation and may exhibit low levels of this post-translational modification. In the VH domain, Asn31 has low potential for deamidation based on sequence and conformation. In CDR-H2, Asn53 has a moderate potential for deamidation, which prevents post-translational modification; it has been experimentally determined that Asn53 can be modified to Gln, Ser, or Ala and maintain binding. In CDR-H3, Trp100 is solvent-exposed and may have potential for oxidation, especially under stress conditions.

[0334] In-solution endoproteinase digestion For mAb sequencing analysis, in-solution endoproteinase digestion of monoclonal antibodies (mAbs) was performed. 50 μg of antibody was reduced with DTT, alkylated using iodoacetamide, acetone precipitated, and reconstituted in water at a concentration of 1 μg / μL. In-solution digestion of antibody samples was performed using five individual enzyme digestions: Asp-N, chymotrypsin, elastase, trypsin, and pepsin, according to the manufacturer's instructions. The samples were then lyophilized, resuspended in 0.1% TFA, and purified using a C18 Zip-Tip. The samples were then dried in a vacuum centrifuge and stored frozen until mass spectrometry analysis.

[0335] mass spectrometry Intact mass measurement The mAb samples were denatured, reduced, and acidified. Proteins were then analyzed using an Agilent 1100 HPLC interfaced to a Waters QToF Ultima Global mass spectrometer (LC-ESI-TOF MS). Appropriate LC-MS spectra were processed (combined, subtracted, smoothed, and deconvoluted) using Waters MassLynx 4.1 software.

[0336] LC-MS / MS analysis The purified peptides were resuspended in 0.1% formic acid, and each half of the digest was analyzed on an Orbitrap analyzer (Q-Exactive, Thermo Fisher Scientific) equipped with a nanospray source and an EASY-nLC 1000 system (Thermo Fisher Scientific). The peptides were loaded onto a 50 cm (75 μm internal diameter) EASY-Spray column packed with PepMap® RSLC 2 μm C18 resin (Thermo Fisher Scientific) at 800 bar pressure. The peptides were eluted at a rate of 250 mL / min using a gradient set from 0% to 30% acetonitrile in 0.1% formic acid over 60 min. The peptides were introduced into the Q-Exactive mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray ion source. The instrument method consisted of one MS full scan (400-1600 m / z) in an Orbitrap mass analyzer with an automatic gain control (AGC) target of 1E6, a maximum ion injection time of 120 ms, and a resolution of 70000, followed by 10 data-dependent MS / MS scans with a resolution of 17500, an AGC target of 5E5, and a maximum ion injection time of 100 ms, and one microscan. The intensity threshold for triggering the MS / MS scan was set to a 1.0% underfill ratio. Fragmentation occurred in the HCD collision cell with a normalized collision energy set to 30. Dynamic exclusion was applied using an 8-second setting.

[0337] Table 2 summarizes the biophysical properties of the humanized clones. Molecular weights and extinction coefficients were estimated for the sum of the contributing protein chains in the quaternary structure. By default, the calculation assumes equal monomer contributions from each chain. The extinction coefficient is M -1 cm -1 The absorbance at 280 nm is the predicted absorbance per mole of protein in units of 1. Potential post-translational modifications such as glycosylation, phosphorylation, and proteolysis are not taken into account in estimating molecular weight or extinction coefficient.

[0338] (Table 2) TIFF2025128164000013.tif35157

[0339] Example 2: Production and characterization of anti-TREM2 antibodies Antibody production and characterization Standard protein expression vectors were transfected into HEK293 cells using standard methods, and the cells were then grown for 7 days and harvested. In addition to HEK293, antibodies were also produced in 293 cells that had been CRISPR / Cas9-edited (Alexander Weiss, University of Toronto) to delete mammalian α1,6 fucosyltransferase (FUT8). The pH of the supernatant was adjusted with 1 M Hepes pH 7.4, and sodium azide was added to prevent microbial growth. Proteins were captured using KanCap A resin, and after washing with PBS containing 1 M sodium chloride and PBS, they were eluted with 50 mM citric acid pH 3.5, 100 mM NaCl. Immediately after elution, the solution was neutralized with 1 M Tris (pH 8) containing 0.5 M arginine. Biophysical characterization of the buffer-exchanged proteins into PBS was performed using standard techniques. Protein was quantified at OD280, and the calculated extinction coefficient was used to determine the amount and concentration. Purity and approximate molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris / glycine / SDS, 4-20%) or a Perkin Elmer GXII capillary electrophoresis system. Aggregation status was determined by HPLC with detection at 280 nm using a Sepax Zenix-C SEC-300, 3 μm, 300 Å, 4.6*150 mm size exclusion column and PBS running buffer.

[0340] Antibody affinity measurement using surface plasmon resonance (SPR) Binding kinetics were determined by surface plasmon resonance using a Biacore T200 (GE Healthcare, UK) with human TREM2 His (Sino Biological, Beijing, People's Republic of China) directly immobilized on the chip by amine coupling or human or TREM2 human IgG1 Fc fusion proteins (in-house SEC purified to 95% ultrapure) captured on Series S CM5 chips via anti-His capture. Serial dilutions of the indicated antibodies were injected at 30 μl / min for 2 minutes. PBS or system buffer was then injected at 30 μl / min for 400 seconds to observe dissociation. Binding responses were corrected by subtracting the response of a blank flow cell. For kinetic analysis, k on value and k off A 1:1 Langmuir model for global fitting of the K values ​​was used. d The value is k on and k off was determined from the ratio of

[0341] Table 3 shows antibody binding affinities to human TREM2-His as measured by SPR. (Table 3) TIFF2025128164000014.tif21154

[0342] Table 4 shows antibody binding affinities to human TREM2-Fc as measured by SPR. (Table 4) TIFF2025128164000015.tif32154

[0343] At low ligand density (RL = 500 RU), the PI37017 binding kinetics to human TREM2-Fc did not provide a good fit. This data indicates that the A at position 97 and the K at position 98 of the sequence of SEQ ID NO: 31 (clone 37017) are closely related to the binding of rat IgG 2BThis indicates that humanization of clone #237920 likely results in a substantial loss of human TREM2 binding. Mutation of these framework residues (A97T and K98R) results in increased human TREM2 binding by the humanized clone. See, for example, clone 37012.

[0344] Example 3: Cell binding of anti-TREM2 antibodies Cell binding (EC50 measurement): 100,000–500,000 Expi293 parental or Expi293 cells overexpressing human or mouse TREM2 were plated in 96-well plates, and dead cells were stained with Zombie Near Infrared (Biolegend). Titrations of the indicated unconjugated antibodies were incubated with these cells at a 1:3 dilution range of 0 μg / ml to 10 μg / ml over 8–10 points. Depending on their isotype (hIgG1 or mIgG2a), these primary unconjugated antibodies were detected with Alexa Fluor 647-conjugated anti-human Fc or anti-mouse Fc secondary antibodies (Jackson Immunoresearch). Alexa Fluor 647 signals were measured by flow cytometry (BD Fortessa X-14, BD Biosciences). EC50 values ​​were calculated in Graphpad Prism (Graphpad Software) by curve fitting the signal generated from antibody binding to overexpressing cells through the background fluorescence generated from the HEK293 parental cells.

[0345] This data shows that A at position 97 and K at position 98 of the sequence of SEQ ID NO: 31 (clone 37017) are involved in the formation of rat IgG 2B This indicates that humanization of clone #237920 likely results in a substantial loss of human TREM2 binding. Mutation of these framework residues (A97T and K98R) results in increased human TREM2 binding by the humanized clone. See, for example, clone 37012.

[0346] Table 5 shows the half-maximal saturation binding of anti-TREM2 antibodies to cell surface TREM2. (Table 5) TIFF2025128164000016.tif71128

[0347] Example 4: PI-7012 improves anti-tumor activity in combination with anti-PD-1 Materials and Methods CT26.WT (CRL-2638) cells were purchased from the American Type Culture Collection (ATCC). All antibodies for in vivo use were tested for endotoxin and used at 0.2 EU / mg protein or less. The amino acid sequence of the anti-mouse PD-1 antibody from clone RMP1-14 (Absolute Antibody Inc., catalog number Ab00813-7.1) was determined by mass spectrometry (LC-MS / MS). (Nimmerjahn and Ravetch 2005 Science 310:1510-1512) 1 As described previously, a single point mutation [D265A] was introduced into the Fc region of the mouse IgG1 version of the RMP1-14 antibody to eliminate binding to FcgR. Mouse IgG1 [clone MOPC-21] and mouse IgG2a [clone C1.18.4] isotype controls were purchased from BioXCell. PI-7012 and Afuc-PI-7012 (which have the CDR sequences of PI37012 and were murine in mouse IgG2a format) were produced in Expi293 cells (Thermo Fisher Scientific) or 293 / FUT8 knockout cells (University of Toronto) in mouse IgG2a format, respectively, and purified using MabSelect Protein A resin (GE Life Sciences). The antibodies were eluted with 0.1 M citrate buffer (pH 3.0) and the buffer was exchanged before use.

[0348] All experimental procedures involving live animals were approved by the Institutional Animal Care and Use Committees of Murigenics. Six- to eight-week-old female BALB / c mice were purchased from Taconic and used after a week of acclimation to the animal facility. After thawing from liquid nitrogen stock, CT26 cells were harvested at passages 3 to 7 and then used for in vivo experiments. The right flank of female Balb / c mice was shaved and prepared for injection the day before. On the day of tumor inoculation, cells were harvested and used within 30 minutes. To establish subcutaneous tumors, 1 × 10 6 CT26 cells were implanted into the mice, and the mice were then monitored for tumor growth. Tumor volume was calculated from caliper measurements of tumor dimensions using the formula (L x W2) / 2, where L is the longer measurement. When tumors reached an average size of 80-100 cubic mm, the mice were randomized into treatment groups as shown in Table 6.

[0349] (Table 6) TIFF2025128164000017.tif93132

[0350] Tumor volume and body weight were monitored twice weekly and graphed for group comparison analysis by one-way ANOVA. Mice were euthanized during the study when tumor volume reached approximately 2000 mm3, body weight loss exceeded 15%, or other health-related concerns.

[0351] result We determined whether glycoengineering the affinity of mAbs to specific FcgRs (i.e., by generating an afucosylated version of an anti-TREM2 mAb) could increase antitumor activity. PI-7012 and afuc-PI-7012 were tested in combination with anti-PD-1 in the CT26 tumor model. PI-7012 and afuc-PI-7012 demonstrated similar levels of tumor growth inhibition (79% vs. 88% TGI). Treatment with afuc-PI-7012 resulted in a 30% cure rate. As seen in Figure 1A, afuc-PI-7012, when combined with anti-PD-1, exhibited increased antitumor activity compared with PI-7012. The effect of afucosylation of PI-7012 on antitumor activity was more clearly seen in analysis of individual mouse tumor volumes (Figures 1B and 1C). This indicates that afucosylation of anti-TREM2 antibodies offers significant therapeutic advantages over co-fucosylated antibodies.

[0352] There was no significant loss of body weight in any treatment group over the course of the study (Figure 2). Weight loss is typically used as a surrogate measure of treatment-related toxicity. This data indicates that short- or long-term treatment with anti-TREM2, either as a single agent or in combination with anti-PD-1, was well tolerated and occurred without any significant toxicity being observed.

[0353] Example 5: No apparent toxicity associated with anti-TREM2 therapy Materials and Methods Tissues (lung, liver, brain, kidney, and heart) from mice treated in the above examples were stored in 10% neutral buffered formalin for at least 24 hours and routinely processed for histology. Sections were cut at 5-6 μm and stained with hematoxylin and eosin. Stained slides were examined using a low-magnification (40-100x) light microscope, and images were acquired using HistoWiz. CD68-positive cells were detected using an anti-CD68 antibody (AbD Serotec) and quantified using a light microscope in 8-9 fields of 40x sections.

[0354] result Gross morphological analysis by H&E staining of mouse tissues (lung, liver, heart, kidney, and brain) after treatment did not reveal any morphological changes in mice treated with PI-7012, afuc-PI-7012, and the anti-PD-1 combination compared to mice treated with the isotype control (Figure 3 shows the staining of lung tissue).

[0355] In addition to H&E staining, tissues were also stained for macrophages using anti-CD68. The intracellular marker CD68 is widely used in the literature as a reliable cytochemical marker for immunostaining monocytes / macrophages in inflamed tissues and tumors. No discernible changes in the number of CD68+ macrophages were observed in the lung (Figure 4A) or in the other tissues analyzed (Figure 4B) in any of the treatment groups compared to controls, indicating that anti-TREM2-mediated depletion occurred specifically in the TME.

[0356] Example 6: Restricted TREM2 expression in healthy mouse tissues Materials and Methods All animal studies were approved by the Murigenics Animal Studies Committee. em2Adiuj / J (hereafter referred to as TREM2KO) and control C57BL / 6J mice were from the Jackson Laboratory. Intact lungs, spleens, and bones were harvested and immediately processed for flow cytometry. In parallel, blood was collected by cardiac puncture. Tissues were processed into single-cell suspensions using a Miltenyi MACS tissue dissociation kit. Red blood cells were lysed using 1x erythrocyte lysis buffer (Biolegend). Prior to processing for cell surface staining, cells were stained with Fixable Viability Dye (ThermoFisher Scientific). Anti-mouse immunophenotypic antibodies were diluted in FACS buffer (2% FBS, 2 mM EDTA, 1x PBS) with Fc block and stained for 30 minutes on ice. After staining, cells were washed twice with FACS buffer and then fixed with 2% paraformaldehyde in PBS for 15 minutes. All data were collected on an LSR Fortessa flow cytometer (BD) or Attune flow cytometer (Thermo Fisher) and analyzed using FlowJo software. TREM2KO cell staining is shown in gray plots, and wild-type cell staining is shown in open plots.

[0357] result TREM2 is expressed on activated macrophages, immature dendritic cells, osteoclasts, and microglia 2,3 Cells expressing high levels of TREM2 are thought to be involved in immune surveillance, cell-cell interactions, clearance of tissue debris, and resolution of latent inflammatory responses. 4 Absence of TREM2 expression in these cells, either by gene knockdown or knockout, impairs their ability to phagocytose cellular debris and increases the production of regulatory cytokines. 5 In physiological settings, TREM2 expression in peripheral blood, spleen, liver, or lung is very low to undetectable, as seen in FACS plots (Figure 5). However, when lung- or liver-resident macrophages are isolated and stained for TREM2 as a pure cell population, TREM2 expression becomes detectable.

[0358] Example 7: TREM2 is primarily expressed on mouse TAMs Materials and Methods Tumor tissue was processed to isolate single-cell suspensions using standard methods. Briefly, tumors were minced with razor blades and digested in RPMI-1640 medium containing the enzymes from the Miltenyi MACS dissociation kit. Tumors were treated with GentleMACS according to the manufacturer's recommendations and incubated at 37°C for approximately 40 minutes. The digestion mixture was quenched with PBS containing 2 mM EDTA and 2% fetal bovine serum. The single-cell suspension was then passed through a 70 μm filter, and the cells were then rinsed with FACS buffer. After centrifugation, the cell pellet was resuspended in FACS buffer and stained with an antibody cocktail to identify tumor-associated macrophages and other immune cell populations. 6 TREM2KO cell staining is shown in grey plots, wild-type cell staining is shown in open plots.

[0359] result T cells, B cells, NK cells, and other non-myeloid cell populations, as well as CD45-negative cells, do not express detectable TREM2 on their cell surfaces. However, myeloid cell subsets, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), express TREM2 to varying degrees on their cell surfaces. Among cell types positive for TREM2 in the tumor microenvironment, the density of receptor expression on TAMs was significantly higher than that of other cell types, regardless of tumor origin (CT26 and MC38, as shown in Figure 6).

[0360] Example 8: Restricted TREM2 expression in human peripheral blood leukocytes Materials and Methods Peripheral blood mononuclear cells (PBMCs) and negatively selected CD14+ monocytes obtained from normal human volunteers were provided by AllCells Inc. CD14+ monocytes were differentiated in vitro using standard protocols. 5CD14 cells were cultured in complete medium consisting of RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 μg of streptomycin per ml, 100 U of penicillin per ml, and 10% heat-inactivated FBS. + Monocytes were cultured. To induce differentiation into macrophages, 50 ng / mL M-CSF was added to the culture medium. The medium was replenished every 2–3 days. After 7 days, macrophages were harvested by pipetting, and adherent cells were collected by subsequent trypsinization. Cells were then centrifuged and resuspended in RPMI-1640 supplemented with antibiotics, 2% FBS, recombinant human IFN-g, and 100 ng / mL LPS. To assess cell surface staining of TREM2 in cell subsets, these macrophages were surface stained simultaneously with PBMCs using a standard myeloid cocktail. Cells stained with a control mAb are shown in gray plots. Cells stained with anti-TREM2 mAb are shown in open plots.

[0361] result As can be seen in Figure 7, ex-vivo differentiated macrophages exhibit significantly higher cell surface receptor density for TREM2 compared to the PBMC-based cell types evaluated. Similar to observations reported in the literature, monocytes and some neutrophils express lower levels of TREM2.

[0362] Example 9: TREM2 is predominantly expressed on human TAMs Materials and Methods Human tumor tissue was obtained from the Cooperative Human Tissue Network (CHTN). Fresh human tumor tissue was dissociated into a single-cell suspension using the Miltenyi MACS isolation kit and the gentleMACS protocol. Single-cell suspensions of human tumor tissue were surface stained using a pre-validated multicolor FACS panel. All data were collected on an LSR Fortessa flow cytometer (BD) or an Attune flow cytometer (Thermo Fisher) and analyzed using FlowJo software. Numbers indicate the staining index for each population, which is defined as anti-TREM2 staining minus isotype control staining.

[0363] result Within the tumor microenvironment, TREM2 expression is differentially expressed at high levels on TAMs compared to other cells (Figure 8), making it a translation-associated marker of TAMs. Representative histograms of TREM2 antibody staining (open) or isotype control staining (gray) in various cell populations of mucinous adenocarcinoma are shown. Collectively, this data supports the hypothesis that TREM2-targeted drugs promote depletion of specific TAMs with relatively little to no concomitant effects on peripheral cells or other tissue-resident immune subsets.

[0364] Example 10: Anti-tumor efficacy of anti-TREM2 antibodies in combination with anti-PD-1 in multiple syngeneic tumor models Materials and Methods CT26.WT (CRL-2638), Py8119 (CRL-3278), 4T1 (CRL-2539), and EMT6 (CTL-2755) cells were purchased from the American Type Culture Collection (ATCC). Panc-02 cells were used at AJES Life Sciences (Stony Brook, NY). All antibodies used in vivo had a protein content of 0.2 EU / mg or less. The amino acid sequence of the anti-mouse PD-1 antibody, clone RMP1-14, was determined by mass spectrometry (LC-MS / MS). To eliminate binding to FcgR, a single point mutation, D265A, was introduced into the Fc region of the RMP1-14 antibody. Mouse IgG1, clone MOPC-21, and mouse IgG2a, clone C1.18.4, were isotype controls purchased from BioXCell. PI-7012 and afuc-PI-7012 (both as mouse IgG2a) were produced in Expi293 cells (Thermo Fisher Scientific) or 293 / FUT8 knockout cells, respectively, and then purified using MabSelect Protein A resin (GE Life Sciences). The mAbs were eluted with 0.1 M citrate buffer (pH 3.0), and the buffer was exchanged before use.

[0365] All experimental procedures involving live animals were approved by the Institutional Animal Care and Use Committees of Murigenics. Female BALB / c or C57BL / 6 mice (6–8 weeks old) were purchased from Taconic Laboratories or The Jackson Laboratory and used after acclimatization for one week in the animal facility. After thawing from liquid nitrogen stock, tumor cells were harvested at passages 3–7 and then used for in vivo experiments. The right flank of female mice was shaved and prepared for injection the day before tumor cell inoculation. On the day of tumor inoculation, cells were harvested and used within 30 minutes. To establish subcutaneous tumors, 1 × 10 6 CT26, EMT6, or Panc-02 cells, or 1 x 10 54T1 cells were implanted into the appropriate strain of mice, and the animals were then monitored for tumor growth. An equal volume of a single cell suspension of Py8119 cells was mixed with Matrigel (Corning Cat. No. 354248 or 354263) and then implanted at 2 x 10 per mouse. 6 The cells were transplanted.

[0366] Caliper measurements of tumor dimensions were used to calculate tumor volume using the formula (L x W2) / 2, where L is the longer measurement. When tumors reached an average size of 80-100 cubic mm, mice were randomized into treatment groups as shown in Table 7.

[0367] Tumor volume and body weight were monitored twice weekly and graphed for intergroup comparison analysis by one-way ANOVA. Mice were euthanized when tumor volume reached approximately 2,000 mm3 or when body weight decreased by more than 15% during the study.

[0368] (Table 7) TIFF2025128164000018.tif35165

[0369] result The results are summarized in Table 8. Tumor growth inhibition (%TGI) was determined at the end of the dosing period (t) by the formula: %TGI=(1-{Tt / T0 / Ct / C0} / 1-{C0 / Ct})×100, where Tt=median combination-treated tumor volume at time t, T0=median combination-treated tumor volume at time 0, Ct=median isotype control tumor volume at time t, and C0=median isotype-treated tumor volume at time 0 (before the start of treatment).

[0370] (Table 8) TIFF2025128164000019.tif63149

[0371] Figures 9A-F show the antitumor activity of anti-TREM2 PI-7012 or afuc-PI7012 in combination with anti-PD-1 in multiple syngeneic mouse tumor models. The anti-TREM2 mAb afuc-PI7012 in combination with anti-PD-1 mAb produced significant antitumor activity in the Panc-02 pancreatic tumor model. Figure 9A shows the mean + / - standard deviation of the average tumor volume of 10 mice in each group. Figures 9B, 9C, 9D, and 9E show the tumor volumes of individual animals in each treatment group over time. Figure 9F shows statistical analysis of group mean tumor volumes at 32 days post-implantation. Differences in tumor volume between groups were assessed using statistical analysis available in GraphPad Prism software. Study data were subjected to one-way ANOVA followed by Sidak's multiple comparison test.

[0372] As seen in Figures 9A and 9D, subcutaneous Panc-02 tumors did not respond to single-agent anti-PD-1 mAb immune checkpoint blockade therapy or anti-TREM2 mAb afuc-PI-7012 therapy alone. However, combined treatment of Panc-02 tumor-bearing animals with anti-TREM2 mAb afuc-PI-7012 and anti-PD-1 mAb resulted in significant tumor growth inhibition.

[0373] A combined myeloid conditioning treatment strategy involving the reversal of immune checkpoint-mediated CD8+ T cell depletion was tested in multiple syngeneic tumor models. As shown in Table 8, the combination of anti-TREM2 and anti-PD-1 mAbs resulted in significant tumor growth inhibition and complete regression in several of the tumor models tested. It is important to note that these syngeneic models were grown in two different mouse strain backgrounds (the typical Th-1 C57BL / 6 and Th-2 BALB / c strains), which are known to have significant differences in the composition of the immune infiltrate of tumors grown in these strains in vivo.

[0374] Example 11: Induction of long-term anti-tumor immune memory in mice in response to combined treatment with anti-TREM2 mAb and anti-PD-1 mAb Materials and Methods Tumor-free BALB / c mice from a previous study after anti-TREM2 mAb and anti-PD-1 mAb treatment as described in Example 9 received 1×10 6 The mice were re-challenged 3 months later with 100 mg of CT26 tumor cells. Tumor volumes were measured 25 days after implantation. Age-matched, treatment-naive mice were administered an equivalent number of CT26 cells and followed for tumor growth throughout the study. No additional treatment was provided during the study.

[0375] result After treatment with a combination of anti-TREM2 mAb afuc-PI-7012 and anti-PD-1 mAb, mice cured of CT26 tumors established effective anti-tumor memory responses (Figure 10). Cured mice were able to reject new tumor growth without additional treatment, indicating long-term immune memory against the original implanted tumor. This form of long-term immune memory relies on the maintenance of vigorous CD8+ effector memory responses.

[0376] Example 12: Antitumor effects of anti-TREM2 antibodies in the ID8 ovarian tumor model Materials and Methods All antibodies for in vivo use were tested for endotoxin and used at 0.2 EU / mg or less of protein. Anti-PD-1 (clone RMP1-14) was purchased from Absolute Antibody Inc. or recombinantly produced in mouse IgG1 D265A format at Pionyr. Mouse IgG1 (clone MOPC-21) and mouse IgG2a (clone C1.18.4) isotype controls were purchased from BioXCell or recombinantly produced in HEK293 cells at Pionyr. A chimeric mouse IgG2a version of the anti-TREM2 antibody (PI-7012) was produced in HEK293 cells at Pionyr and assessed for monodispersity and purity by SEC and CE-SDS, as well as endotoxin testing.

[0377] All experimental procedures involving live animals were approved by the Animal Care and Use Committee of AJES Life Sciences LLC. Six- to eight-week-old female B6(Cg)-Tyrc-2J / J or B6-albino mice were purchased from The Jackson Laboratory and used after a one-week acclimation to the animal facility. After thawing from liquid nitrogen stock, mouse ovarian surface epithelial cells overexpressing firefly luciferase, known as ID8-Luc (AJES Life Sciences LLC, Stony Brook, NY), were harvested during passages 3 to 7 and used for in vivo experiments. On the day of tumor inoculation, cells were harvested and used within 45 minutes. To establish intraperitoneal tumors, 5 × 10 6 ID8-Luc cells were injected into the right lower abdominal wall. For study recruitment, all mice were imaged for in vivo luciferase activity 20 days after injection. Based on mean luminescence readings as a surrogate for tumor burden, 40 tumor-bearing animals were recruited into the study. The mean tumor radiance (p / s / cm2 / sr) for each group was 4.7 x 10 4 Ten animals were randomly assigned to four treatment groups to achieve a tumor-bearing tumor size of 1000. Tumor-bearing animals were treated intraperitoneally with the indicated antibodies every 5 days, and luminescence images were acquired weekly. Mice were intraperitoneally injected with 0.2 mL of 15 mg / mL D-luciferin (Promega). Ten minutes after D-luciferin injection, mice were imaged using an instrument equipped with a charge-coupled device camera (IVIS, Xenogen, Alameda, CA). Data were analyzed using Living Image software (Xenogen) and presented as tumor radiance (p / s / cm2 / sr) relative to the peritoneal covering area of ​​interest.

[0378] result As shown in Figure 11, treatment with anti-TREM2 antibody alone resulted in a significant reduction in tumor burden, demonstrating the efficacy of TREM2 antibody treatment as a monotherapy. Treatment with anti-PD1 antibody resulted in a comparable reduction in tumor burden, as did the combination of anti-TREM2 and anti-PD1 antibodies.

[0379] Example 13: TREM2 expression increases with disease grade in multiple cancers Materials and Methods Get Organization Tumor microarrays (TMAs) spanning different tumor indications and disease grades were purchased from Reveal Biosciences (San Diego, CA) and included 48 duplicate patient cases or over 96 single cases as 2mm cores. TMAs at Reveal Biosciences were generated by acquiring tissue fixed in 10% neutral buffered formalin for 24 hours and processed using the same SOP. Sections were collected onto Superfrost Plus or Startfrost Adhesive slides, and all TMAs were freshly cut into 4µm sections at the time of order and stored at 4°C.

[0380] TREM2 IHC staining The IHC assay for detecting TREM2-positive cells in formalin-fixed, paraffin-embedded (FFPE) tissues was standardized for all TMA staining. First, slides were baked in a 60°C oven for 45 minutes, followed by deparaffinization with xylene three times for 5 minutes each. Next, slides were rehydrated in a gradient ethanol series from 100% to 70% ethanol and finally washed with distilled water. A decloaking pressure chamber (Biocare) was used for a heat-induced antigen retrieval step at 110°C for 15 minutes in sodium citrate buffer (Sigma, C9999), pH 6.0. To block endogenous peroxidase activity, a blocker solution (Vector Labs) was applied to the slides for 15 minutes, followed by rinsing with PBS buffer containing Tween (Alfa Aesar-J63596). Nonspecific binding was blocked by incubating the tissue sections overnight at 4°C with blocking solution containing goat serum (Vector Labs). PIT2D, also known as recombinant anti-TREM2 antibody clone EPR20243 (Abcam, ab209814), was used as the primary antibody at a concentration of 5 μg / ml in PBS for 60 minutes at room temperature. Next, slides were washed twice for 5 minutes each with PBS-Tween and then incubated with a 1:500 dilution of HRP polymer-conjugated anti-rabbit secondary antibody (Vector Labs MP-7500 Detection Kit) for 20 minutes. Slides were washed twice for 5 minutes each with PBS-Tween before proceeding to the detection step. DAB substrate was prepared according to the manufacturer's instructions (Abcam, ab64238) and applied to the slides for 3 minutes, followed by thorough rinsing with distilled water. Slides were counterstained with hematoxylin for 30 seconds, rinsed in running water, dehydrated in a gradient ethanol series from 70% to 100% ethanol, and dried in xylene. The stained slides were finally mounted in culture medium and covered with a coverslip to dry overnight.

[0381] Imaging and Scoring TMA slides were imaged on a Vectra microscope (Perkin Elmer-Akoya Biosciences) using the bright-field setting. After scanning the entire slide at 10x magnification, the quantification of TREM2+ cells was assessed using the following IHC scoring system: 0: no staining in the interstitial region within the core; 1: approximately 25% positive cells in the interstitial region within the core; 2: 50% positive cells in the interstitial region within the core; and 3: 75% positive cells in the interstitial region within the core. The percentage of positive cells between these groups was scored as 0.5 (approximately 12%), 1.5 (approximately 37.5%), 2.5 (approximately 62.5%), and 3.5 (90%). The intensity of TREM2 staining was determined as low (score 1), moderate (score 2), and strong (score 3). Cores with deformation, missing, or collapsed areas of more than half the area were excluded from the analysis and were not scored. Each core was simultaneously inspected by two operators, and the scores for each patient case (averaged across duplicate cores, if available) were plotted in a Prism file. Cores that were deformed by more than half their area, missing, or collapsed were excluded from the analysis and were not scored.

[0382] The H-score for each core was calculated as follows: % of TREM2+ cells × intensity score. For example, if a core has an IHC score of 2 (50%) and moderate staining intensity (2), the calculated H-score would be 50 × 2 = 100.

[0383] result Figure 12A shows that TREM2 expression is elevated in ovarian cancer at grades II and III using IHC staining and scoring. Figure 12B shows that TREM2 expression is elevated in ovarian cancer at grades II and III using H-score staining and scoring. 16 of 30 ovarian cancer cases (53.3%) were TREM2-positive. Figure 12C shows that TREM2 expression is elevated in gastric cancer, as well as signet ring cell carcinoma and gastric undifferentiated carcinoma at grades I, II, and III. Figure 12D shows that TREM2 expression is elevated in liver cancer and cholangiocarcinoma at grades I, II, and III. Figure 12E shows that TREM2 expression is elevated in prostate cancer at grades II and III. Figure 12F shows that TREM2 expression is elevated in ductal adenocarcinoma of pancreatic cancer. Figure 12G shows that TREM2 expression is elevated in bladder cancer at grades I to II. Figure 12H shows significant TREM2 expression in lung cancer. Figure 12I shows significant TREM2 expression in colon cancer. Figure 12J shows significant TREM2 expression in kidney cancer. Figure 12K shows increased TREM2 expression in all grades of breast cancer. Figure 12L shows TREM2 expression in TNBC cancer. Figure 12M shows significant TREM2 expression in endometrial cancer, with approximately 85% of the TMA samples tested positive across all grades. As seen in Figure 12N, lung adenocarcinoma samples expressed slightly higher levels of TREM2 compared to lung squamous cell carcinoma samples. Therefore, to improve the reliability of this study, 77 additional lung adenocarcinoma samples were tested. Figure 12O shows significant TREM2 expression in lung adenocarcinoma and additional lung cancer subtypes. Table 9 provides a summary of TREM2 expression, which is positively correlated with disease grade and negatively correlated with survival. TREM2 expression was determined using internal IHC scores for 48–150 subjects per indication.

[0384] (Table 9) TIFF2025128164000020.tif80165

[0385] Example 14: Anti-TREM2 antibodies deplete M2-like TAMs Materials and Methods Female BALB / c mice were given 1 × 10 6 Subcutaneous tumors were established as described in Example 11 by injecting EMT6 cells into mice. When the average tumor volume reached more than 200 cubic mm, mice received two doses of 30 mg / kg of isotype antibody or afuc-PI-7012, 5 days apart. Tumor volume was calculated as described above. Tumors were harvested two days after the second dose. Fat and fibrous material were removed from the tumor by excision. Tumors were then weighed and processed for single-cell suspension by a combination of mechanical and enzymatic dissociation. After mincing the tissue, tumors were enzymatically digested using an optimized enzyme cocktail (Miltenyi Biotec, Tumor Dissociation Kit, mouse 130-096-830) in gentleMACS C tubes (Miltenyi Biotec, 130-093-235) in a gentleMACS Octo Dissociator (Miltenyi Biotec, 130-095-937). After dissociation, samples were applied to filters to remove any remaining large particles. For flow cytometry, a panel of antibodies was used to surface stain single-cell suspensions of tumor tissue. The antibody panel for assessing myeloid subsets included antibodies specific for CD45, XCR1, F4 / 80, CD64, CD11c, Ly6C, CD11b, Ly6G, CD24, MHC class II, and lineage markers within the dump channel (CD45R, CD90.2, CD3e, NKp46, CD19, and Siglec F). The antibody panel for assessing lymphoid subsets included antibodies specific for CD45, CD4, CD25, B220, NKp46, CD44, CD90.2, CD8a, CD11b, and CD49b. All data were collected on an Attune flow cytometer (Thermo Fisher) and analyzed using FlowJo software.

[0386] result As shown in Figure 13A, anti-TREM2 antibody treatment resulted in a reduction in tumor size in vivo. In addition, anti-TREM2 antibody therapy significantly reduced MHCII expression compared to isotype antibody alone. low Reduces the number of TAMs and MHCII high Anti-TREM2 antibody therapy also increased the number of CD8+ T cells and NKp46+ NK cells compared to isotype antibodies alone (Figure 13C).

[0387] MHCII low These TAMs, also known as "M2-like" TAMs, have immunosuppressive activities, such as blocking the infiltration of CD8+ T cells and the expression and secretion of the immunosuppressive factors IL-10 and TGFβ. high TAMs are also known as "M1-like" TAMs. Thus, as shown in this example, treatment with a TREM2 antibody resulted in a reduction of all immunosuppressive M2-like TAMs, as well as cells associated with anti-tumor immunity, such as CD8+ T cells, NKp46+ NK cells, and M1-like MHCII TAMs. high This resulted in an increase in TAM.

[0388] Example 15: Cytokine and CD8 responses with anti-TREM2 and anti-PD1 antibodies + T cell stimulation Female BALB / c mice were given 1 × 10 6 CT26 cells were injected to establish subcutaneous tumors as described in Example 11. Tumors were approximately 1000 mm 3Once established, mice were administered two doses of isotype antibodies, anti-PD1 antibodies (5 mg / kg), afuc-PI-7012 (15 mg / kg), or anti-PD1 and anti-TREM2 antibodies, 5 days apart. Tumors were harvested two days after the second dose. Fat and fibrous material were removed from the tumors by excision. Tumors were then weighed and processed for single-cell suspension by a combination of mechanical and enzymatic dissociation. After mincing the tissue, tumors were enzymatically digested using an optimized enzyme cocktail (Miltenyi Biotec, Tumor Dissociation Kit, mouse 130-096-830) in gentleMACS C tubes (Miltenyi Biotec, 130-093-235) in a gentleMACS Octo Dissociator (Miltenyi Biotec, 130-095-937). After dissociation, the samples were gently spun to pellet the cells, and the clarified supernatant was then collected and treated with a protease inhibitor to inactivate the tumor dissociation enzyme (Thermo Fisher Scientific, Halt Protease Inhibitor Cocktail 100X, 78429). After collecting the supernatant, the pelleted sample was applied to a filter to remove any remaining large particles. For intratumoral cytokine levels, the supernatant was then analyzed for cytokine composition using the Meso Scale Discovery analyte detection platform (Meso Scale Discovery, V-PLEX Mouse Cytokine 19-plex Kit, K-15255D-1). For lymphocyte cytokine analysis, single-cell suspensions were stimulated with phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P8139), ionomycin (Thermo Fisher Scientific, I24222), and brefeldin A (BFA) (Sigma-Aldrich, B7651-5MG) for 4 hours. Stimulated cells were then surface stained for flow cytometry using a panel of antibodies, including antibodies specific for CD45, CD90.2, CD44, CD8α, CD4, and CD44.Cells were then fixed and permeabilized (Thermo Fisher Scientific, FoxP3 / Transcription Factor Staining Buffer Set, 00-5523-00) and stained with antibodies against granzyme B (BioLegend, 515406), TNF-α (BioLegend, 506328), and IFN-γ (BioLegend, 50584), as well as FOXP3 (BioLegend, 126406). Cells were analyzed by flow cytometry on an Attune flow cytometer and analyzed using FlowJo software.

[0389] As shown in Figure 14A, combined treatment with anti-TREM2 and anti-PD1 antibodies induced the production of the pro-inflammatory cytokines IFN-γ, TNF-α, and CXCL1. Treatment with anti-TREM2 antibody alone did not induce pro-inflammatory cytokines in this experiment, but the combination of TREM2 and PD1 antibodies increased cytokine production more than PD-1 antibody alone.

[0390] In addition, combined treatment with anti-TREM2 and anti-PD-1 antibodies enhanced anti-tumor CD8+ T cells in mice. As shown in Figure 14B, anti-TREM2 and anti-PD-1 antibodies alone had minimal effect on the number of CD8+ T cells expressing granzyme B (GrzB), TNF-α, or IFN-γ. However, combined treatment with both anti-TREM2 and anti-PD-1 antibodies increased the number of CD8+ T cells expressing granzyme B (GrzB), TNF-α, or IFN-γ.

[0391] Example 16: TREM2 antibodies induce CXCL10 secretion C57BL / 6 bone marrow mononuclear cells were harvested and differentiated into bone marrow-derived macrophages (BMDMs) by incubation with 25 ng / ml CSF-1 for 6 days. BMDMs were incubated for 24 hours with increasing concentrations of PI-7012, afuc-PI-7012, or an isotype control antibody. After incubation, cell supernatants were then analyzed for cytokine composition using the Meso Scale Discovery analyte detection platform (Meso Scale Discovery, V-PLEX Mouse Cytokine 19-plex Kit, K-15255D-1).

[0392] As shown in Figure 15, incubation with the anti-TREM2 antibodies PI-7012 and afucosylated-PI-7012 induced a dose-dependent increase in CXCL10 secretion by BMDMs. The afucosylated TREM2 antibody had the greatest CXCL10 response. Thus, the TREM2 antibodies induced BMDMs to secrete increased levels of the chemokine.

[0393] Example 17: TREM2 expression in gastric cancer Level 2 RNA-seq data for The Cancer Genome Atlas cohort of gastric cancer samples were downloaded from the Broad Institute using firehose_get. TREM2 RSEM values ​​from both tumor and adjacent normal samples were transformed to log2 counts per million and plotted in R.

[0394] Pre-normalized TREM2 expression profiles and associated clinical data for 192 gastric patients were downloaded from the NCBI GEO website (accession GSE15459). Expression profiles were divided into two cohorts based on median TREM2 levels. Kaplan-Meier survival curves were plotted for each cohort, and associated log-rank tests were performed using the survival and survminer packages in R.

[0395] As shown in the left panel of Figure 16, gastric cancer patients had increased expression of TREM2 compared to normal tissue samples. In addition, TREM2 expression levels in gastric cancer were inversely correlated with the patient's chances of survival (right panel of Figure 16). Thus, TREM2 expression is elevated in gastric cancer patients, and high TREM2 expression is associated with poorer survival outcomes.

[0396] Example 18: TREM2 expression in ovarian cancer Materials and Methods Normalized TREM2 expression profiles and associated clinical data for 285 ovarian patients were downloaded from the NCBI GEO website (accession GSE9899). Expression profiles were divided into three cohorts based on median, tertile, and quartile levels of TREM2 expression. Kaplan-Meier survival curves for each cohort were plotted, and associated log-rank tests were performed using the survival and survminer packages in R. The tertile cohort was the cohort with the top 33% of TREM2 expression compared with the bottom 33%, and the quartile cohort was the cohort with the top 25% of TREM2 expression compared with the bottom 25%.

[0397] Normalized TREM2 expression and clinical data from the ovarian dataset were also used to compare TREM2 expression and malignancy likelihood. Tumors were divided into two cohorts based on tumor phenotype and pathological examination for malignancy likelihood. TREM2 expression levels were compared between the two cohorts using the Wilcoxon rank-sum test implemented in the R statistical programming language.

[0398] result Figure 17A shows the median cohort survival curve, Figure 17B shows the tertile cohort survival curve, and Figure 17C shows the TREM2 low and TREM highFigure 17 shows the survival curves of quartile cohorts of patients with expression. TREM2 expression levels in ovarian cancer are inversely correlated with the likelihood of patient survival. In addition, higher TREM2 expression is associated with poorer survival outcomes, as shown by the survival probability of the median split cohort (top 50% TREM2 expression, p=0.0041, Figure 17A) compared with the tertile split cohort (top 33% TREM2 expression, p=0.0007, Figure 17B) or quartile split cohort (top 25% TREM2 expression, p=0.00049, Figure 17C).

[0399] Additionally, TREM2 expression correlated with ovarian tumor grade (Figure 18). Low TREM2 expression was associated with tumors of low malignant potential (LMP), whereas tumors with high TREM2 expression were more likely to be malignant (MAL) (p=0.0018).

[0400] Example 19: TREM2 is primarily expressed in ovarian tumor TAMs Dissociated single human ovarian patient tumor cells (DTCs) were purchased from Discovery Life Sciences. Cells were sorted by flow cytometry for CD45 positivity to enrich for immune cells and then encapsulated for single-cell RNA sequencing using the 10X Genomics Chromium controller. The resulting raw data were sequentially processed using the 10X CellRanger program and the Seurat module in the R programming language. Cell type-specific marker genes were used to manually annotate the resulting t-SNE dimensionality-reduced cell populations, and TREM2 expression was plotted. TREM2+ cells were defined as any cells with TREM2 expression.

[0401] Single-cell sequencing derived from CD45+ immune infiltrates sorted from dissociated human ovarian tumor cells showed that TREM2 is primarily expressed in tumor-associated macrophages (TAMs) (Figure 20, TREM2+ cell percentages are shown in dark gray).

[0402] Example 20: Soluble TREM2 levels in ovarian cancer Materials and Methods Plasma concentrations of soluble human TREM2 in ovarian cancer samples were determined as described below.

[0403] Human soluble TREM2 (hsTREM2) in plasma samples obtained from a commercial supplier was quantified using a sandwich immunoassay developed on the MSD platform. A biotinylated mouse monoclonal antibody (clone 7245, an in-house developed antibody) directed against human TREM2 was used as a capture agent and bound to a precoated streptavidin plate. After washing, plasma samples from ovarian cancer patients (n = 28) or non-cancer patients (n = 32) were added to the plate. Soluble TREM2 protein in the samples bound to the capture antibody and was detected with a sulfo-tagged mouse monoclonal antibody (clone 7222, an in-house developed antibody) directed against hTREM2. Bound soluble TREM2 was then quantified by electrochemiluminescence (ECL).

[0404] MSD's hsTREM2 assay has a dynamic range of 30 ng / mL to 41 pg / mL. The LLOQ and LOD of this assay are 41 pg / mL and 2.4 pg / mL, respectively. This hsTREM2 assay can detect circulating TREM2 protein in plasma samples.

[0405] result Elevated levels of soluble human TREM2 were detected in ovarian cancer plasma samples. Figure 19 shows a comparison of the amount of soluble TREM2 (ng / ml) in ovarian plasma samples and normal non-cancer plasma samples. Ovarian cancer patients had more soluble TREM2 in their serum. The mean TREM2 plasma concentration in ovarian samples was 11.11 ng / ml, while the mean TREM2 plasma concentration in non-cancer samples was 4.8 ng / ml (p=<0.0001).

[0406] While the present invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will recognize that various changes in form and detail can be made herein without departing from the spirit and scope of the invention.

[0407] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0408] References TIFF2025128164000021.tif78162

[0409] array TIFF2025128164000022.tif209170TIFF2025128164000023.tif225170TIFF2025128164000024.tif229170TIFF2025128164000025.tif149170

[0410] Sequence information SEQUENCE LISTING <110> PIONYR IMMUNOTHERAPEUTICS, INC. <120> METHODS OF USING ANTI-TREM2 ANTIBODIES <150> US 62 / 889,990 <151> 2019-08-21 <150> PCT / US2018 / 065026 <151> 2018-12-11 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Asn Arg Phe Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Ile Tyr Asn Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Tyr Thr Ser Asn Arg Phe Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Ile Tyr Asn Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Asn Arg Phe Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Ile Tyr Asn Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Asn Arg Phe Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Ile Tyr Asn Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Phe Ser Asn Tyr Tyr Met Ala 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Glu Trp Ala Gly Ser Gly Tyr 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Asn Val Gly Asn Asn Leu Ala 1 5 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Tyr Thr Ser Asn Arg Phe Thr 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Arg Ile Tyr Asn Ser Pro Trp 1 5 <210> 15 <211> 230 <212> PRT <213> Homo sapiens <400> 15 Met Glu Pro Leu Arg Leu Leu Ile Leu Leu Phe Val Thr Glu Leu Ser 1 5 10 15 Gly Ala His Asn Thr Thr Val Phe Gln Gly Val Ala Gly Gln Ser Leu 20 25 30 Gln Val Ser Cys Pro Tyr Asp Ser Met Lys His Trp Gly Arg Arg Lys 35 40 45 Ala Trp Cys Arg Gln Leu Gly Glu Lys Gly Pro Cys Gln Arg Val Val 50 55 60 Ser Thr His Asn Leu Trp Leu Leu Ser Phe Leu Arg Arg Trp Asn Gly 65 70 75 80 Ser Thr Ala Ile Thr Asp Asp Thr Leu Gly Gly Thr Leu Thr Ile Thr 85 90 95 Leu Arg Asn Leu Gln Pro His Asp Ala Gly Leu Tyr Gln Cys Gln Ser 100 105 110 Leu His Gly Ser Glu Ala Asp Thr Leu Arg Lys Val Leu Val Glu Val 115 120 125 Leu Ala Asp Pro Leu Asp His Arg Asp Ala Gly Asp Leu Trp Phe Pro 130 135 140 Gly Glu Ser Glu Ser Phe Glu Asp Ala His Val Glu His Ser Ile Ser 145 150 155 160 Arg Ser Leu Leu Glu Gly Glu Ile Pro Phe Pro Pro Thr Ser Ile Leu 165 170 175 Leu Leu Leu Ala Cys Ile Phe Leu Ile Lys Ile Leu Ala Ala Ser Ala 180 185 190 Leu Trp Ala Ala Ala Trp His Gly Gln Lys Pro Gly Thr His Pro Pro 195 200 205 Ser Glu Leu Asp Cys Gly His Asp Pro Gly Tyr Gln Leu Gln Thr Leu 210 215 220 Pro Gly Leu Arg Asp Thr 225 230 <210> 16 <211> 693 <212> Ms <213> Homo sapiens <400> 16 atggagcctc tccggctgct catcttactc tttgtcacag agctgtccgg agcccacaac accacagtgt tccagggcgt ggcgggccag tccctgcagg tgtcttgccc ctatgactcc 120 atgaagcact gggggaggcg caaggcctgg tgccgccagc tgggagaga gggcccatgc 180 cagcgtgtgg tcagcacgca caacttgtgg ctgctgtcct tcctgaggag gtggaatggg 240 agcacagcca tcacagacga taccctgggt ggcactctca ccattacgct gcggaatcta caaccccatg atgcgggtct ctaccagtgc cagagcctcc atggcagtga ggctgacacc 360 ctcaggaagg tcctggtgga ggtgctggca gaccccctgg atcaccgggga tgctggagat 420 ctctggttcc ccggggagtc tgagagcttc gaggatgccc atgtggagca cagcatctcc 480 aggagcctct tggag aatccccttc ccccctt cctccttct cctcctggcc 540 tgcatctttc tcatcaagat tctagcagcc agcgccctct gggctgcagc ctggcatgga cagaagccag ggacacatcc acccagtgaa ctggactgtg gccatgaccc agggtatcag ctccaaactc tgccagggct gagagacacg tga <210> 17 <211> 227 <212> PRT <213> Mus musculus <400> 17 Met Gly Pro Leu His Gln Phe Leu Leu Leu Leu Ile Thr Ala Leu Ser 1 5 10 15 Gln Ala Leu Asn Thr Thr Val Leu Gln Gly Met Ala Gly Gln Ser Leu 20 25 30 Arg Val Ser Cys Thr Tyr Asp Ala Leu Lys His Trp Gly Arg Arg Lys 35 40 45 Ala Trp Cys Arg Gln Leu Gly Glu Glu Gly Pro Cys Gln Arg Val Val 50 55 60 Ser Thr His Gly Val Trp Leu Leu Ala Phe Leu Lys Lys Arg Asn Gly 65 70 75 80 Ser Thr Val Ile Ala Asp Asp Thr Leu Ala Gly Thr Val Thr Ile Thr 85 90 95 Leu Lys Asn Leu Gln Ala Gly Asp Ala Gly Leu Tyr Gln Cys Gln Ser 100 105 110 Leu Arg Gly Arg Glu Ala Glu Val Leu Gln Lys Val Leu Val Glu Val 115 120 125 Leu Glu Asp Pro Leu Asp Asp Gln Asp Ala Gly Asp Leu Trp Val Pro 130 135 140 Glu Glu Ser Ser Ser Phe Glu Gly Ala Gln Val Glu His Ser Thr Ser 145 150 155 160 Arg Asn Gln Glu Thr Ser Phe Pro Pro Thr Ser Ile Leu Leu Leu Leu 165 170 175 Ala Cys Val Leu Leu Ser Lys Phe Leu Ala Ala Ser Ile Leu Trp Ala 180 185 190 Val Ala Arg Gly Arg Gln Lys Pro Gly Thr Pro Val Val Arg Gly Leu 195 200 205 Asp Cys Gly Gln Asp Ala Gly His Gln Leu Gln Ile Leu Thr Gly Pro 210 215 220 Gly Gly Thr 225 <210> 18 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 19 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 20 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Leu Thr Asn Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Trp Ala Gly Ser Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 21 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ...

Claims

1. 1. A pharmaceutical composition for treating ovarian cancer in a subject in need thereof, comprising an isolated antibody that specifically binds to TREM2, the antibody comprising a variable heavy domain (VH) sequence comprising CDR-H1, CDR-H2, and CDR-H3, and a variable light domain (VL) sequence comprising CDR-L1, CDR-L2, and CDR-L3; a. the CDR-H1 comprises the sequence set forth in SEQ ID NO: 39; b. the CDR-H2 comprises the sequence set forth in SEQ ID NO: 40; c. the CDR-H3 comprises the sequence set forth in SEQ ID NO:41; d. the CDR-L1 comprises the sequence set forth in SEQ ID NO:42; e. the CDR-L2 comprises the sequence set forth in SEQ ID NO: 43; and f. the CDR-L3 comprises the sequence set forth in SEQ ID NO:44 Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the antibody comprises a VH sequence set forth in SEQ ID NO: 1, 3, or 5.

3. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5; and a VL sequence shown in SEQ ID NO: 2, 4, or 6.

4. The pharmaceutical composition of claim 1 , wherein the antibody comprises the VH sequence shown in SEQ ID NO:

1.

5. The pharmaceutical composition of claim 1 , wherein the antibody comprises the VL sequence shown in SEQ ID NO:

2.

6. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO:

2.

7. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises a heavy chain sequence set forth in SEQ ID NO:25 and a light chain sequence set forth in SEQ ID NO:

26.

8. The antibody has a 1, 2, 3, 4, 5, or 5.12 x 10 －９ K below M Ｄ The pharmaceutical composition of claim 1, which binds to human TREM2 at the

9. 10. The pharmaceutical composition of claim 1, wherein the antibody is capable of specifically killing, depleting, or neutralizing TREM2+ myeloid cells.

10. 2. The pharmaceutical composition of claim 1, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-mediated cellular phagocytosis (ADCP) activity, or complement-dependent cytotoxicity (CDC) activity.

11. The pharmaceutical composition of claim 1 , wherein the antibody is a monoclonal antibody.

12. The pharmaceutical composition of claim 1 , wherein the antibody is afucosylated.

13. The pharmaceutical composition of claim 1 , wherein the antibody comprises a heavy chain constant region of human IgG1.

14. 10. The pharmaceutical composition of claim 1, wherein the subject has previously undergone, is concurrently undergoing, or will subsequently undergo immunotherapy.

15. 15. The pharmaceutical composition of claim 14, wherein the immunotherapy is a therapy with a therapeutic agent comprising at least one of an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; atezolizumab; avelumab; durvalumab; nivolumab; pembrolizumab; cemiplimab; ipilimumab; or tremelimumab.

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