Methods of using anti-trem2 antibodies

Abstract

Provided herein are anti-TREM2 antibodies and related methods of making and using anti-TREM2 antibodies. Also provided are methods and compositions for enhancing an immune response and / or treating an immune-related disorder, e.g., cancer, in a subject using an anti-TREM2 antibody or antigen-binding fragment thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits of international application PCT / US2018 / 065026, filed December 11, 2018, and U.S. provisional application 62 / 889,990, filed August 21, 2019, both of which are incorporated herein by reference in their entirety for all purposes.

[0003] sequence list

[0004] This application contains a sequence list of documents submitted via EFS and incorporated herein by reference in their entirety. The ASCII copy was created on December 19, 2019, named PII-011WO_SL.txt, and is 65,993 bytes in size. Background Technology

[0005] Immunity plays a role in preventing extratumor growth. Complex microenvironments can arise within lesions, and despite T cell recruitment, effective control over developing masses is often lacking. Understanding the balance between tumor elimination and tumor escape depends on understanding the differential roles played by myeloid cells within the tumor microenvironment.

[0006] The myeloid population in the tumor microenvironment mainly includes monocytes and neutrophils (sometimes loosely grouped as myeloid-derived suppressor cells), macrophages, and dendritic cells. Although the myeloid population within a tumor has long been considered as a whole to be non-stimulatory or suppressive, it has more recently become clear that not all tumor-infiltrating myeloid cells are equivalent.

[0007] In normal tissues, many of these myeloid cells are essential for both innate and adaptive immunity to function properly, and in particular for wound repair. However, in the context of cancer, macrophages are often described in significant excess and the populations of these and other cell types are in a functionally abnormal or skewed state. "Macrophage" infiltration, when considered as a collective population defined by a single marker such as CD68 or CD163, is associated with worse outcomes in subjects across 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)). But the phenotypic and functional sub- environment of macrophages from the tumor microenvironment is complicated by the similarity of macrophages and dendritic cells, and is problematic in tumor biology. Morphological criteria have often been applied to the problem; one method to attempt to distinguish dendritic cells from macrophages is based on the more spiny or dendritic morphology of dendritic cells, while macrophages are more cryptic or globular in morphology (Bell et al., J Exp Med 555, 1999; 190: 1417-26). Other groups are attempting to distinguish based on genetic and cell surface markers.

[0008] There is diversity in the antigen presenting compartments within tumors, and T cells can distinguish the characteristics of antigen presenting cells (APCs). Because T cells are the main drivers of tumor immunity, it would be important to understand the exact characteristics of their cognate APCs. Among the cells that are able to present tumor-derived antigens to T cells, and thereby maintain T cells in an activated state, myeloid cells are prominent. Antigen presentation occurs within the tumor itself, and can influence the function of tumor cytotoxic T lymphocytes (CTLs). T cell activation by antigen presenting cells (APCs) is an important component in antigen-specific immune responses and tumor cell killing. Because these myeloid populations represent the main T cell interaction partners and antigen presenting cells to arrive at the ensuing tumor reactive cytotoxic T lymphocytes, understanding their differences can lead to therapeutic approaches.

[0009] Related patent applications include: PCT / US2015 / 052682, filed September 28, 2015; PCT / US2016 / 054104, filed September 28, 2016; and PCT / US2018 / 065026, filed December 11, 2018; each of which is incorporated by reference herein in their entirety for all purposes.

[0010] All patents, patent applications, publications, files and articles cited herein are incorporated by reference in their entirety. SUMMARY

[0011] In one aspect, provided herein is a method of 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 for binding to mouse TREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31 and 32). In one aspect, provided herein is a method of treating solid 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 for binding to mouse TREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31 and 32).

[0012] In some embodiments, the antibody contains 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 shown in SEQ ID NO: 1; a VL sequence shown in SEQ ID NO: 2; and an active human IgGl 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 a sequence shown in SEQ ID NO: 1. In some embodiments, the antibody comprises a sequence shown in SEQ ID NO: 2. In some embodiments, the antibody comprises a sequence shown in SEQ ID NO: 1 and a sequence shown in SEQ ID NO: 2.

[0013] In some embodiments, the antibody contains 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. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5. In some embodiments, 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. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1.

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

[0016] In another aspect, provided herein is a method of treating a solid cancer in a subject in need thereof, 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. In another aspect, provided herein is a method of treating an 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), 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.

[0017] In another aspect, provided herein are methods of treating a solid cancer in a subject in need thereof, 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 are methods of treating an ovarian cancer in a subject in need thereof, 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.

[0018] In some embodiments, the antibody contains 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. In some embodiments, the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3, or 5. In some embodiments, the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3, or 5 and the VL sequence shown in SEQ ID NO: 2, 4, or 6. In some embodiments, the antibody comprises the VH sequence shown in SEQ ID NO: 1. The method of claim 16, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown in SEQ ID NO: 2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises the heavy chain sequence shown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO: 26.

[0019] In some embodiments, the antibody binds to human TREM2 with a KD of less than or equal to about 1, 2, 3, 4, or 5 x 10 -9 K of M D binds to human TREM2 as measured by surface plasmon resonance (SPR) assay.

[0020] In some embodiments, the antibody is capable of specifically killing, depleting, or disabling a TREM2+ myeloid cell; optionally a non-stimulatory myeloid cell; optionally an intratumoral myeloid cell.

[0021] 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.

[0022] In some embodiments, the antibody is at least one of a monoclonal antibody, a neutralizing antibody, an antagonistic antibody, an agonistic antibody, a polyclonal antibody, an IgGl antibody, an IgG3 antibody, a 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.

[0023] In some embodiments, the antibody is an antigen-binding fragment, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecule, dual variable domain antibody, single variable domain antibody, linear antibody, or V domain antibody thereof.

[0024] In some embodiments, the antibody comprises a framework, optionally wherein the framework is an Fc, optionally a human Fc. In some embodiments, the antibody comprises a heavy chain constant region selected from the class of 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 IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody comprises a heavy chain constant region of IgGl.

[0025] In some embodiments, the Fc comprises one or more modifications, wherein the one or more modifications results in an increase in half-life, an increase in ADCC activity, an increase in ADCP activity, or an increase in CDC activity compared to the Fc without the one or more modifications. In some embodiments, the Fc binds an Fc gamma receptor selected from the group consisting of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.

[0026] In some embodiments, the antibody binds to an extracellular domain of TREM2 on a TREM2+ myeloid cell, optionally wherein the myeloid cell is intratumoral.

[0027] In some embodiments, the antibody binds to an extracellular domain of TREM2 on a myeloid cell, wherein the myeloid cell is a CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3- non-stimulatory myeloid cell, wherein the antibody kills, incapacitates, or depletes the non-stimulatory myeloid cell by ADCC, CDC, and / or ADCP to a level less than the level of non-stimulatory myeloid cells present in a cancer prior to contacting the non-stimulatory myeloid cell with the antibody, wherein the non-stimulatory myeloid cell is present in an immune cell population comprising CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+ stimulatory myeloid cells and the non-stimulatory myeloid cell, and wherein killing, incapacitating, or depleting the non-stimulatory myeloid cell treats the cancer.

[0028] In some embodiments, the contacting 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 subject has previously received, is concurrently receiving, or will subsequently receive an immunotherapy.

[0029] In some embodiments, the immunotherapy is at least one of: a checkpoint inhibitor; a checkpoint inhibitor of T cells; 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; a chemotherapy; a 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.

[0030] In another aspect, provided herein is a method of treating a 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 for binding to mouse TREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31 and 32).

[0031] 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 a VH sequence shown in SEQ ID NO: 1; a VL sequence shown in SEQ ID NO: 2; and an active human IgGl Fc region.

[0032] In some embodiments, the antibody comprises 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. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5. In some embodiments, 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. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1.

[0033] In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26.

[0034] In another aspect, provided herein is a method of treating a 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.

[0035] In another aspect, provided herein are methods of treating a gastric cancer in a subject in need thereof, 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.

[0036] In some embodiments, the antibody contains 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. In some embodiments, the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3, or 5. In some embodiments, the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3, or 5 and the VL sequence shown in SEQ ID NO: 2, 4, or 6. In some embodiments, the antibody comprises the VH sequence shown in SEQ ID NO: 1. The method of claim 16, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown in SEQ ID NO: 2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises the heavy chain sequence shown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO: 26.

[0037] In some embodiments, the antibody is capable of specific killing, depletion, or disabling of TREM2+ myeloid cells; optionally non-stimulatory myeloid cells; optionally intratumoral myeloid cells, at a concentration of less than or equal to about 1, 2, 3, 4, or 5 x 10 -9 K of M D binds human TREM2, as measured by surface plasmon resonance (SPR) assay.

[0038] In some embodiments, the antibody is capable of specific killing, depletion, or disabling of TREM2+ myeloid cells; optionally non-stimulatory myeloid cells; optionally intratumoral myeloid cells, at a concentration of less than or equal to about 1, 2, 3, 4, or 5 x 10

[0039] 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.

[0040] In some embodiments, the antibody is at least one of a monoclonal antibody, a neutralizing antibody, an antagonistic antibody, an agonistic antibody, a polyclonal antibody, an IgGl antibody, an IgG3 antibody, a 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.

[0041] In some embodiments, the antibody is an antigen-binding fragment thereof, a Fab, a Fab', a F(ab')2, a Fv, a scFv, a (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.

[0042] In some embodiments, the antibody comprises a backbone, optionally wherein the backbone is an Fc, optionally a human Fc. In some embodiments, the antibody comprises a heavy chain constant region selected from the class of 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 IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody comprises a heavy chain constant region of IgGl.

[0043] In some embodiments, the Fc comprises one or more modifications, wherein the one or more modifications results in an increase in half-life, an increase in ADCC activity, an increase in ADCP activity, or an increase in CDC activity compared to an Fc not having the one or more modifications. In some embodiments, the Fc binds an Fc gamma receptor selected from the group consisting of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.

[0044] In some embodiments, the antibody binds to an extracellular domain of TREM2 on a TREM2+ myeloid cell, optionally wherein the myeloid cell is within a tumor.

[0045] In some embodiments, the antibody binds to an extracellular domain of TREM2 on a myeloid cell, wherein the myeloid cell is a CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3- non-stimulatory myeloid cell, wherein the antibody kills, incapacitates, or depletes the non-stimulatory myeloid cell by ADCC, CDC, and / or ADCP to a level less than the level of non-stimulatory myeloid cells present in a cancer prior to contacting the non-stimulatory myeloid cell with the antibody, wherein the non-stimulatory myeloid cell is present in an immune cell population comprising CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+ stimulatory myeloid cells and the non-stimulatory myeloid cell, and wherein killing, incapacitating, or depleting the non-stimulatory myeloid cell treats the cancer.

[0046] In some embodiments, the contacting 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 subject has previously received, is concurrently receiving, or will subsequently receive an immunotherapy.

[0047] In some embodiments, the immunotherapy is at least one of: a checkpoint inhibitor; a checkpoint inhibitor of T cells; 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; a chemotherapy; a 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.

[0048] In another aspect, provided herein are methods of enhancing an immune response, the method comprising administering to a subject an isolated humanized antibody that binds to human TREM2 (SEQ ID NO: 15) and competes for binding to mouse TREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31 and 32). 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.

[0049] In some embodiments, the antibody comprises: CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or 39, CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or 40, CDR-H3 comprising the sequence set forth in SEQ ID NO: 11 or 41, CDR-L1 comprising the sequence set forth in SEQ ID NO: 12 or 42, CDR-L2 comprising the sequence set forth in SEQ ID NO: 13 or 43, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 14 or 44.

[0050] In some embodiments, the 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 IgGl Fc region. In some embodiments, the antibody contains 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.

[0051] In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5. In some embodiments, 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. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2. In some embodiments, the antibody is the 37012 antibody. In some embodiments, the antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26.

[0052] In another aspect, provided herein are methods 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.

[0053] In another aspect, provided herein are methods of enhancing an immune response, the method comprising administering to a 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 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.

[0054] In some embodiments, the antibody contains 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.

[0055] In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1, 3, or 5. In some embodiments, 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. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1. In some embodiments, the antibody comprises a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2.

[0056] In some embodiments, the antibody is a 37012 antibody. In some embodiments, the antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 26. In some embodiments, the antibody binds human TREM2 with a Kd of less than or equal to about 1, 2, 3, 4, or 5 x 10 -9 K of M D binds human TREM2, as measured by surface plasmon resonance (SPR) assay.

[0057] In some embodiments, the subject has ovarian cancer. In some embodiments, the subject has gastric cancer.

[0058] In some embodiments, the subject has previously received, is concurrently receiving, or will subsequently receive an immunotherapy. In some embodiments, the immunotherapy is at least one of: a checkpoint inhibitor; a checkpoint inhibitor of T cells; 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; a chemotherapy; a 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.

[0059] In some embodiments, the antibody is capable of specifically killing, depleting, or disabling a TREM2+ myeloid cell; optionally a non-stimulatory myeloid cell; optionally an intratumoral myeloid cell.

[0060] 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.

[0061] In some embodiments, the antibody has receptor-ligand blocking, agonizing, or antagonizing activity. In some embodiments, the antibody has agonizing activity.

[0062] In some embodiments, the antibody induces an increase in expression of at least one cytokine or chemokine in a cell, as compared to an isotype control antibody. In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of: IFN-g, TNF-a, CXCL1, or CXCL10. In some embodiments, the cytokine or chemokine is CXCL10.

[0063] 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.

[0064] 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.

[0065] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1A Anti-tumor activity mediated by anti-TREM2 PI-7012 in combination with anti-PD-1 antibody is shown in a CT-26 syngeneic mouse tumor model. The anti-tumor activity is improved with afucosylation of PI-7012 in combination with anti-PD-1 antibody. Shown is the mean tumor volume (10 mice / group). FIG. 1B Individual tumor volumes are shown for PI-7012. FIG. 1C Individual tumor volumes are shown for afucosylated PI-7012 (afuc-PI-7012).

[0067] FIG. 2 No significant weight loss with combination treatment. Ten mice in each group were treated with the indicated antibodies and body weight was recorded at frequent intervals. The mean body weight of each group is plotted against the study days.

[0068] FIG. 3 In addition to H&E staining, macrophages of the tissue were stained using an anti-CD68 antibody. The intracellular marker CD68 has been used in the literature as a reliable cytochemical marker for immunostaining of monocytes / macrophages in inflamed tissues and tumors. In the lung (panel E), as well as in the other tissues analyzed, no discernible change in the number of CD68+ macrophages was observed in any of the treatment groups compared to control, indicating that the depletion mediated by anti-TREM2 specifically occurs in the TME.

[0069] FIG. 4A Anti-CD68 staining of FFPE lung tissue from the indicated treatment groups is shown. FIG. 4B Eight to nine fields per section were used for quantification by light microscopy.

[0070] FIG. 5A TREM2 expression is either absent or very low on cells in the selected tissues is shown. FIG. 5B TREM2 expression is either absent or very low on cells in the selected tissues is shown. The hatched histograms are from TREM2 KO and the open histograms are from wild-type mice. The antibody used for anti-TREM2 staining is clone 237920 from R&D Systems.

[0071] FIG. 6 Cell surface expression of TREM2 (open histograms) is significantly higher on TAMs compared to granulocytic or monocytic MDSCs within both MC38 and CT26 tumors. Lymphocytes do not express TREM2. Isotype control staining is shown in gray-filled histograms.

[0072] FIG. 7Cell surface expression of TREM2 (open histograms) was significantly higher on CD14-derived macrophages compared to any PBMC subset. Human PBMCs or macrophages were surface stained for TREM2 (open histograms) or isotype control (gray histograms). PBMC subsets were distinguished as neutrophils, monocytes, or T cells using a pre- validated, multi-color FACS panel.

[0073] FIG. 8 Cell surface expression of TREM2 (open histograms) was significantly higher on TAMs compared to other infiltrates or non-CD45 positive cells. Single cell suspensions from human tumor tissue were surface stained for TREM2 (open histograms) or isotype control (gray histograms). Immune and non-immune subsets were distinguished using a pre- validated, multi-color FACS panel.

[0074] FIG. 9A Anti-TREM2 mAb afuc-PI7012 in combination with anti-PD-1 mAb was shown to result in significant anti-tumor activity in a Panc-02 pancreatic tumor model. Tumor volume in female C57BL / 6J mice implanted with Panc-02 tumor cells and treated with the indicated mAbs was tracked over time. The Y-axis represents the mean + / - standard deviation of the mean tumor volume in 10 mice per group. FIG. 9B Tumor volume from individual animals treated with isotype control mAb is shown. FIG. 9C Tumor volume from individual animals treated with anti-TREM2 mAb afuc-PI7012 is shown. FIG. 9D Tumor volume from individual animals treated with anti-PD-1 antibody is shown. FIG. 9E Tumor volume from individual animals treated with anti-TREM2 mAb afuc-PI7012 and anti-PD-1 antibody is shown. FIG. 9F Statistical analysis of group mean tumor volume on day 32 post-implantation for each treatment group is shown.

[0075] FIG. 10 Tumor-free BALB / c mice following anti-TREM2 mAb plus anti-PD-1 mAb treatment were re-challenged with CT26 tumor cells three months later (square symbols). Age-matched treatment-naive mice (circle symbols) received an equivalent number of CT26 cells and tumor growth was tracked during the study period. Mice were not provided additional treatment during the study period.

[0076] FIG. 11Anti-TREM2 mAb afuc-PI7012 in combination with anti-PD-1 mAb was shown to result in significant anti-tumor activity in the ID8 ovarian tumor model. Luminescence of tumor cells 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 mean tumor luminescence of 10 mice in each group.

[0077] FIG. 12A TREM2 expression in ovarian cancer was shown. FIG. 12B TREM2 expression in ovarian cancer was shown. FIG. 12C TREM2 expression in gastric cancer was shown. FIG. 12D TREM2 expression in liver cancer was shown. FIG. 12E TREM2 expression in prostate cancer was shown. FIG. 12F TREM2 expression in pancreatic cancer was shown. FIG. 12G TREM2 expression in bladder cancer was shown. FIG. 12H TREM2 expression in lung cancer was shown. FIG. 12I TREM2 expression in colon cancer was shown. FIG. 12J TREM2 expression in kidney cancer was shown. FIG. 12K TREM2 expression in breast cancer was shown. FIG. 12L TREM2 expression in TNBC cancer was shown. FIG. 12M TREM2 expression in melanoma was shown. FIG. 12N TREM2 expression in endometrial cancer was shown. FIG. 12O TREM2 expression in lung adenocarcinoma was shown.

[0078] FIG. 13A Anti-TREM2 antibody treatment was shown to result in reduced tumor size in vivo in the EMT6 model. FIG. 13B Anti-TREM2 antibody therapy was shown to reduce the number of MHCII 低 TAMs and increase the number of MHCII 高 TAMs compared to isotype antibody alone. FIG. 13C Anti-TREM2 antibody therapy was shown to increase the number of CD8+ T cells and NKp46+ NK cells compared to isotype antibody alone.

[0079] FIG. 14A Production of pro-inflammatory cytokines IFN-g, TNF-a, and CXCL1 was shown following treatment with anti-TREM2 antibody and PD1 antibody. FIG. 14B The number of CD8+ T cells expressing granzyme B (GrzB), TNF-a, or IFN-g was shown to increase following treatment with both anti-TREM2 antibody and anti-PD-1 antibody.

[0080] FIG. 15 Dose-dependent increase in CXCL10 secretion by BMDM shown after incubation with anti-TREM2 antibodies.

[0081] FIG. 16 TREM2 expression levels in gastric cancer shown to inversely correlate with patient survival probability.

[0082] FIG. 17A TREM2 expression levels in ovarian cancer shown to inversely correlate with patient survival probability. FIG. 17B TREM2 expression levels in ovarian cancer shown to inversely correlate with patient survival probability. FIG. 17C TREM2 expression levels in ovarian cancer shown to inversely correlate with patient survival probability.

[0083] FIG. 18 TREM2 expression levels in ovarian cancer shown to inversely correlate with tumor malignancy.

[0084] FIG. 19 Comparison of the amount of soluble TREM2 (ng / ml) in ovarian cancer plasma samples and normal non-cancer plasma samples shown.

[0085] FIG. 20 TREM2 shown to be primarily expressed on tumor-associated macrophages (TAMs). Bars indicate % of CD45+ immune infiltrate cell types isolated from ovarian tumors. Inserted dark gray bars indicate percentage of TREM2+ cells in each cell subset. TREM2 positive cells are predominantly found in TAM and macrophage populations. DETAILED DESCRIPTION

[0086] DEFINITIONS

[0087] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural; and vice versa. In the event that any definition provided in the following contradicts any document incorporated by reference herein, the definition provided above shall control.

[0088] It should be appreciated that aspects and embodiments of the application described herein include those "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0089] For all compositions described herein, and for all methods using the compositions described herein, the compositions can comprise the recited components or steps, or can "consist essentially of the recited components or steps. When a composition is described as "consisting essentially of the recited components, the composition contains the recited components, and can contain other components that do not materially affect the treated condition, but does not contain any additional components that materially affect the treated condition other than those explicitly recited; or if the composition does contain additional components that materially affect the treated condition other than those recited, the composition does not contain said additional components in a concentration or in an amount sufficient to materially affect the treated condition. When a method is described as "consisting essentially of the recited steps, the method contains the recited steps, and can contain other steps that do not materially affect the treated condition, but the method does not contain any additional steps that materially affect the treated condition other than those explicitly recited. As a non-limiting specific example, when a composition is described as "consisting essentially of a certain component, the composition can additionally contain any number of pharmaceutically acceptable carriers, vehicles, or diluents, and other such components that do not materially affect the treated condition.

[0090] The term "optionally," when used in conjunction with the term "comprising," is intended to mean that one or more of the listed components are optionally present, and that the entire list of components is contemplated, along with every subset thereof.

[0091] "Effective amount" or "therapeutically effective amount" as used herein refers to the amount of a therapeutic compound, such as an anti-TREM2 antigen binding agent or an anti-TREM2 antibody, administered to a subject, either as a single dose or as part of a series of doses, alone or in combination with another therapeutic modality, that is effective in producing or promoting the desired therapeutic effect. Examples of desired therapeutic effects are enhancing an immune response, slowing or delaying tumor development; stabilizing the disease; improving one or more symptoms. An effective amount can be given in one or more doses.

[0092] The term "treat" as used herein refers to delaying or reversing the progression of a condition, such as cancer. The term "treat" as used herein refers to the act of treating a condition, such as cancer.

[0093] "Individual" or "subject" as used herein 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, guinea pigs, rats, cats, etc. In some embodiments, the individual is a human. In some embodiments, the individual is a mouse.

[0094] The term "modulate" or "modulation" refers to decreasing or inhibiting, or alternatively, activating or increasing, the recited variable.

[0095] The terms "increase" and "activation" mean an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, an increase to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more in the stated variable.

[0096] The terms "decrease" and "inhibition" mean a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, a decrease to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or more in the stated variable.

[0097] The term "agonize" means to activate receptor signaling to induce a biological response associated with activation of the receptor. An "agonist" is an entity that binds to a receptor and agonizes the receptor.

[0098] The term "antagonize" means to inhibit receptor signaling to inhibit a biological response associated with activation of the receptor. An "antagonist" is an entity that binds to a receptor and antagonizes the receptor.

[0099] The term "about" as used herein means the usual error range of a person skilled in the art for the respective value. An exemplary error range is plus or minus 5%. Reference herein to "about" a value or parameter includes (and describes) embodiments with that value or parameter per se.

[0100] It must be noted that, as used herein, the singular articles "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0101] Methods for determining any of the structural and functional features described herein are known in the art.

[0102] Antibodies

[0103] Structures

[0104] The present application provides antibodies and compositions comprising antibodies that bind to TREM2 proteins, including antibodies that incapacitate non-stimulatory myeloid cells.

[0105] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that comprise 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 multi-specific antibodies.

[0106] 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 its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g and m, respectively.

[0107] An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids that is 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 IgGl heavy chain comprises, from N- to C-terminus, a VH, a CHI, a CH2 and a CH3 domain. A light chain comprises, from N- to C-terminus, a VL and a CL domain. An IgGl heavy chain comprises a hinge between the CHI and CH2 domains. In certain embodiments, an immunoglobulin construct comprises at least one immunoglobulin domain from an IgG, IgM, IgA, IgD or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are from or derived from an immunoglobulin-based construct such as a minibibody or nanobody. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody or any chimeric antibody.

[0108] In some embodiments, the antibodies provided herein comprise a heavy chain. In one embodiment, the heavy chain is an IgA heavy chain. In one embodiment, the heavy chain is an IgD heavy chain. In one embodiment, the heavy chain is an IgE heavy chain. In one embodiment, the heavy chain is an IgG heavy chain. In one embodiment, the heavy chain is an IgM heavy chain. In one embodiment, the heavy chain is an IgGl heavy chain. In one embodiment, the heavy chain is an IgG2 heavy chain. In one embodiment, the heavy chain is an IgG3 heavy chain. In one embodiment, the heavy chain is an IgG4 heavy chain. In one embodiment, the heavy chain is an IgAl heavy chain. In one embodiment, the heavy chain is an IgA2 heavy chain.

[0109] The term "hypervariable region" or "HVR" as used herein refers to each of the regions in an antibody variable domain which are highly variable in sequence and / or form structurally defined loops ("hypervariable loops") "hypervariable loop"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). HVRs typically comprise amino acid residues from the hypervariable loops and / or from the Complementarity Determining Regions (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. Except for CDR1 in the VH, the CDRs typically 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 domains that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), with the definitions including overlapping or subsets of amino acid residues when compared to each other. However, application of either definition to refer to the CDRs of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact number of residues encompassed by a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can determine in a routine manner which residues comprise a particular CDR in light of the variable region amino acid sequence of the antibody.

[0110] The amino acid sequence boundaries of CDRs can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al. (supra) ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme); each of which is incorporated by reference herein in its entirety.

[0111] Table A provides the location of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat scheme and the Chothia scheme. For CDR-H1, residue numbering using both the Kabat numbering scheme and the Chothia numbering scheme is provided.

[0112] CDRs can be assigned, for example, using antibody numbering software such as Abnum, available at www.bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated herein by reference in its entirety.

[0113] Table A. Residues in CDRs according to the Kabat numbering scheme and the Chothia numbering scheme.

[0114]

[0115]

[0116] The C-terminal end of CDR-H1 varies between H32 and H34, depending on the length of the CDR, when numbered using the Kabat numbering convention.

[0117] When referring to residues in an antibody heavy chain constant region, the "EU numbering scheme" is generally used (e.g., as reported in Kabat et al. (supra)). The EU numbering scheme is used to refer to residues in the antibody heavy chain constant regions described herein, unless otherwise stated.

[0118] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In a particular described embodiment, in a single chain Fab molecule, the C-terminal end of the Fab light chain is linked to the N-terminal end of the Fab heavy chain. As described in more detail herein, a scFv has the variable domain of the light chain (VL) connected from its C-terminal end by a polypeptide chain to the N-terminal end of the variable domain of the heavy chain (VH). Alternatively, a scFv comprises a polypeptide chain in which the C-terminal end of the VH is connected by a polypeptide chain to the N-terminal end of the VL.

[0119] A "Fab fragment" (also referred to as an antigen binding fragment) contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), as well as the variable domains VL and VH on the light chain and heavy chain, respectively. The variable domains contain the complementarity determining loops (CDRs, also referred to as hypervariable regions) involved in antigen binding. A Fab' fragment differs from a Fab fragment only 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.

[0120] An "F(ab')2" fragment contains two Fab' fragments that are joined by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of whole antibodies. F(ab') fragments can be dissociated, for example, by treatment with beta-mercaptoethanol.

[0121] An "Fv" fragment comprises a noncovalent heterodimer of one heavy chain variable domain and one light chain variable domain.

[0122] A "single-chain Fv" or "scFv" includes the VHand VLdomains 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 VHand VLdomains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv 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.

[0123] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H ), the Fc domain can be after V H or V L . Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0124] The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen in the absence of another variable domain. 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 very stable and are easily expressed 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).

[0125] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure, and having heavy chains that include an Fc region. For example, a "full-length antibody" when used in reference to an IgG molecule is an antibody that includes two heavy chains and two light chains.

[0126] The term "epitope" means a portion of an antigen to which a specific binding antibody binds. Epitopes are often composed of contiguous amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics as well as specific charge characteristics. The distinction between conformational and linear epitopes is based on the availability of the epitope for binding to an antibody in different conditions. Binding to a linear epitope, but not to a conformational epitope, can be lost in the presence of a denaturing solvent. An epitope can include amino acid residues that are directly involved in binding as well as other amino acid residues that are not directly involved in binding. An epitope to which an antibody binds can be determined using known techniques for epitope determination, such as, for example, testing the binding of the antibody to a TREM2 variant with different point mutations or to a chimeric TREM2 variant.

[0127] 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 expressed by the same cell, or a TREM2 molecule and a non-TREM2 molecule). In some aspects, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some aspects, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody").

[0128] A "monospecific antibody" is an antibody comprising one or more binding sites that specifically bind a single epitope. An example of a monospecific antibody is a naturally occurring IgG molecule, which, although bivalent (i.e., has two antigen binding domains), recognizes the same epitope at each of the two antigen binding domains. Binding specificity can be present in any suitable valency.

[0129] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. The population of substantially homogeneous antibodies contains only minor amounts of

[0130] "Effector function" refers to those biological activities mediated by the Fc region of an antibody, which can vary depending on the antibody isotype. Examples of antibody effector functions include Clq 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.

[0131] Anti-TREM2 antibodies can include those described herein, such as the clones set forth 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 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 among TREM2 proteins from different species.

[0132] 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.

[0133] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human antibodies (recipient antibody) 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, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, that has a desired specificity, affinity, or biological effect. Humanized antibodies are less likely to induce an immune response, and / or induce a less severe immune response, when administered to a human subject, compared to the non-human species antibody. In some cases, selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. The humanized antibody can also comprise residues not found in the recipient antibody or donor antibody. These modifications can be made to further improve antibody function. Examples of how to make 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 additional 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.

[0134] In one embodiment, one or more constant domains from a human antibody are fused to one or more variable domains of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the possible immunogenicity of the non-human antibody when it is administered to a human subject, wherein the altered amino acid residues are not critical to the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen.

[0135] A "human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes a human antibody repertoire or a human antibody-encoding sequence (e.g., obtained from a human source or redesigned). Human antibodies specifically exclude humanized antibodies. In one embodiment, both the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be produced in a variety of ways, including by immunizing a mouse that has been genetically modified to express antibodies produced from human heavy and / or light chain-encoding genes with the antigen of interest.

[0136] 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 a scFv (sFv) fragment. In some embodiments, the antibody fragment is a scFv-Fc fragment. In some embodiments, the antibody fragment is a fragment of a single domain antibody.

[0137] Sequences of TREM2 antibodies

[0138] V H Domain

[0139] In some embodiments, the antibodies provided herein comprise a V H sequence selected from SEQ ID NOs: 1, 3, 5, and 7. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 1. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 3. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 5. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 7.

[0140] In some embodiments, the antibodies provided herein comprise a V H sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the illustrative V H sequences provided as SEQ ID NOs: 1, 3, 5, and 7. In some embodiments, the antibodies provided herein comprise a V HIn some embodiments, the antibody provided herein comprises a V

[0141] V L domain

[0142] In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 2. In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 4. In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 6. In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 8. In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 8. In some embodiments, the antibody provided herein comprises a V

[0143] In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 2, 4, 6, and 8 with at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity. In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 2, 4, 6, and 8 with at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity. In some embodiments, the antibody provided herein comprises a V L sequence of SEQ ID NO: 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, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0144] V H -V L combination

[0145] In some embodiments, the antibodies provided herein comprise a V H sequence selected from SEQ ID NO: 1, 3, 5, and 7; and a V L sequence selected from SEQ ID NO: 2, 4, 6, and 8.

[0146] In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 1 and a V L sequence of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 3 and a V L sequence of SEQ ID NO: 4. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 5 and a V L sequence of SEQ ID NO: 6. In some embodiments, the antibodies provided herein comprise a V H sequence of SEQ ID NO: 7 and a V L sequence of SEQ ID NO: 8. In certain aspects, any one of SEQ ID NOs: 1, 3, 5, and 7 can be combined with any one of SEQ ID NOs: 2, 4, 6, and 8. For example, SEQ ID NO: 1 can be combined with any one of SEQ ID NOs: 2, 4, 6, or 8. As another example, SEQ ID NO: 2 can be combined with any one of SEQ ID NOs: 1, 3, 5, or 7.

[0147] In some embodiments, the antibodies provided herein comprise a V H sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the illustrative V H sequence provided as SEQ ID NO: 1, 3, 5, and 7; and a V LIn some embodiments, an antibody provided herein comprises a VH sequence provided as SEQ ID NO: 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 a VL sequence provided as SEQ ID NO: 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0148] CDR

[0149] In some embodiments, an antibody provided herein comprises one to three CDRs of a VH domain selected from SEQ ID NO: 1, 3, 5, and 7. H In some embodiments, an antibody provided herein comprises two to three CDRs of a VH domain selected from SEQ ID NO: 1, 3, 5, and 7. H In some embodiments, an antibody provided herein comprises three CDRs of a VH domain selected from SEQ ID NO: 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.

[0150] In some embodiments, the CDR is a CDR that is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to a CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 1, 3, 5, and 7. In some embodiments, the CDR-H1 is a CDR-H1 of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is a 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 a 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0151] 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.

[0152] The CDRs of the VH and VL as determined using Kabat, Chothia, AbM, contact, and IMGT are shown in Table A1 below.

[0153]

[0154]

[0155] In some embodiments, the CDRs are CDRs that are at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, the CDR-L1 is a CDR-L1 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-L2 is a 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, the CDR-L3 is a 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0156] 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.

[0157] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H3 of SEQ ID NO: 11 or 41. In some embodiments, the CDR-H3 is the CDR-H3 of 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, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0158] In some embodiments, the antibodies provided herein comprise a CDR-H2 of SEQ ID NO: 10 or 40. In some aspects, the CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H2 of SEQ ID NO: 10 or 40. In some embodiments, the CDR-H2 is the 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, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0159] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 9 or 39. In some aspects, the CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H1 of SEQ ID NO: 9 or 39. In some embodiments, the CDR-H1 is the 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0160] In some embodiments, an antibody provided herein comprises a CDR-H3 of SEQ ID NO: 11 or 41 and a CDR-H2 of SEQ ID NO: 10. In some embodiments, an antibody provided herein comprises 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H3 of SEQ ID NO: 11 or 41, the CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H2 of SEQ ID NO: 10 or 40, and the CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H1 of SEQ ID NO: 9 or 39. In some embodiments, the CDR-H3 is the 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 the 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 the 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0161] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 14 or 44. In some aspects, the CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L3 of SEQ ID NO: 14 or 44. In some embodiments, the CDR-L3 is the 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, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0162] In some embodiments, the antibodies provided herein comprise a CDR-L2 of SEQ ID NO: 13 or 43. In some aspects, the CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L2 of SEQ ID NO: 13 or 43. In some embodiments, the CDR-L2 is the 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, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0163] In some embodiments, the antibodies provided herein comprise a CDR-L1 of SEQ ID NO: 12 or 42. In some aspects, the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-L1 is the 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0164] In some embodiments, an antibody provided herein comprises a CDR-L3 of SEQ ID NO: 14 or 44 and a CDR-L2 of SEQ ID NO: 13 or 43. In some embodiments, an antibody provided herein comprises 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L3 of SEQ ID NO: 14 or 44, the CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L2 of SEQ ID NO: 13 or 43, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-L3 is the 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 the 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 the 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein, and can be, for example, re-isolated according to the methods provided herein for obtaining antibodies.

[0165] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H3 of SEQ ID NO: 11 or 41, the CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H2 of SEQ ID NO: 10 or 40, the CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-H1 of SEQ ID NO: 9 or 39, the CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L3 of SEQ ID NO: 14 or 44, the CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L2 of SEQ ID NO: 13 or 43, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L1 of SEQ ID NO: 12 or 42. In some embodiments, the CDR-H3 is the 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 the 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 the 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 the 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 the 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 the 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, the variants are obtained from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, the variant is not obtained from a sequence provided herein and can be, for example, re-isolated according to a method provided herein for obtaining an antibody.

[0166] In some embodiments, an antibody provided herein comprises CDR-H1 of SEQ ID NO: 9, CDR-H2 of SEQ ID NO: 10, CDR-H3 of SEQ ID NO: 11, CDR-L1 of SEQ ID NO: 12, CDR-L2 of SEQ ID NO: 13, and CDR-L1 of SEQ ID NO: 14. In some embodiments, an antibody provided herein comprises CDR-H1 of SEQ ID NO: 39, CDR-H2 of SEQ ID NO: 40, CDR-H3 of SEQ ID NO: 41, CDR-L1 of SEQ ID NO: 42, CDR-L2 of SEQ ID NO: 43, and CDR-L1 of SEQ ID NO: 44.

[0167] Fc region

[0168] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called 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. "Fc polypeptide" as used herein in reference to a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, an Fc polypeptide of a dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences. Fc can be of class IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.

[0169] The terms “Fc receptor” and “FcR” are used to describe receptors that bind to the Fc region of an antibody. For example, an FcR can be a naturally occurring human FcR. Typically, an FcR is an FcR (γ receptor) that binds to IgG antibodies and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Other isotypes of immunoglobulins can also be bound by certain FcRs (see, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). The activating receptor FcγRIIA contains the immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (reviewed in...). (In 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 covered by the term “FcR” in this paper. The term also includes neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J.Immunol.117:587(1976); and Kim et al., J.Immunol.24:249(1994)).

[0170] In some implementations, the antibody is an IgG1 antibody.

[0171] In some implementations, the antibody is an IgG3 antibody.

[0172] In some implementations, the antibody is an IgG2 antibody.

[0173] In some implementations, the antibody is an IgG4 antibody.

[0174] Modifications in the CH2 domain can affect FcR binding to Fc. Numerous amino acid modifications in the Fc region for selectively altering Fc affinity for different Fc-gamma (Fc-gamma) receptors are known in the art. In one embodiment, the Fc comprises one or more modifications to promote selective binding to Fc-gamma receptors.

[0175] Exemplary mutations that alter FcR binding to Fc are listed below:

[0176] 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);

[0177] 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);

[0178] 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);

[0179] S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S, et al. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10);

[0180] S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S, et al. Mol Immunol. 2008 Sep;45(15):3926-33);

[0181] 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 others listed in WO2011 / 120134 and WO2011 / 120135, incorporated herein by reference. Therapeutic Antibody Engineering (by William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine, Volume 11, ISBN 1 907568 37 9, October 2012) lists mutations on page 283.

[0182] In some embodiments, the antibodies described herein include modifications to improve its ability to mediate effector functions. Such modifications are known in the art and include afucosylation, or engineering the Fc for affinity to the activating receptors, primarily FCGR3a for ADCC, and to Clq for CDC. Table B below outlines various designs reported in the literature for engineering effector functions.

[0183] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of positions 298, 333, and 334 of the Fc region. 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, incorporated by reference in its entirety.

[0184] In some embodiments, the antibodies provided herein comprise one or more alterations that improve or diminish 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.

[0185] Accordingly, in one embodiment, the antibodies described herein can include a dimeric Fc comprising one or more amino acid modifications conferring improved effector function as indicated in Table B. In another embodiment, the antibodies can be made afucosylated to improve effector function.

[0186] Table B: CH2 Domain and Effector Function Engineering

[0187]

[0188]

[0189] Fc modifications that reduce FcyR 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 silent 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", Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp 225-249). These strategies include reducing effector function by altering glycosylation, using an IgG2 / IgG4 backbone, or introducing mutations in the hinge or CH2 region of the Fc. For example, U.S. Patent Publication No. 2011 / 0212087 (Strohl), International Patent Publication No. WO 2006 / 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 specific modifications to reduce FcyR or complement binding to Fc.

[0190] Specific non-limiting examples of known amino acid modifications to reduce FcyR or complement binding to Fc include those identified in Table C below:

[0191] Table C: Modifications to Reduce FcyR or Complement Binding to Fc

[0192]

[0193]

[0194] Methods to produce antibodies with little or no fucose on the Fc glycosylation site (Asn 297, EU numbering) without changing the amino acid sequence are well known in the art. The ProBioGen AG technology is based on the introduction of a gene for an enzyme that deflects the cellular fucose biosynthesis pathway into the cells used for antibody production. This prevents the addition of the sugar "fucose" to the N-linked antibody carbohydrate moiety by the 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 stably overexpressing the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) (see Henning von Horsten et al, Glycobiol 2010, 20: 1607-1618) or Lec13 CHO cells which are deficient in protein fucosylation (see Ripka et al, Arch. Biochem. Biophys., 1986, 249: 533-545; U.S. Patent Publication No. 2003 / 0157108; WO 2004 / 056312; each of which is incorporated by reference herein in its entirety), and knockout cell lines such as a-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (see Yamane-Ohnuki et 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 herein in its entirety). Another method to obtain antibodies with reduced level of fucosylation can be found in U.S. Patent 8,409,572, which teaches selecting cell lines for producing antibodies that are capable of producing lower levels of fucosylation on the antibodies.

[0195] The antibodies can be completely afucosylated (which means that they contain no detectable fucose), or they can be partially afucosylated, which means that the amount of fucose contained by the isolated antibodies is less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose typically detected for similar antibodies produced by a mammalian expression system.

[0196] In some respects, the antibody provided herein comprises an IgG1 domain having a reduced trehalose content at position Asn 297 compared to the naturally occurring IgG1 domain. The Fc domain is known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, which is incorporated herein by reference in its entirety. In some respects, the antibody does not contain any trehalose at position Asn 297. The amount of trehalose can be determined using any suitable method, such as that described in WO 2008 / 077546, which is incorporated herein by reference in its entirety.

[0197] In some embodiments, the antibodies provided herein comprise bimeric oligosaccharides, such as biantennary oligosaccharides bimeric with GlcNAc linked to the Fc region of the antibody. The antibody variants may have reduced trehalose sizing 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 these patents is incorporated herein by reference in its entirety.

[0198] Other illustrative glycosylation variants that may be incorporated into the antibodies provided herein are described, for example, in U.S. Patent Publications 2003 / 0157108, 2004 / 0093621, 2003 / 0157108, 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0132140, 2004 / 0110704, 2004 / 0110282, and 2004 / 0109865; and International Patent Publications 2000 / 61739 and 2001 / 292. 46, 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 the aforementioned patents and documents is incorporated herein by reference in its entirety.

[0199] In some embodiments, the antibodies provided herein comprise an Fc region having at least one galactose residue in an oligosaccharide linked to the Fc region. The antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO 1999 / 22764; each of which is incorporated herein by reference in its entirety.

[0200] Examples of cell lines capable of producing de-trehalosylation antibodies include CHO-DG44, which stably overexpresses the bacterial oxidoreductase GDP-6-deoxy-D-lythreo-4-hexylose reductase (RMD) (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells that are defective in protein trehalosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; US Patent Publication No. 2003 / 0157108; WO 2004 / 056312; each of the aforementioned documents and patents is incorporated herein by reference in its entirety, and knockout cell lines such as α-1,6-trehalosyltransferase gene or FUT8 knockout CHO cells (see Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87:614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107; each of the aforementioned documents and patents is incorporated herein by reference in its entirety).

[0201] In some implementations, the antibody exhibits 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 carrying Fc receptors on their cell surface, including monocytes and macrophages, recognize and bind to the Fc region of the antibody bound to the target cell. Phagocyte phagocytosis of the target cell can be initiated after the Fc receptor binds to the antibody-bound target cell. ADCP can be considered a form of ADCC.

[0202] In some implementations, antibodies can form immune complexes. For example, immune complexes can be tumor cells covered by antibodies.

[0203] In some respects, anti-TREM2 antibodies do not substantially bind to myeloid cells present on the exterior of cancerous tissue. In other respects, anti-TREM2 antibodies do not substantially bind to stimulating myeloid cells present within cancerous tissue.

[0204] In some implementations, the antibody is a monoclonal antibody.

[0205] In some implementations, the antibody is a polyclonal antibody.

[0206] In some embodiments, antibodies are produced by hybridomas. In other embodiments, antibodies are produced by recombinant cells engineered to express the desired variable and constant domains.

[0207] In some implementations, the antibody may be a single-chain antibody or other antibody derivative that retains antigen specificity and the lower hinge region or a variant thereof.

[0208] In some embodiments, the antibody may be a multifunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment thereof, or a variant thereof. In a particular embodiment, the antibody fragment or a derivative thereof is selected from Fab fragments, Fab′2 fragments, CDRs, and ScFvs.

[0209] In some embodiments, the antibody is specific to a surface antigen such as the TREM2 protein. In some embodiments, the therapeutic antibody is specific to 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 motif.

[0210] Combination

[0211] Regarding antibody binding to target molecules, the terms "binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "specific binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "specific binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "specific to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen," "selective binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, and "selective to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen" mean that the binding is measurably different from nonspecific or nonselective interactions (e.g., interactions with non-target molecules). Specific binding can be measured, for example, by measuring the binding to the target molecule and comparing it to the binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics an epitope recognized on the target molecule. In that case, if the binding of the antibody to the target molecule is competitively inhibited by the control molecule, then specific binding is indicated.

[0212] “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 indicated, as used herein, “affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of molecule X for its partner Y can be determined by the dissociation equilibrium constant (K0). D The following describes in more detail the dynamic components that contribute to the dissociation equilibrium constant. Affinity can be measured by commonly used methods known in the art, including those described herein, such as surface plasmon resonance (SPR) techniques (e.g., ) or biolayer interferometry (e.g.) ).

[0213] As used in this article, the term "k" d (s) -1 () refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as k. 解离 value.

[0214] As used in this article, the term "k" a (M) -1 ×s -1 () refers to the association rate constant of a specific antibody-antigen interaction. This value is also known as k. 缔合 value.

[0215] As used in this article, the term "K" D (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. K D =k d / k a In some implementations, the affinity of an antibody is expressed as a Kelvin-weighted average (K0) relating the interaction between the antibody and its antigen. D Description. For clarity, as is known in the art, smaller K... D A value indicates a higher affinity interaction, while a larger K value indicates a lower affinity interaction. D The value indicates a lower affinity interaction.

[0216] As used in this article, the term "K" A (M) -1 K refers to the association equilibrium constant of a specific antibody-antigen interaction. A =k a / k d .

[0217] When used herein in the context of two or more antibodies, the terms “competing with” or “cross-competing with” indicate that the two or more antibodies compete to bind to an antigen (e.g., TREM2). In one exemplary assay, TREM2 is coated on a surface and brought into contact 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 brought into contact with TREM2, followed by the addition of a second TREM2 antibody. If, in either assay, the presence of the first TREM2 antibody reduces the binding of the second TREM2 antibody, then the antibodies compete with each other. The term “competing with” also includes combinations of antibodies in which one antibody reduces the binding of another antibody, but in which no competition is observed when the antibodies are added in 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%. Skilled technicians can select the concentration of an antibody to be used in a competitive assay based on the antibody's affinity for TREM2 and its titer. The assays described in this definition are illustrative, and skilled technicians can use any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., “Immunoassay Methods”, 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 these references is incorporated herein by reference in its entirety.

[0218] In some implementations, the antibodies provided herein are in quantities less than or equal to 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. -9 M of K D Binding to human TREM2, as measured by a Biacore assay. In some embodiments, the K of the antibody provided herein...D In 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 5-10x10 -9 Between M, as measured by Biacore.

[0219] In some implementations, the antibodies provided herein are in quantities less than or equal to 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 10⁻⁶. -9 M or smaller K D Binding to human TREM2, as measured by a Biacore assay. In some embodiments, the antibodies provided herein are in concentrations of 1.9–1.8, 1.8–1.7, 1.7–1.6, 1.6–1.5, or 1.9–1.5 x 10⁻⁶. -9 K between M D Binds to human TREM2, as measured by a Biacore assay. In some embodiments, the antibodies provided herein are in doses less than or equal to 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 1x10. -4 (1 / s) or smaller K d Binding to human TREM2, as measured by a Biacore assay. In some embodiments, the antibodies provided herein are available in 7-10, 7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8.5-8.5, 8.5-9, 9-9.5, or 9.5-10x10. -4 K between (1 / s) d Binds to human TREM2, as measured by a Biacore assay. In some embodiments, the antibodies provided herein are in quantities greater than or equal to 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 10^9. 5 (1 / Ms) or larger K aBinds to human TREM2, as measured by a Biacore assay. In some embodiments, the antibodies provided herein are in concentrations 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 9-10x10. 5 K between (1 / Ms) a Combined with human TREM2, as measured by Biacore assays.

[0220] In some embodiments, the antibodies provided herein bind to human TREM2 at EC50 levels less than or equal to 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, as measured by flow cytometry. In some embodiments, the antibodies bind to human TREM2 at EC50 levels between 0.6 and 1.4 nM, as measured by flow cytometry. In some embodiments, the antibodies bind to human TREM2 at EC50 levels 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.

[0221] In some embodiments, the antibodies provided herein bind mouse TREM2 at EC50 levels less than or equal to 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, as measured by flow cytometry. In some embodiments, the antibodies bind mouse TREM2 at EC50 levels between 0.6 and 1.4 nM, as measured by flow cytometry. In some embodiments, the antibodies bind mouse TREM2 at EC50 levels 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.

[0222] In some embodiments, the antibodies provided herein do not bind human TREM2 at an EC50 greater than or equal to 20 nM, as measured by flow cytometry. In some embodiments, the antibodies provided herein do not bind mouse TREM2 at an EC50 greater than or equal to 3 nM, as measured by flow cytometry.

[0223] To screen for antibodies that bind to epitopes on target antigens (e.g., TREM2) bound by the target antibody, routine cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow and David Lane (1988). Alternatively, epitope localization can be performed using methods known in the art.

[0224] Competition between antibodies can be determined by an assay in which the antibody being tested inhibits or blocks the specific binding of a reference antibody to a common antigen (see, for example, Junghans et al., Cancer Res. 50:1495, 1990; Fendly et al., Cancer Research 50:1550-1558; US 6,949,245). As measured in a competitive binding assay, if an excess of the test antibody (e.g., at least 2, 5, 10, 20, or 100 times) inhibits or blocks the binding of the reference antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, then the test antibody competes with the reference antibody. Antibodies identified by a competitive binding assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to a neighboring epitope sufficiently adjacent to the epitope bound by the reference antibody to achieve steric hindrance. For example, a second competitive antibody can be identified that competes with the first antibody described herein for binding to TREM2. In some cases, such as as measured in a competitive binding assay, the second antibody may block or inhibit the binding of the first antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some cases, the second antibody may displace more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the first antibody.

[0225] Function

[0226] In some embodiments, the antibody has antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC can occur when the antibody binds to an antigen on the surface of pathogenic or tumorigenic target cells. Effector cells carrying Fcγ 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 to the Fc region of the antibody bound to the target cell. This binding can trigger activation of intracellular signaling pathways, leading to cell death. In certain embodiments, the immunoglobulin Fc region isotypes (isotypes) of the antibody include human IgG1 and IgG3. As used herein, ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize the antibody bound to the target cell and subsequently cause the lysis of the target cell. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of the target molecule, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be performed. Effector cells that can be used for said assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the target molecule can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0227] In some embodiments, the antibody possesses complement-dependent cytotoxic (CDC) activity. Antibody-induced CDC is mediated by proteins in the classical complement cascade and is triggered by the binding of the complement protein Clq to the antibody. Binding of the antibody's Fc region to Clq induces activation of the complement cascade. In certain embodiments, the immunoglobulin Fc region isotypes (isotypes) of the antibody include human IgG1 and IgG3. As used herein, CDC refers to the ability of a molecule to dissolve a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (e.g., a polypeptide (e.g., an antibody)) that is complexed with a homologous antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0228] 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 the cell. An agonist antibody can bind to and activate NSM, thereby leading to changes in cell proliferation or altered antigen-presenting capacity. An agonist antibody can bind to and activate NSM, thereby triggering intracellular signaling pathways that lead to alterations in cell growth or apoptosis.

[0229] In some embodiments, the antibody is an antagonistic antibody. The antagonistic antibody can block (e.g., reduce) one or more activities or functions of NSM after the antibody binds to the TREM2 protein expressed on the cell. For example, the antagonistic antibody can bind to one or more NSM proteins and block the binding of ligands to one or more NSM proteins, thereby preventing cell differentiation and proliferation or altering antigen presentation capacity. The antagonistic antibody can bind to the TREM2 protein and prevent activation of the TREM2 protein by ligands, thereby altering intracellular signaling pathways that promote cell growth and survival.

[0230] In some implementations, the antibody is a subtractive antibody. Subtractive antibodies are antibodies that, upon contact, kill non-stimulated myeloid cells through interactions with other immune cells containing various molecules. For example, when binding to cells carrying the TREM2 protein, the antibody can bind complement proteins and induce complement-dependent cell lysis. When binding to cells carrying the TREM2 protein, the antibody can also trigger neighboring cells carrying Fc receptors to kill them through antibody-dependent cytotoxicity (ADCC).

[0231] In some embodiments, the antibody is a neutralizing antibody, and said antibody neutralizes one or more biological activities of NSM. In some embodiments, the TREM2 protein is expressed on the surface of unstimulated myeloid cells, and the antibody recognizes the extracellular domain of the TREM2 protein.

[0232] In some embodiments, the antibody is selective for NSM (preferentially binding to TREM2). In some embodiments, the antibody selectively binding to NSM has a dissociation constant (Kd) in the range of 0.0001 nM to 1 μM. In some embodiments, the antibody specifically binds to a conserved epitope on the TREM2 protein among proteins from different species. In another embodiment, selective binding includes, but is not required to be, exclusive binding.

[0233] In one embodiment, the anti-TREM2 antibody bound to its target is responsible for causing the reduction of non-stimulated myeloid cells it binds to in vivo. In some embodiments, effector proteins induced by clustered antibodies can trigger a variety of responses, including the release of inflammatory cytokines, regulation of antigen production, endocytosis, or cell killing. In one embodiment, the antibody can recruit and activate complement in vivo or mediate antibody-dependent cytotoxicity (ADCC), or mediate phagocytosis in vivo by binding to Fc receptors. The antibody can also reduce non-stimulated myeloid cells after binding by inducing apoptosis or necrosis of the non-stimulated myeloid cells.

[0234] In some implementations, the inactivation of unstimulated myeloid cells is achieved in vitro and by: a) killing the unstimulated myeloid cells; b) reducing the unstimulated myeloid cells with magnetic beads; or c) sorting the unstimulated myeloid cells using fluorescence-activated cell sorting (FACS).

[0235] In some embodiments, the antibody binds to or is conjugated to an effector molecule. In a particular embodiment, the antibody is conjugated to at least one therapeutic agent selected from the group consisting of: radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, antiangiogenic agents, pro-apoptotic agents, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, secondary antibodies, and secondary antibody fragments.

[0236] In some embodiments, the antibody is conjugated to a drug, such as a toxin, chemotherapeutic agent, immunomodulator, or radioisotope. Several methods for preparing ADCs (antibody-drug conjugates) are known in the art and described, for example, in U.S. Patents 8,624,003 (pot method), 8,163,888 (one-step method), and 5,208,020 (two-step method). An antibody or its antigen-binding fragment may be conjugated to at least one drug agent, including radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, anti-angiogenic agents, pro-apoptotic agents, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, secondary antibodies, and antigen-binding secondary antibody fragments.

[0237] Non-stimulated myeloid cells (NSM)

[0238] This article provides methods and compositions for inactivating and / or detecting unstimulated myeloid cells (NSM), including the use of anti-TREM2 antibodies. This article also provides methods and compositions for targeting and / or detecting unstimulated myeloid cells expressing NSM proteins.

[0239] This article also provides methods and compositions for inactivating and / or detecting non-stimulated myeloid cells, including the use of antibodies against non-human homologs of human NSM proteins in that non-human individual.

[0240] As used herein, non-stimulated myeloid cells are myeloid cells that are insufficiently effective at stimulating an immune response (e.g., less effective at stimulating an anti-tumor response in the tumor microenvironment compared to stimulated myeloid cells). In some embodiments, non-stimulated myeloid cells are less effective at presenting antigens (e.g., tumor antigens) to T cells or less effective at stimulating tumor-specific T cell responses compared to stimulated myeloid cells. In some embodiments, non-stimulated myeloid cells may exhibit a reduced ability to take up tumor-associated antigens, process tumor-associated antigens, and / or present tumor-associated antigens to T cells compared to stimulated myeloid cells. Non-stimulated myeloid cells may contain a reduced ability to resensitize cytotoxic T lymphocytes or may not contain such ability, or in some cases, may not stimulate effective tumor cell killing. Compared to stimulated myeloid cells, non-stimulated myeloid cells may exhibit lower expression of genes and cell surface markers involved in antigen processing, antigen presentation, and / or antigen co-stimulation, including but not limited to CD80, CD86, MHC1, and MHCII.

[0241] Compared to stimulated myeloid cells, non-stimulated myeloid cells may exhibit lower expression of genes associated with cross-presentation, co-stimulation, and / or stimulating 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. In some embodiments, non-stimulated myeloid cells achieve differentiation and survival dependent on the transcription factor IRF4 and the cytokines GM-CSF or CSF-1. In some implementations, non-stimulated myeloid cells can promote tumor angiogenesis by secreting vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS), and support tumor growth by secreting epidermal growth factor (EGF).

[0242] In some embodiments, the non-stimulated myeloid cells are tumor-associated macrophages (TAMs), neutrophils, monocytes, or dendritic cells (DCs). In some embodiments, the non-stimulated myeloid cells are not dendritic cells (DCs). In some embodiments, the non-stimulated myeloid cells are neutrophils.

[0243] In some implementations, the non-stimulated myeloid cells are tumor-associated macrophages (TAMs). TAMs are macrophages that are present near or inside cancerous tumors and are derived from circulating monocytes or resident tissue macrophages.

[0244] In some implementations, non-stimulated and stimulated myeloid cells are distinguished based on the markers they express or the markers they selectively express. The expression of a cell surface marker can be described as '+' or 'positive'. The absence of a cell surface marker can be described as '-' or 'negative'. The expression of a cell surface marker can be further described as 'high' (high level of marker expression) or 'low' (low level of marker expression). The level of the marker can be determined by various methods known in the art, such as immunostaining and FACS analysis, or gel electrophoresis and Western blotting.

[0245] In some embodiments, the non-stimulated myeloid cells are dendritic cells (DCs). In some embodiments, dendritic cells can be distinguished by spikes or dendritic morphology. In one embodiment, the non-stimulated dendritic cells are at least CD45+, HLA-DR+, CD14-, CD11c+, and BDCA1+ (also referred to as DC1 cells). In one embodiment, the non-stimulated 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 stimulated myeloid cells.

[0246] In some embodiments, the non-stimulated myeloid cells are tumor-associated macrophages. In some embodiments, such as in humans, the non-stimulated tumor-associated macrophages are at least CD45+, HLA-DR+, and CD14+. In some embodiments, the non-stimulated tumor-associated macrophages are at least CD45+. + HLA-DR + CD14 + CD11b + In some implementations, non-stimulatory tumor-associated macrophages are at least CD45. + HLA-DR + CD14 + CD11c+ In some implementations, non-stimulatory tumor-associated macrophages are at least CD45. + HLA-DR + CD14 + BDCA3 - In some implementations, non-stimulatory tumor-associated macrophages are at least CD45. + HLA-DR + CD14 + BDCA3 - CD11b + In some implementations, non-stimulatory tumor-associated macrophages are at least CD45. + HLA-DR + CD14 + BDCA3 - CD11c + In some implementations, non-stimulatory tumor-associated macrophages are at least CD45. + HLA-DR + CD14 + CD11b + and CD11c + In some implementations, non-stimulatory tumor-associated macrophages are at least CD45. + HLA-DR + CD14 + BDCA3 - CD11b + and CD11c + .

[0247] In some embodiments, the methods and compositions of the present invention can be used to target TAMs and DCs in other mammals, such as mice. In said embodiments, mouse TAMs and DCs are contacted with the TREM2 antibody. In one embodiment, for example in mice, tumor-associated macrophages are at least CD45+, HLA-DR+, CD14+, and CD11b. 高 and CD11c 低 (Also known as TAM1). In one implementation, for example in mice, the tumor-associated macrophages are at least CD45+, HLA-DR+, CD14+, and CD11b. 低 and CD11c 高 (Also known as TAM2). As used in this article, the term "CD11b" 高 "Macrophages" refers to macrophages that express high levels of CD11b. The term "CD11b" is used as it is understood herein. 低 "Macrophages" involve expressing a contrast agent to CD11b on their surface.高 The CD11b level in macrophages, actually lower levels in macrophages than in macrophages. As used in this article, the term "CD11c" is used... 高 "This involves macrophages expressing high levels of CD11c. As used in this article, 'CD11c'..." 低 "Macrophages" involve expressing, on their surface, a contrast agent compared to Cd11c. 高 The CD11c level of macrophages, actually lower levels of CD11c in macrophages.

[0248] In some embodiments, the non-stimulated myeloid cells of the present invention include one or more of TAM and DC1 cells.

[0249] In some embodiments, such as in mice, the non-stimulated myeloid cells of the present invention include one or more of TAM1, TAM2, and DC1 cells. In said embodiments, the non-stimulated myeloid cells of the present invention are contacted with the TREM2 antibody.

[0250] In some implementations, non-stimulated myeloid cells are myeloid cells within the tumor.

[0251] In some embodiments, non-stimulated myeloid cells are localized within the periphery of the tumor lesion or in the transforming tumor duct, where they come into contact with homologous T cells. In one embodiment, the localization of non-stimulated myeloid cells is altered such that the cells are no longer localized at the tumor periphery or no longer in contact with T cells.

[0252] In some embodiments, non-stimulated myeloid cells are present in an immune cell population comprising both stimulated and non-stimulated myeloid cells. In some embodiments, non-stimulated myeloid cells are present in an immune cell population comprising only non-stimulated myeloid cells. The immune cell population of the present invention may be pure, homogeneous, heterogeneous, derived from multiple sources (e.g., diseased tissue, tumor tissue, healthy tissue, cell bank), maintained in primary cell culture, maintained in immortalized culture, and / or maintained in in vitro culture.

[0253] In some implementations, non-stimulated myeloid cells are tumor-associated macrophages.

[0254] In some implementations, the non-stimulated myeloid cells are dendritic cells.

[0255] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 - CD11c + and BDCA1 +In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 - CD11c + and BDCA1 + The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 - CD11c + and BDCA1 + The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 - CD11c + and BDCA1 + The cellular composition.

[0256] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + BDCA3 - In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + BDCA3 - The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + BDCA3 - The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + BDCA3 - The cellular composition.

[0257] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + CD11b + In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + CD11b +The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + CD11b + The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + CD11b + The cellular composition.

[0258] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + CD11c + In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + CD11c + The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + CD11c + The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + CD11c + The cellular composition.

[0259] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + BDCA3 - and CD11c + In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + BDCA3 - and CD11c + The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + BDCA3 - and CD11c +The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + BDCA3 - and CD11c + The cellular composition.

[0260] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + BDCA3 - CD11b + In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + BDCA3 - CD11b + The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + BDCA3 - CD11b + The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + BDCA3 - CD11b + The cellular composition.

[0261] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + CD11b + and CD11c + In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + CD11b + and CD11c + The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + CD11b + and CD11c +The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + CD11b + and CD11c + The cellular composition.

[0262] In some implementations, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + BDCA3 - CD11b + and CD11c + In some implementations, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + BDCA3 - CD11b + and CD11c + The cells. In some implementations, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + BDCA3 - CD11b + and CD11c + The cells are composed of CD45. In some implementations, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + BDCA3 - CD11b + and CD11c + The cellular composition.

[0263] In some implementations, non-stimulated myeloid cells are not CD45. + HLA-DR + CD14 - CD11c + and BDCA3 + In some implementations, non-stimulated myeloid cells contain cells that are not CD45. + HLA-DR + CD14 - CD11c + and BDCA3 + . cells.

[0264] In some implementations, such as in mice, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + CD11b 高 and CD11c 低 In some implementations, such as in mice, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + CD11b 高 and CD11c 低 The cells. In some implementations, such as in mice, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + CD11b 高 and CD11c 低 The cells are composed of CD45. In some implementations, such as in mice, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14 + CD11b 高 and CD11c 低 The cellular composition. In the described embodiment, unstimulated mouse myeloid cells are contacted with the TREM2 antibody.

[0265] In some implementations, such as in mice, non-stimulated myeloid cells are CD45. + HLA-DR + CD14 + CD11b 低 and CD11c 高 In some implementations, such as in mice, non-stimulated myeloid cells contain CD45. + HLA-DR + CD14 + CD11b 低 and CD11c 高 The cells. In some implementations, such as in mice, non-stimulated myeloid cells are composed of CD45. + HLA-DR + CD14 + CD11b 低 and CD11c 高 The cells are composed of CD45. In some implementations, such as in mice, non-stimulated myeloid cells are primarily composed of CD45 cells. + HLA-DR + CD14+ CD11b 低 and CD11c 高 The cellular composition. In the described embodiment, unstimulated mouse myeloid cells are contacted with the TREM2 antibody.

[0266] In some implementations, non-stimulated myeloid cells are present in cancerous tissue.

[0267] In some implementations, immune cell populations are present in cancerous tissue.

[0268] In some implementations, non-stimulated cells and stimulated myeloid cells are present in cancerous tissue.

[0269] In some implementations, the biological sample contains a population of immune cells, including both non-stimulated and stimulated myeloid cells.

[0270] NSM cells can generally refer to DC1, TAM1, and TAM2 cells present in tumor tissue and distinguishable from other cell types based on their expression of NSM cell markers. For example, genes and related proteins expressed or translated in greater abundance 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 their cell surface. In some respects, NSM cells do not express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1.

[0271] In one embodiment, one or more of the NSM marker genes listed in NSM cell expression table A. In another embodiment, 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 NSM cell expression table D. In another embodiment, most or all of the NSM markers listed in NSM cell expression table D. In yet another embodiment, NSM cells are identified as expressing MRC1, MS4A7, C1QC, APOE, C1QB, C1QA, and C5AR1.

[0272] Table D

[0273]

[0274] Stimulated myeloid cells

[0275] As used herein, stimulated myeloid cells (also referred to in some respects as SDCs) are myeloid cells that effectively stimulate immune responses (e.g., more effectively stimulating antitumor responses in the tumor microenvironment compared to non-stimulated myeloid cells). In some embodiments, stimulated myeloid cells effectively present antigens (e.g., tumor antigens) to T cells or effectively stimulate tumor-specific T cell responses compared to non-stimulated myeloid cells. In some embodiments, stimulated myeloid cells may exhibit an increased ability to take up tumor-associated antigens, process tumor-associated antigens, and / or present tumor-associated antigens to T cells compared to non-stimulated myeloid cells. Compared to non-stimulated myeloid cells, stimulated myeloid cells may have an increased ability to resensitize cytotoxic T lymphocytes or, in some cases, stimulate effective tumor cell killing. Compared to non-stimulated myeloid cells, stimulated myeloid cells may exhibit higher expression of genes and cell surface markers involved in antigen processing, antigen presentation, and / or antigen co-stimulation, including but not limited to CD80, CD86, MHC1, and MHCII.

[0276] Exemplary markers of stimulatory myeloid cell identity are listed in Table A. For example, in human SDCs, expression of Xcr1, Clec9a, and BDCA3 (CD141) is a marker of SDC identity. It should be noted that in mice, CD103 can also be used as a strong marker of SDC identity, but it is not expressed in human SDCs.

[0277] In one embodiment, SDCs are tumor-infiltrating myeloid cells that are dendritic cells and also express one or more of the SDC markers listed in Table A. In another embodiment, SDCs are tumor-infiltrating myeloid cells that are dendritic cells and also express two, three, four, five, six, seven, eight, nine, or all of the SDC markers listed in Table A. In yet another embodiment, SDCs are identified as tumor-infiltrating myeloid dendritic cells expressing 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, SDCs do not substantially express TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and / or TMEM119 on their cell surface. In some embodiments, 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, along with other assays recognized in the art, can be used to assess the expression of the biomarkers disclosed herein.

[0278] Stimulated myeloid cells can be CD45 + HLA-DR + CD14 - CD11c + and BDCA3 + Stimulated myeloid cells can be CD45. + HLA-DR + and BDCA3 + Stimulated myeloid cells can be CD45. + HLA-DR + CD14 - and BDCA3 + Stimulated myeloid cells can be CD45. + HLA-DR + CD11c + and BDCA3 + .

[0279] Proteins, nucleotides and homologs

[0280] This document provides methods and compositions for inactivating and / or detecting unstimulated human myeloid cells expressing NSM proteins. In some embodiments, the invention relates to inactivating and / or detecting unstimulated myeloid cells expressing NSM protein homologs from non-human mammalian cells. For example, NSM proteins in mice can exhibit a defined expression pattern similar to its human homologs. Therefore, in one embodiment, this document provides methods and compositions for inactivating and / or detecting unstimulated mouse myeloid cells expressing NSM proteins. This document also provides similar methods and compositions for inactivating and / or detecting unstimulated cells from any individual expressing NSM proteins with a similar expression pattern to the NSM protein expression pattern.

[0281] NSM proteins or nucleotides may include at least one or more of C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, MERTK, C1QC, TMEM119, MS4A7, APOE, CYP4F18, TREM2, TLR7, and LILRB4, and their homologs. SDC proteins or nucleotides may include at least one or more of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1, and their homologs. Cell surface NSM proteins may include at least one or more of TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and TMEM119. Cell surface NSM proteins can be detected by one or more anti-TREM2 antibodies, either alone or in combination. Typically, NSM is positive for NSM protein or nucleotides and negative for SDC protein or nucleotides; conversely, SDC is typically positive for SDC protein or nucleotides and negative for NSM protein or nucleotides.

[0282] The antibodies described herein contain at least one polypeptide, but they typically contain an HC / LC dimer, i.e., four polypeptides. Polynucleotides encoding the polypeptides described herein are also described. Antibodies are typically isolated.

[0283] As used herein, “isolated” means that the agent (e.g., a polypeptide or polynucleotide) has been identified and isolated from and / or recovered from components of its native cell culture environment. Contaminant components of its native environment are substances that will interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. “Isolated” also means that the agent has been produced synthetically, for example, through human intervention.

[0284] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers containing amino acid residues. That is, a description relating to a polypeptide is equally applicable to a description of a peptide and a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are not naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0285] The term "amino acid" refers to naturally occurring amino acids and non-naturally occurring amino acids, as well as amino acid analogs and amino acid simulants that function in a similar manner to naturally occurring amino acids. Naturally encoded amino acids are 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolidone and selenocysteine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., the presence of an α-carbon bound to a hydrogen, carboxyl, amino, or R group, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylthionium. These analogs have modified R groups (such as ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. References to amino acids include, for example, naturally occurring L-amino acids that form proteins; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-protein-forming amino acids such as β-alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art as characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methyl amino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine), amino acids having an additional methylene group in the side chain (“homo” amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group (e.g., sulfoalanine). Incorporating non-natural amino acids, including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids, into the proteins of the present invention can be advantageous in many different respects. Peptides containing D-amino acids, etc., exhibit increased in vitro or in vivo stability compared to their counterparts containing L-amino acids. Therefore, peptides, etc., constructed with D-amino acids, can be particularly useful when greater intracellular stability is required or demanded. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability and extended lifespan in vivo when these properties are desired. Furthermore, D-peptides and the like cannot be efficiently processed to achieve class II major histocompatibility complex-defined presentation to T helper cells, and therefore have a lower likelihood of inducing humoral immune responses throughout the organism.

[0286] Amino acids may be referred to in this article by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0287] This invention also includes polynucleotides encoding polypeptides of antibodies. The terms "polynucleotide" or "nucleotide sequence" are intended to refer to a continuous segment having two or more nucleotide molecules. Nucleotide sequences may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.

[0288] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise explicitly limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioides, aminophosphates, etc.) used in antisense technologies. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses its conserved modified variants (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. In particular, degenerate codon substitution can be achieved by producing a sequence in which the third position of one or more of the selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (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)).

[0289] The term "conserved variant" applies to both amino acid sequences and nucleic acid sequences. Regarding a specific nucleic acid sequence, a "conserved variant" refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a certain codon, that codon can be changed to any of the corresponding codons without altering the encoded polypeptide. This nucleic acid variation is a type of "silent variant" of conserved variant. Each possible silent variant of the nucleic acid is also described herein for each nucleic acid sequence encoding a polypeptide. Those skilled in the art will recognize that each codon in a nucleic acid (except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variant of the nucleic acid encoding a polypeptide is implicit in each of these sequences.

[0290] Regarding amino acid sequences, those skilled in the art will recognize that alterations, additions, or deletions of a single amino acid or a small percentage of amino acids in the sequence encoded by a nucleic acid, peptide, polypeptide, or protein sequence are “conserved variants”, wherein the alteration results in the deletion, addition, or substitution of an amino acid by a chemically similar amino acid. Conservation of functionally similar amino acids is known to those skilled in the art. The conserved variants are those added to the polymorphic variants, interspecific homologs, and alleles described herein, and do not exclude the polymorphic variants, interspecific homologs, and alleles described herein.

[0291] Conservative substitutions of functionally similar amino acids are known to those skilled in the art. The following eight groups each contain amino acids that are conserved substitutes for each other: 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

[0139] 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)).

[0292] The term "identical" or "identity percentage" in the context of two or more nucleic acid or polypeptide sequences means that two or more sequences or subsequences are identical. If sequences have a certain percentage of identical amino acid residues or nucleotides when compared and aligned to achieve maximum correspondence across a comparison window or designated region (i.e., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity across a designated region), then they are "substantially identical." Alignments for the purpose of determining the percentage of amino acid sequence identity can be performed in various ways within the skill of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. This definition also relates to the complementary sequence of the test sequence. Identity may traverse a region of at least about 50 amino acids or nucleotides, or a region of 75-100 amino acids or nucleotides, or, when not specified, span the entire sequence of a polynucleotide or polypeptide. The polynucleotide encoding the polypeptide of the present invention (including homologs from species other than humans) can be obtained by a method comprising the steps of: screening a library under strict hybridization conditions with a labeled probe having the polynucleotide sequence or a fragment thereof described herein, and isolating a full-length cDNA and a genomic clone containing the polynucleotide sequence. The hybridization techniques are well known to those skilled in the art.

[0293] For sequence comparisons, a reference sequence is typically used as the test sequence for comparison. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, subsequence coordinates are specified if necessary, and the sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters.

[0294] As used herein, a “comparison window” includes a segment having any number of consecutive positions selected from groups of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, wherein, after optimal alignment of two sequences, the sequence can be compared with a reference sequence having the same number of consecutive positions. Methods for aligning sequences for comparison are known to those skilled in the art. This may include, but is not limited to, the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, the computerized execution programs of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package (Genetics Computer Group, 575 Science Dr., Madison, Wis.), or the manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)) to perform optimal alignment of the sequences used for comparison.

[0295] An example of an algorithm suitable for determining the percentage of sequence identity and the percentage of sequence similarity is the BLAST and BLAST 2.0 algorithms, 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 analysis is publicly available from the National Center for Biotechnology Information at ncbi.nlm.nih.gov on the World Wide Web. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and compares two strands as default values. For amino acid sequences, the BLASTP program uses a word length of 3 and an expected value (E) of 10, along with a BLOSUM62 score matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915) with an alignment value (B) of 50, an expected value (E) of 10, M=5, N=-4, and comparing two strands as default values. The BLAST algorithm is typically performed with the "low complexity" filter turned off.

[0296] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability when comparing a tested nucleic acid to a reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0297] The phrase "selective (or specific) hybridization" means that when a specific nucleotide sequence is present in a complex mixture (including but not limited to total cellular or library DNA or RNA), under strict hybridization conditions, the molecule binds to, doubles, or hybridizes only with that sequence.

[0298] The phrase “strict hybridization conditions” refers to the hybridization of sequences of DNA, RNA, or other nucleic acids, or combinations thereof, under conditions of low ionic strength and high temperature, as known in the art. Typically, under strict conditions, a probe will hybridize to its target sequence in a complex mixture of nucleic acids (including, but not limited to, total cellular or library DNA or RNA), but not to other sequences in said complex mixture. Strict conditions are sequence-dependent and will vary under different conditions. Longer sequences hybridize specifically at higher temperatures. A comprehensive 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).

[0299] As used herein, the term "engineering" is considered to include any manipulation of the peptide backbone or post-translational modification of naturally occurring peptides or recombinant peptides or fragments thereof. Engineering includes modifications to the amino acid sequence, alterations to glycosylation patterns, or modifications to the side chain groups of individual amino acids, as well as combinations of these methods. Engineered proteins are expressed and produced using standard molecular biology techniques.

[0300] The term "isolated nucleic acid molecule or polynucleotide" refers to a nucleic acid molecule, i.e., DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include those present in cells that typically contain polynucleotide molecules, but which are located extrachromosomally or at a chromosomal location different from their native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts, and in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids described herein also include molecules produced synthetically, for example by PCR or chemical synthesis. Furthermore, in some embodiments, the polynucleotide or nucleic acid includes regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0301] The term "polymerase chain reaction" or "PCR" generally refers to a method for amplifying desired nucleotide sequences in vitro, as described, for example, in U.S. Patent No. 4,683,195. Generally, PCR methods involve repeated cycles of primer extension synthesis using oligonucleotide primers capable of preferentially hybridizing to template nucleic acids.

[0302] For a nucleic acid or polynucleotide to have a nucleotide sequence that is at least, for example, 95% "identical" to the reference nucleotide sequence of the present invention, this means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, with the exception that for every 100 nucleotides of the reference nucleotide sequence, the polynucleotide sequence may include up to five point mutations. 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 substituted with another nucleotide, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5' or 3' ends of the reference nucleotide sequence or anywhere between those ends, individually scattered among residues in the reference sequence, or scattered in one or more consecutive groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention can be routinely determined using known computer programs such as the computer programs discussed above with respect to polypeptides (e.g., ALIGN-2).

[0303] If the amino acid sequence of a derivative or variant of a polypeptide shares at least 50% identity with a sequence of 100 amino acids from the original peptide, then the derivative or variant is referred to as sharing "homology" or "homology" with the peptide. In some embodiments, the derivative or variant is at least 75% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the derivative or variant is at least 85% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 90% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the derivative or variant is at least 99% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative.

[0304] As used herein, the term "modification" refers to any change made to a given polypeptide, such as changes to the length, amino acid sequence, or chemical structure of the polypeptide, or co-translational or post-translational modifications. The formal term "(modified)" indicates that the polypeptide in question is optionally modified, meaning that the polypeptide in question may or may not be modified.

[0305] In some aspects, the polypeptide comprises an amino acid sequence or fragment thereof that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a related (e.g., polypeptide and / or antibody) amino acid sequence set forth in one or more of the tables disclosed herein or set forth in one or more of the accessions disclosed herein. In some aspects, the 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 a related nucleotide sequence or fragment thereof set forth in one or more of the tables disclosed herein or set forth in one or more of the accessions disclosed herein. In some respects, the nucleotide sequence includes those that are at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the nucleotide sequences disclosed herein, such as those set forth in one or more of the tables disclosed herein or by one or more accessions disclosed herein.

[0306] Pharmaceutical Composition

[0307] This application provides compositions comprising antibodies, including pharmaceutical compositions comprising any or more of the antibodies described herein and one or more pharmaceutically acceptable excipients. In some embodiments, the composition is sterile. Pharmaceutical compositions typically contain an effective amount of antibody.

[0308] In addition to one or more of the antibodies disclosed herein, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances well 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 exact nature of the carrier or other substance may depend on the route of administration, such as oral, intravenous, transdermal or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0309] Pharmaceutical compositions intended for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may include solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions typically include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. They may include physiological saline solutions, dextran or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0310] For intravenous, transdermal, or subcutaneous injection, or injection at the site of pain, the active ingredient will be in a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using isotonic media such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0311] Whether the substance administered to an individual is a peptide, antibody (e.g., anti-TREM2 antibody), nucleic acid, small molecule, or other pharmaceutically useful compound, administration is preferably at a “therapeutic effective amount” or a “preventive effective amount” (as the case may be, although prevention can be considered treatment), sufficient to demonstrate benefit to the individual. The actual dosage, as well as the rate and timing of administration, will depend on the nature and severity of the protein aggregation disorder being treated. The designation of treatment, such as the determination of dosage, is within the purview of the general practitioner and other physicians, and generally takes into account the condition to be treated, the individual subject's condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0312] Depending on the condition to be treated, the composition may be used alone or in combination with other treatments, either simultaneously or sequentially.

[0313] method

[0314] Preparation method

[0315] The antibodies described herein can be generated using, for example, the recombinant methods and compositions described in U.S. Patent No. 4,816,567.

[0316] In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. The nucleic acid may encode an amino acid sequence comprising a VL containing the antibody and / or an amino acid sequence comprising a VH containing the antibody (e.g., the light chain and / or heavy chain of the antibody) or an amino acid sequence comprising a VHH containing a single-domain antibody. In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In one embodiment, the nucleic acid is provided in a polycistronic vector. In another embodiment, a host cell comprising the nucleic acid is provided. In one embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL containing the antibody and an amino acid sequence comprising a VH containing an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL containing an antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH containing an antigen-binding polypeptide construct. In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for preparing an antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0317] For recombinant antibody production, the nucleic acid encoding the antibody, such as that described above, is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The nucleic acid can be isolated and sequenced using routine procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

[0318] The term "substantially purified" means that the construct or variant thereof described herein is substantially or substantially free of components that are typically associated with or interact with proteins as found in their natural environment, i.e., native cells or, in the case of recombinant heteropolymers. In some embodiments, the construct or variant thereof described herein is substantially free of cellular material, including protein formulations 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 proteins. When the heteropolymer or variant thereof is recombinantly generated from host cells, in some embodiments, the protein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the cell dry weight. When the heteropolymer or its variants are recombined from host cells, in some embodiments the protein is present in the culture medium at a cell dry weight of 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. In some embodiments, the “substantially purified” heteropolymers produced by the methods described herein have a purity level 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%, or at least about 70%, particularly, at least about 75%, 80%, or 85%, and more particularly, at least about 90%, at least about 95%, at least about 99%, or greater, as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0319] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.

[0320] "Recombinant host cell" or "host cell" refers to a cell containing exogenous polynucleotides, independent of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for creating recombinant host cells. The exogenous polynucleotides may be maintained in the form of a non-integrating vector, such as a plasmid, or may be integrated into the host genome. Host cells may include CHO, CHO derivatives, NSO, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0321] As used in this article, the term "eukaryote" refers to organisms belonging to the phylogenetic domain eukaryotic domain, 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, protozoa, etc.

[0322] As used in this article, the term "prokaryote" refers to a prokaryotic organism. For example, non-eukaryotic organisms may belong to the phylogenetic domains of eubacteria (including but not limited to Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) or archaea (including but not limited to Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium (such as Haloferax volcanii and Halobacterium species NRC-1), Archaeoeglobus fulgidus, Pyrococcus furiosus, and Pyrococcus huskae). (e.g., horikoshii, Aeuropyrum pernix) phylogenetic domain.

[0323] For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (ed. BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.) After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0324] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0325] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting fall armyworm (Spodoptera frugiperda) cells.

[0326] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0327] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth can be useful. Other examples of useful mammalian host cell lines include: monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); and mouse mammary tumors (MMT). 060562); TRI cells, such as those described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0328] In one embodiment, the antibody described herein is generated in stable mammalian cells by: transfecting at least one stable mammalian cell with a nucleic acid encoding the antibody at a predetermined ratio; and expressing the nucleic acid in at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acid is determined in a transient transfection experiment to determine the relative ratio of the input nucleic acid that results in the highest percentage of antibody in the expressed product.

[0329] One embodiment is a method of generating antibodies in stable mammalian cells as described herein, wherein the expression product of at least one stable mammalian cell contains a greater percentage of the desired glycosylated antibody compared to a single heavy chain or light chain polypeptide or other antibody.

[0330] In some embodiments, a method for generating glycosylated antibodies in stable mammalian cells as described herein includes identifying and purifying the desired glycosylated antibody. In some embodiments, the identification is achieved by one or both of liquid chromatography and mass spectrometry.

[0331] If necessary, the antibody can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques performed at atmospheric or high pressure using systems such as FPLC and HPLC, including ion exchange, hydrophobic interactions, affinity, size fractionation, or gel filtration and reversed-phase chromatography. Purification methods also include electrophoresis, immunoassay, precipitation, dialysis, and chromatographic focusing. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, many natural proteins bind to Fc and antibodies, and these proteins can be used in this invention to purify antibodies. 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 achieved using specific fusion couplers. For example, if GST fusion is used, then glutathione resin can be used to purify the antibody; if a His tag is used, then Ni... +2Antibodies can be purified using affinity chromatography, or, if a flag tag is used, by immobilized anti-flag antibodies. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Edition, Scopes, Springer-Verlag, NY, 1994, which is incorporated herein by reference in its entirety. The degree of purification required will vary depending on the intended use of the antibody. In some cases, purification is unnecessary.

[0332] In some embodiments, antibodies are purified using anion exchange chromatography methods including, but not limited to, the following: Q-agarose, DEAE agarose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, and Fractogel Q and DEAE column chromatography.

[0333] In certain embodiments, the proteins described herein are purified using cation exchange chromatography including, but not limited to, the following: SP-agarose, CM agarose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and analogues.

[0334] Furthermore, the antibodies described herein can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY, and Hunkapiller et al., Nature, 310:105-111 (1984)). For instance, peptides corresponding to fragments of a polypeptide can be synthesized using a peptide synthesizer. Additionally, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions. Non-classical amino acids include, but are not limited to, D-isomers of 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, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, sulfoalanine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, and designed amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and generally amino acid analogs. Furthermore, amino acids can be D-(dextral) or L-(levorotatory) amino acids.

[0335] How to use

[0336] In one aspect, this application provides a method for contacting unstimulated myeloid cells with an anti-TREM2 antibody, such as a human antibody, said contact resulting in the inactivation of the unstimulated myeloid cells.

[0337] On the other hand, this application provides a method for contacting unstimulated myeloid cells with an anti-TREM2 mouse antibody, said contact resulting in the inactivation of the unstimulated myeloid cells.

[0338] In some implementations, the non-stimulated cells are one or more of DC1 cells and TAM cells.

[0339] In some embodiments, this application provides a method for incapacitating unstimulated myeloid cells, the method comprising contacting the unstimulated myeloid cells with a TREM2 antibody, thereby killing the unstimulated myeloid cells. Incapacitating means causing the cells to be partially or completely nonfunctional. In some embodiments, incapacitating unstimulated myeloid cells leads to the induction of growth arrest in the cells. In some embodiments, incapacitating unstimulated myeloid cells leads to apoptosis of the cells. In some embodiments, incapacitating unstimulated cells leads to cell lysis, such as by complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC). In some embodiments, incapacitating unstimulated myeloid cells leads to cell necrosis. In some embodiments, incapacitating unstimulated myeloid cells leads to the induction of growth arrest in the cells. In some embodiments, incapacitating unstimulated myeloid cells leads to cell inactivation. In some embodiments, incapacitating unstimulated myeloid cells leads to the neutralization of the activity of the TREM2 protein in the cells. In some embodiments, incapacitating unstimulated myeloid cells leads to reduced cell proliferation. In some embodiments, deactivation of unstimulated myeloid cells leads to cell differentiation. In some embodiments, deactivation of unstimulated myeloid cells results in a reduced ability of cells to act as inhibitory antigen-presenting cells or an increased ability of cells to act as activating antigen-presenting cells. In some embodiments, deactivation of unstimulated myeloid cells leads to mislocalization of cells within tumor tissue or the tumor microenvironment (TME). In some embodiments, deactivation of unstimulated myeloid cells leads to spatial organizational changes of cells within tumor tissue or the tumor microenvironment. In some embodiments, deactivation of unstimulated myeloid cells leads to temporal expression changes of cells within tumor tissue or the TME. In some embodiments, the method further includes the removal of unstimulated myeloid cells.

[0340] Any increase, decrease, or alteration in any and all aspects of the loss of capacity in non-stimulated myeloid cells as described herein, relative to cells not exposed to the anti-TREM2 antibody.

[0341] In another aspect, this application provides a method for contacting unstimulated myeloid cells with an anti-TREM2 antibody, said contact resulting in modulation of the function of the unstimulated myeloid cells. Modulation may 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 the inactivation of the unstimulated myeloid cells. In some embodiments, the modulation of the function of the unstimulated myeloid cells results in an increased ability of the cells to stimulate both native CD8+ T cells and activated CD8+ T cells, for example, by increasing the ability of the unstimulated cells to cross-present tumor antigens on MHCI molecules to native CD8+ T cells. In some embodiments, the modulation increases the T-cell stimulation function of the unstimulated myeloid cells, including, for example, the ability of the cells to trigger T-cell receptor (TCR) signaling, T-cell proliferation, or T-cell cytokine production. In one embodiment, the survival of the unstimulated cells is reduced, or the proliferation of the unstimulated cells is reduced. In one embodiment, the ratio of stimulated myeloid cells to unstimulated myeloid cells is increased.

[0342] Any increase, decrease, or alteration in any and all aspects that reduce the function of non-stimulated myeloid cells as described herein, relative to cells not exposed to the anti-TREM2 antibody.

[0343] In some embodiments, this application provides a method for killing (also referred to as inducing cell death) unstimulated myeloid cells, the method comprising contacting the unstimulated myeloid cells with an anti-TREM2 antibody, thereby killing the unstimulated myeloid cells. In some embodiments, the killing is increased relative to unstimulated myeloid cells that have not been contacted with the anti-TREM2 antibody. In some embodiments, contact induces apoptosis of the unstimulated myeloid cells. In some embodiments, contact induces apoptosis of the unstimulated myeloid cells. In some embodiments, the unstimulated myeloid cells are in an immune cell population comprising both unstimulated and stimulated myeloid cells. In some embodiments, the method further includes removing the unstimulated myeloid cells. In some embodiments, 10%-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.

[0344] In some embodiments, this application provides a method for increasing the ratio of stimulated myeloid cells to non-stimulated myeloid cells in an immune cell population comprising stimulated and non-stimulated myeloid cells, the method comprising contacting the immune cell population with an anti-TREM2 antibody. In some embodiments, the ratio is increased relative to a cell population that has not yet been 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 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 of DC2 cells to DC1+TAM1+TAM2 cells is increased. In some implementations, the ratio is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0345] In some embodiments, prior to exposure, the ratio of stimulated myeloid cells to non-stimulated myeloid cells is in the range of 0.001:1 to 0.1:1. In some embodiments, after exposure, the ratio of stimulated myeloid cells to non-stimulated myeloid cells is in the range of 0.1:1 to 100:1.

[0346] In some embodiments, the number of non-stimulated myeloid cells is reduced. In some embodiments, the stimulated myeloid cells are DC2 cells. In some embodiments, non-stimulated myeloid cells are killed, for example, due to necrosis or apoptosis. In some embodiments, non-stimulated myeloid cells are induced to undergo growth arrest. In some embodiments, non-stimulated myeloid cells cease to proliferate. In some embodiments, the spatial localization of non-stimulated myeloid cells is altered, and their proportion in specific regions of the tumor medulla oblongata (TME) is increased. In some embodiments, the temporal expression of non-stimulated myeloid cells is altered, and their proportion is increased during specific time periods during tumor development.

[0347] In some embodiments, contact is performed in vitro. In some embodiments, contact is performed in vivo. In certain embodiments, contact is performed in vivo in a human. In some embodiments, contact is achieved by administering an anti-TREM2 antibody. In some embodiments, the individual receiving the antibody (such as a human) has cancer.

[0348] In another aspect, the present invention provides a method for treating an immune-related disorder (e.g., cancer) in an individual, the method comprising administering to the individual an effective amount of a composition comprising an anti-TREM2 antibody. In yet another aspect, the present invention provides a method for enhancing an immune response in an individual, the method comprising administering to the individual an effective amount of a composition comprising an anti-TREM2 antibody. In some embodiments, these methods are also provided in combination with other co-therapies such as PDL blockade therapy, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, CTLA4 blockade therapy, anti-CTLA4 antibody, extensive 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 therapies, and conventional chemotherapy.

[0349] In some embodiments, the method further includes determining the expression level of TREM2 protein in a biological sample from an 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 includes the mRNA expression level of the mRNA encoding the TREM2 protein. In some embodiments, the TREM2 protein expression level includes the protein expression level of NSM. In some embodiments, the expression level of TREM2 protein in the sample is detected using a method selected from the group consisting of: FACS, Western blotting, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunoassay methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplexed qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques and FISH, and combinations thereof.

[0350] In another aspect, this application provides a method for determining the presence or absence of non-stimulated myeloid cells in general, or for determining the presence or absence of specific non-stimulated myeloid cells (e.g., DC1 cells, TAM1 cells, and / or TAM2 cells), the method comprising: contacting a cell population containing non-stimulated myeloid cells with an anti-TREM2 antibody; and quantifying the number of non-stimulated myeloid cells. In another aspect, this application provides a method for determining the presence or absence of non-stimulated myeloid cells, the method comprising: contacting a population of immune cells containing both non-stimulated and stimulated myeloid cells with an anti-TREM2 antibody; detecting a complex or portion indicating binding of the antibody to the cells; and optionally, quantifying the number of non-stimulated myeloid cells in the population. In another aspect, a method is provided for determining the relative ratio of non-stimulated myeloid cells to stimulated myeloid cells, the method comprising: contacting an immune cell population containing both non-stimulated and stimulated myeloid cells with an anti-TREM2 antibody; quantifying the number of stimulated and non-stimulated myeloid cells; and determining the relative ratio of non-stimulated to stimulated myeloid cells.

[0351] In the implementation methods described herein for detection and / or quantification, the anti-TREM2 antibody binds to the TREM2 protein, but does not necessarily affect biological responses such as ADCC, although it may have an impact on biological responses.

[0352] In another aspect, the present invention provides a method for identifying an individual who may respond to an immunotherapy (e.g., using an anti-TREM2 antibody) for treating an immune-related disorder (e.g., cancer), the method comprising: detecting the expression level of a TREM2 protein in a biological sample from the individual; and determining, based on the expression level of the TREM2 protein, whether the individual is responsive to the immunotherapy, wherein an elevated level of the TREM2 protein in the individual relative to the TREM2 protein level in a healthy individual indicates that the individual is responsive to the immunotherapy. In some embodiments, these methods can also be used to diagnose an individual's immune-related disorder (e.g., cancer), and based on the expression level of the TREM2 protein, wherein an elevated level of the TREM2 protein in the individual relative to the TREM2 protein level in a healthy individual indicates that the individual has cancer. In some embodiments, the expression level includes the mRNA expression level of an mRNA encoding the TREM2 protein. In other embodiments, the expression level of the TREM2 protein includes the protein expression level of the TREM2 protein. In some embodiments, methods selected from the group consisting of: FACS, Western blotting, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunoassay methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplexed qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques and FISH, and combinations thereof. In these embodiments, anti-TREM2 antibodies bind to the TREM2 protein, but do not necessarily affect biological responses 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.

[0353] This article also discloses a method for enhancing a subject's immune response to tumors or enhancing the efficacy of immunotherapy. Generally, treatments that increase the abundance of SDCs will improve subject outcomes, such as recurrence-free survival, and will enhance the efficacy of cancer immunotherapy. Treatment can increase the relative or absolute abundance of SDC cells in a subject's tumor. Treatment can decrease the relative or absolute abundance of NSM cells in a subject's tumor.

[0354] Exemplary approaches to general treatment strategies include increasing the number of SDCs by systemic introduction of Flt3L. Another approach is to treat the subject's autologous bone marrow or blood cells with Flt3L while blocking CSF1. SDC transcription factors such as IRF8, Mycl1, or BATF3 or ZBTB46, expressed via retroviruses in bone marrow or blood progenitor cell populations, can also be used to drive SDC development. Another treatment strategy involves systemic elimination of NSM cells while selectively pardoning SDCs. This can produce a generally favorable change in the ratios of these populations. Elimination of NSM cells can be achieved by any means, including the administration (systemic or localized to the tumor) of antibodies targeting the TREM2 surface protein.

[0355] In some embodiments, the treatment enhancing SDC is administered as a therapeutic treatment to better enable the subject's natural immune system to control or eradicate cancer. In another embodiment, the treatment enhancing SDC of the present invention is administered in combination with therapeutic treatments such as immunotherapy (the administration prior to, concurrent with, or after the immunotherapy), wherein the treatment enhancing SDC acts as an adjunct or adjuvant treatment to increase the efficacy of the therapeutic treatment.

[0356] Enhancing the immune response includes increasing, maintaining, initiating, or inducing an immune response after administration of a TREM2-bound isolated antibody, compared to the immune response following administration of an isotype control antibody. In some embodiments, enhancing the immune response includes increasing the immune response compared to an isotype control antibody. In some embodiments, enhancing the immune response includes maintaining immunity compared to an isotype control antibody. In some embodiments, enhancing the immune response includes initiating an immune response compared to an isotype control antibody. In some embodiments, enhancing the immune response includes inducing an immune response compared to an isotype control antibody.

[0357] In some implementations, the treatment enhances the immune response in the subject. In some implementations, the enhanced immune response is an adaptive immune response. In some implementations, the enhanced immune response is an innate immune response. In some implementations, the antibody induces a memory immune response.

[0358] In some implementations, the antibody induces an increase in the expression of at least one cytokine or chemokine in cells compared to an isotype control antibody.

[0359] In some embodiments, at least one cytokine or chemokine is selected from the group consisting of IFN-γ, TNF-α, CXCL1, or CXCL10.

[0360] In some implementations, the cytokine or chemokine is CXCL10.

[0361] Application method

[0362] In some embodiments, the methods provided herein can be used to treat immune-related disorders 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.

[0363] In some implementations, for in vivo administration of the anti-TREM2 antibody described herein, the normal dose may range from about 10 ng per kg of individual body weight per day to about 100 mg or more, preferably varying from about 1 mg / kg / day to 10 mg / kg / day, depending on the route of administration. For repeated administration over several days or longer, treatment may continue until the desired suppression of symptoms is achieved, depending on the severity of the disease or condition being treated. An exemplary dosing regimen includes an initial dose of about 2 mg / kg of the anti-TREM2 antibody, followed by a weekly maintenance dose of about 1 mg / kg every other week. Other dosing regimens may be useful depending on the pharmacokinetic decay pattern desired by the physician. For example, dosing to individuals once a week to twenty-one times a week is considered herein. In some embodiments, administration may be performed in the range of about 3 μg / kg to about 2 mg / kg (such as 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). In some embodiments, the dosing frequency is three times daily, twice daily, once daily, every other day, once weekly, 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 monthly, once every two months, once every three months, or longer. Progression of therapy can be easily monitored using routine techniques and assays. The dosing regimen, including the administration of the anti-TREM2 antibody, may vary over time independently of the dose used.

[0364] In some implementations, the methods described herein (such as enhancing the immune response or achieving methods to disenhance the ability of non-stimulated myeloid cells) can be used to treat cancer, thus individuals receiving anti-TREM2 antibodies or anti-TREM2 antibodies who have cancer.

[0365] The antibodies provided in this article can be used to treat any cancer that is suitable for treatment. Cancer can be any carcinoma, adenocarcinoma, soft tissue cancer, sarcoma, teratoma, melanoma, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or brain cancer known in the medical field. In some implementations, the cancer is a solid cancer. In some implementations, the cancer is a liquid cancer. In some implementations, the cancer is immune-evading. In some implementations, the cancer is immune-responsive. In some implementations, the cancer is melanoma, kidney cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer, kidney cancer, bladder cancer, esophageal cancer, kidney cancer, melanoma, leukemia, lymphoma, or mesothelioma. In some implementations, the cancer is colon cancer, pancreatic cancer, or breast cancer.

[0366] In some embodiments, the immune-related disorder is an immune-related disorder associated with the expression of TREM2 protein on non-stimulated myeloid cells (in humans) or the expression of a homolog of TREM2 protein in a non-human species. In some embodiments, the immune-related disorder is an immune-related disorder associated with the overexpression of TREM2 protein on non-stimulated myeloid cells compared to stimulated myeloid cells. In some embodiments, the overexpression of TREM2 mRNA or TREM2 protein is about at least 2-, 5-, 10-, 25-, 50-, or 100-fold higher than that of stimulated myeloid cells.

[0367] In some implementations, antibodies are administered intravenously, intramuscularly, subcutaneously, superficially, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecal, intraventricular, or intranasal routes. An effective amount of anti-TREM2 antibody can be administered to treat cancer. The appropriate dose of anti-TREM2 antibody can be determined based on the type of cancer to be treated, the type of anti-TREM2 antibody, the severity and progression of the cancer, the individual's clinical condition, the individual's clinical history and response to treatment, and the attending physician's judgment.

[0368] Combination therapy

[0369] In some embodiments, the antibodies provided herein are administered in combination with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered in combination 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; mononuclear cell vaccines; antigen-binding proteins that bind both T cells and antigen-presenting cells; BiTE dual antigen-binding proteins; Toll-like receptor ligands; cytokines; cytotoxic therapy; chemotherapy; cell inhibitors; radiotherapy; small molecule inhibitors; small molecule agonists; immunomodulators; and epigenetic regulators, combinations thereof.

[0370] In some implementations, the additional therapeutic agent is an antibody. In some implementations, the additional therapeutic agent is an antibody that binds to one or more proteins on the surface of tumor cells.

[0371] For cancer treatment, anti-TREM2 antibodies can be combined with one or more antibodies that inhibit immune checkpoint proteins. Of particular interest are immune checkpoint proteins displayed on the surface of tumor cells. The most actively studied immune checkpoint receptors in clinical cancer immunotherapy, namely 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 blocking either of these receptors suggests that anti-tumor immunity can be enhanced at multiple levels, and that combination strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.

[0372] PD-1 has two ligands: 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 inhibits T cell activation / function by binding to its receptor PD-1 on T cells. Inhibitors that block the interaction between PD-1 and its homologous ligands PD-L1 and PD-L2 on cancer cells lead to an increase in both T cell activation and function, and prevent cancer cells from escaping the immune system.

[0373] 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.

[0374] 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 the following: atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1), nivolumab (PD-1), pembrolizumab (PD-1), cimiplimab (PD-1), ipilimumab (CTLA4), tremelimumab (CTLA4), or any combination thereof.

[0375] The additional therapeutic agent may be administered by any suitable means. In some embodiments, the antibody and additional therapeutic agent provided herein are included in the same pharmaceutical composition. In some embodiments, the antibody and additional therapeutic agent provided herein are included in different pharmaceutical compositions.

[0376] In embodiments in which the antibodies and additional therapeutic agents provided herein are included in different pharmaceutical compositions, administration of the antibody may occur before, simultaneously with, and / or after administration of the additional therapeutic agent. In some embodiments, administration of the antibodies and additional therapeutic agents provided herein occurs within approximately one month of each other. In some embodiments, administration of the antibodies and additional therapeutic agents provided herein occurs within approximately one week of each other. In some embodiments, administration of the antibodies and additional therapeutic agents provided herein occurs within approximately one day of each other. In some embodiments, administration of the antibodies and additional therapeutic agents provided herein occurs within approximately twelve hours of each other. In some embodiments, administration of the antibodies and additional therapeutic agents provided herein occurs within approximately one hour of each other.

[0377] Medicine boxes and products

[0378] This application provides a kit comprising any one or more of the antibody compositions described herein. In some embodiments, the kit further comprises a component selected from secondary antibodies, reagents for immunohistochemical analysis, pharmaceutically acceptable excipients, and instruction manuals, and any combination thereof. In a particular embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein and one or more pharmaceutically acceptable excipients.

[0379] This application also provides articles comprising any of the antibody compositions or kits described herein. Examples of articles include vials (including sealed vials).

[0380] Example

[0381] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should, of course, be taken into account.

[0382] Unless otherwise indicated, the present invention will be practiced using conventional protein chemistry, biochemistry, recombinant DNA techniques and pharmacological methods that are of skill in the art. These techniques are well described in the literature. See, for example, Tetreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); Allehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods in Enzymology (edited by S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd edition (Plenum Press), Volumes A and B (1992).

[0383] Example 1: Humanization of anti-TREM2 antibody.

[0384] Humanization of clone #237920

[0385] Monoclonal rat IgG specific to mouse TREM2 and human TREM2 2B Clone #237920 (R&D Systems catalog number MAB17291) was used for sequencing and humanization. Briefly, the disulfide bonds in the antibody were reduced with dithiothreitol (DTT), and the free sulfhydryl groups were alkylated with iodoacetamide. The alkylated antibody was digested with sequencing-grade endonuclease, purified using a spin column, and sequenced by LC-MS / MS analysis. The sequence is shown below.

[0386]

[0387]

[0388] The VH and VL sequences were compared with libraries of known human germline sequences available 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 VLκ gene (F+ORF, 74 germline sequences).

[0389] For 237920VH, the human germline IGHV3-23 (allele 1) was selected as the acceptor sequence, and the human heavy chain IGHJ4 (allele 1) junction region (J gene) was selected from... the international ImMunoGeneTicsinformation The sequence of human junctions compiled at www.imgt.org (creator and leader: Marie-Paule Lefranc, Montpellier, France).

[0390] For 237920VL, the human germline IGKV1-39 (allele 1) was selected as the acceptor sequence, and the human light chain IGKJ2 (allele 1) conjugate region (J gene) was selected from... the international ImMunoGeneTicsinformation The sequence of human junctions compiled at www.imgt.org (creator and leader: Marie-Paule Lefranc, Montpellier, France).

[0391] CDRs are determined according to the exemplary or AbM definitions (see Dr. Andrew C.R. Martin's website www.bioinf.org.uk / abs / for a table comparing CDR definitions). The positions of non-CDR residues in the human germline framework (i.e., VH and VL) are altered to the corresponding parental mouse sequences, for example, to optimize the binding of humanized antibodies.

[0392] Table 1A shows the VL, VH, and complete heavy and light chain sequences of the humanized forms created from mAb 237920. Other humanized forms of the parental clone were created from mAb 37017 through additional mutations. Table 1B shows the CDR sequences.

[0393]

[0394]

[0395]

[0396]

[0397] Table 1B – CDRs of humanized antibodies according to exemplary systems

[0398]

[0399] Table 1C – CDRs of humanized antibodies based on the AbM system

[0400]

[0401] Framework comparison of humanized antibodies

[0402] CDR-H1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY60

[0403] CDR-H2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY60

[0404] CDR-H3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWV A SLTNSGGSTYY60

[0405] 3-23*01 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY60

[0406] CDR-H1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDYWGQGTLVTVSS119

[0407] CDR-H2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TR EWAGSGYFDYWGQGTLVTVSS119

[0408] CDR-H3 ADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYC TR EWAGSGYFDYWGQGTLVTVSS119

[0409] 3-23*01 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK----------WGQGTLVTVSS109

[0410] CDR-L1 DIQMTQSPSSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPS60

[0411] CDR-L2 DIQMTQSPSSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNRFTGVPS60

[0412] 1-39*01 DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS60

[0413] CDR-L1 RFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK 107

[0414] CDR-L2 RFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELK 107

[0415] 1-39*01 RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP---PFGQGTKLEIK 106

[0416] Within the VL domain, in the CDR, Asn28, Asn31, Asn32, and Asn53 exhibit low deamidation potential based on sequence and conformation. Asn93 exhibits low to moderate deamidation potential and shows low levels of this post-translational modification. Within the VH domain, Asn31 exhibits low deamidation potential based on sequence and conformation. In CDR-H2, Asn53 exhibits moderate deamidation potential; to prevent post-translational modification, Asn53 can be replaced with Gln, Ser, or Ala, and the maintenance of binding was determined experimentally. In CDR-H3, Trp100 is exposed to solvents and exhibits oxidation potential, especially under stress conditions.

[0417] Endopeptide digestion in solution

[0418] Solution-digestion of monoclonal antibodies (mAbs) with restriction enzymes was performed for mAb sequencing analysis. 50 μg of antibody was reduced with DTT, alkylated with iodoacetamide, precipitated with acetone, and reconstituted in water at a concentration of 1 μg / μL. Solution-digestion of the antibody samples was performed using five single enzymes: Asp-N, chymotrypsin, elastase, trypsin, and pepsin, following 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 by vacuum centrifugation and kept frozen until mass spectrometry analysis.

[0419] Mass spectrometry

[0420] Complete quality measurement

[0421] The mAb samples were denatured, reduced, and acidified. Proteins were then analyzed using an Agilent 1100 HPLC (LC-ESI-TOF MS) system connected to a Waters QToF Ultima Global mass spectrometer. Appropriate LC-MS chromatograms were processed (combined, subtracted, smoothed, and deconvoluted) using Waters MassLynx 4.1 software.

[0422] LC-MS / MS analysis

[0423] The purified peptides were resuspended in 0.1% formic acid, and half of each 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 substrate containing... The peptide was loaded onto a 50 cm (75 μm inner diameter) EASY-Spray column of 2 μm C18 resin (Thermo Fisher Scientific). Elution was performed using a gradient of 0.1% formic acid in 0%–30% acetonitrile over 60 min at a rate of 250 nl / min. The peptide was then introduced into a Q-Exactive mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray ionization source. The instrumentation method consisted of a full MS scan (400–1600 m / z) at 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 70,000. This was followed by 10 data-dependent MS / MS scans at a resolution of 17,500, an AGC target of 5E5, and a maximum ion time of 100 ms, and a microscan. The intensity threshold used to trigger the MS / MS scans was set to 1.0% of the fill rate. Disintegration occurs in the HCD collision pool, where the normalized collision energy is set to 30. Dynamic exclusion is applied using an 8-second setting.

[0424] Table 2 summarizes the biophysical characteristics of the humanized clones. The molecular weight and extinction coefficient of the sum of protein chains functioning in the quaternary structure were estimated. By default, the calculations assumed that contributions from each chain were equal and achieved by monomers. The extinction coefficient is the predicted absorbance of the protein at 280 nm per volume molar concentration, expressed in M0.05. -1 cm -1 The units are expressed as [units]. Potential post-translational modifications such as glycosylation, phosphorylation, and proteolysis are not considered in the molecular weight or extinction coefficient estimates.

[0425]

[0426] Example 2: Generation and characterization of anti-TREM2 antibody

[0427] Antibody production and characterization

[0428] Standard protein expression vectors were transfected into HEK293 cells using standard methods. Cells were then allowed to grow for 7 days and collected. In addition to HEK293, antibodies were also generated in 293 cells (Alexander Weiss, University of Toronto) lacking mammalian α1,6-trehalosyltransferase (FUT8) through CRISPR / Cas9 editing. The pH of the supernatant was adjusted with 1M PBS (pH 7.4), and sodium azide was added to inhibit microbial growth. KanCap A resin was used to capture proteins, and antibodies were eluted with 50 mM citrate (pH 3.5) and 100 mM NaCl after washing with PBS and PBS containing 1 M sodium chloride. Immediately after elution, the solution was neutralized with 1 M Tris (pH 8) containing 0.5 M arginine. Proteins exchanged for PBS were biophysically characterized using standard techniques. Proteins were quantified by OD280, and the number and concentration were determined using calculated extinction coefficients. Reduced and non-reduced SDS-PAGE (Biorad guideline Tris / glycine / SDS, 4–20%) or the Perkin Elmer GXII capillary electrophoresis system were used to determine purity and approximate molecular weight. Aggregation status was determined by HPLC, using a Sepax Zenix-C SEC-300, 3µm. Detection was performed at 280 nm using a 4.6*150 mm size exclusion column and PBS run buffer.

[0429] Antibody affinity measurement using surface plasmon resonance (SPR)

[0430] Using a Biacore T200 (GE Healthcare, UK), binding kinetics were determined by surface plasmon resonance using human TREM2 His (Sino Biological, Beijing, PR China) artificially captured on an S-series CM5 chip via anti-His trapping, or TREM2 human IgG1 Fc fusion protein directly immobilized on the chip via amine conjugation (internally SEC purified to >95% purity). Serial dilutions of the indicated antibody were injected at 30 μL / min for 2 minutes. This was followed by injection of PBS or system buffer at 30 μL / min for 400 seconds to observe dissociation. The binding response was corrected by subtracting the response from that in a blank flow cell. For kinetic analysis, kJ / kJ was used. 缔合 value and k 解离 The overall fit of the values ​​is a 1:1 Langmuir model. K d The value is determined by k 缔合 and k 解离 The ratio is determined.

[0431] Table 3 shows the binding affinity of the antibody to human TREM2-His as measured by SPR.

[0432]

[0433] Table 4 shows the binding affinity of the antibody to human TREM2-Fc as measured by SPR.

[0434]

[0435] At low ligand densities (RL = 500 RU), the binding kinetics of PI37017 to human TREM2-Fc did not lead to a good fit. This data indicates that in rat IgG... 2B Following humanization of clone #237920, the A at position 97 and the K at position 98 of the sequence in SEQ ID NO:31 (clone 37017) may result in a substantial loss of human TREM2 binding. Mutations in these framework residues (A97T and K98R) lead to increased human TREM2 binding achieved by the humanized clone. See, for example, clone 37012.

[0436] Example 3: Cell binding of anti-TREM2 antibody

[0437] Cell binding (measured by EC50):

[0438] 100,000 to 500,000 Expi 293 parental cells or Expi 293 cells overexpressing human or mouse TREM2 were plated in 96-well plates, and dead cells were stained with Zombie Near Infrared (Biolegend). These cells were incubated with a titrant of the indicated unconjugated antibody, obtained at a concentration of 0 to 10 μg / ml, across 8-10 spots at a 1:3 dilution range. These primary unconjugated antibodies were detected using either an Alexa Fluor 647-conjugated anti-human Fc secondary antibody or an anti-mouse Fc secondary antibody (Jackson Immunoresearch), depending on their isotype (hIgG1 or mIgG2a). Alexa Fluor 647 signal was measured by flow cytometry (BD Fortessa X-14, BD Biosciences). In Graphpad Prism (Graphpad Software), the EC50 value is calculated by curve fitting the signal generated by antibody-bound overexpressing cells, which exceeds the background fluorescence generated by HEK293 parental cells.

[0439] This data indicates that in rat IgG 2BFollowing humanization of clone #237920, the A at position 97 and the K at position 98 of the sequence in SEQ ID NO:31 (clone 37017) may result in a substantial loss of human TREM2 binding. Mutations in these framework residues (A97T and K98R) lead to increased human TREM2 binding achieved by the humanized clone. See, for example, clone 37012.

[0440] Table 5 shows the half-maximum saturation binding of anti-TREM2 antibody to TREM2 on the cell surface.

[0441]

[0442]

[0443] Example 4: Combination of PI-7012 and anti-PD-1 antibody improves anti-tumor activity

[0444] Materials and methods

[0445] CT26.WT (CRL-2638) cells were purchased from the American Type Culture Collection (ATCC). All antibodies intended for in vivo use were tested for endotoxin and used at or below 0.2 EU / mg protein. 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). A single point mutation [D265A] was introduced into the Fc region of the mouse IgG1 form of the RMP1-14 antibody to eliminate binding to FcgR, as described in the literature (Nimmerjahn and Ravetch 2005 Science 310:1510-1512). 1 As described in [reference needed]. Mouse IgG1 [clone MOPC-21] and mouse IgG2a [clone C1.18.4] isotype controls were purchased from BioXCell. PI-7012 and Afuc-PI-7012 (containing the CDR sequence of PI37012 and murine-derived in the form of mouse IgG2a) were generated in Expi293 cells (ThermoFisher Scientific) or 293 / FUT8 knockout cells (University of Toronto) as mouse IgG2a and purified using MabSelect Protein A resin (GE Life Sciences). Antibodies were eluted with 0.1M citrate buffer (pH 3.0) and the buffer was exchanged before use.

[0446] All experimental procedures involving live animals were approved by Murigenics’ Institutional Animal Care and Use Committee. Six- to eight-week-old female BALB / c mice were purchased from Taconic and used after a week of acclimatization at the animal facility. CT26 cells were collected after 3 to 7 subcultures following thawing from liquid nitrogen stock, followed by in vivo experiments. The right ventral region of the female Balb / c mice was shaved one day in advance and prepared for injection. Cells were collected on the day of tumor inoculation and used within 30 minutes. To establish subcutaneous tumors, 1x10 cells were implanted. 6 CT26 cells were collected, and tumor growth in mice was then monitored. Tumor volume was calculated using the formula (L×W2) / 2 from the tumor size measurements taken with a caliper, where L is the longer measurement. When the tumor reached an average size of 80–100 cubic millimeters, mice were randomly assigned to the treatment groups shown in Table 6.

[0447]

[0448] Tumor volume and body weight were monitored twice weekly, and plotted for cohort comparison analysis using univariate ANOVA. Mice were euthanized when tumor volume reached approximately 2000 cubic millimeters, when body weight decreased by more than 15% during the study period, or due to other health-related concerns.

[0449] result

[0450] We determined whether the affinity of the mAb for certain FcgRs could increase antitumor activity through glycosylation engineering (i.e., by generating a trehalose-free form of the anti-TREM2 mAb). PI-7012 and afuc-PI-7012 in combination with anti-PD-1 antibodies were tested in a CT26 tumor model. PI-7012 and afuc-PI-7012 showed similar levels of tumor growth inhibition (79% vs. 88% TGI). Treatment with afuc-PI-7012 resulted in a 30% cure rate. FIG. 1A As observed, compared to PI-7012, afuc-PI-7012 exhibited increased antitumor activity when combined with anti-PD-1 antibodies. The effect of trehalose-free PI-7012 on antitumor activity was more clearly observed in the analysis of tumor volume in individual mice. FIG. 1B and FIG. 1C This demonstrates that the absence of trehalosylation in anti-TREM2 antibodies provides a significant therapeutic advantage over core trehalosylated antibodies.

[0451] During the study, there was no significant weight loss in either treatment group. FIG. 2Weight loss is often used as a surrogate measure of treatment-related toxicity. This data indicates that short- or long-term treatment with anti-TREM2 antibodies, either as a single agent or in combination with anti-PD-1 antibodies, is well tolerated and occurs without any significant toxicity being observed.

[0452] Example 5: No significant toxicity associated with anti-TREM2 therapy

[0453] Materials and methods

[0454] Tissues (lung, liver, brain, kidney, and heart) from mice treated in the above examples were preserved in 10% neutral buffered formalin for at least 24 hours, processed using standard histological methods, cut at 5-6 μm, and stained with hematoxylin and eosin. The stained slides were examined using low-power (40-100x) optical microscopy, and images were obtained using HistoWiz. CD68-positive cells were detected using an anti-CD68 antibody (AbD Serotec), and quantification was performed on 8-9 fields of view of 40x sections using an optical microscope.

[0455] result

[0456] Compared to isotype-controlled mice, overall morphological analysis of H&E staining of treated mouse tissues (lung, liver, heart, kidney, and brain) revealed no morphological changes in mice treated with the combination of PI-7012, afuc-PI-7012, and anti-PD-1 antibody. FIG. 3 (Showing staining of lung tissue).

[0457] In addition to H&E staining, anti-CD68 antibody was used to stain the macrophages in the tissue. The intracellular marker CD68 has been documented in the literature as a reliable cytochemical marker for immunostaining of monocytes / macrophages in inflamed tissues and tumors. In the lungs ( FIG. 4A In the study, and in other tissues analyzed, no discernible changes in the number of CD68+ macrophages were observed in any treatment group compared to the control. FIG. 4B This indicates that anti-TREM2-mediated attenuation specifically occurs in the TME.

[0458] Example 6: Limited TREM2 expression in healthy mouse tissues

[0459] Materials and methods

[0460] All animal research was approved by the Murigenics Animal Research Committee. C57BL / 6J-Trem2 em2Adiuj / J (hereinafter referred to as TREM2KO) and control C57BL / 6J mice were obtained from The Jackson Laboratory. Intact lungs, spleens, and bones were collected and immediately processed for flow cytometry. Blood was collected in parallel via cardiac puncture. Tissues were processed into single-cell suspensions using the MiltenyiMACS Tissue Dissociation Kit. Red blood cells were lysed using 1x erythrocyte lysis buffer (Biolegend). Cells were stained with a fixable viability dye (ThermoFisher Scientific) and subsequently processed for cell surface staining. Anti-mouse immunophenotypic analysis antibodies, along with Fc blocking agents, were diluted in FACS buffer (2% FBS, 2mM EDTA, 1×PBS) and stained on ice for 30 min. After staining, cells were washed twice with FACS buffer and then fixed in PBS containing 2% paraformaldehyde for 15 min. All data were collected on an LSR Fortessa flow cytometer (BD) or an Attune flow cytometer (Thermo Fisher) and analyzed using FlowJo software. TREM2KO cell staining is shown in the shaded image, while wild-type cell staining is shown in the hollow image.

[0461] result

[0462] 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, tissue debris clearance, and the resolution of underlying inflammatory responses. 4 Knocking down or removing TREM2 expression from these cells impairs their ability to phagocytose cellular debris and also increases their production of regulatory cytokines. 5 In physiological settings, TREM2 expression is present at very low to no levels in peripheral blood, spleen, liver, or lungs, as seen in the FACS diagram (Figure 5). However, TREM2 expression becomes detectable if lung or liver-resident macrophages are isolated and stained with TREM2 as a pure cell population.

[0463] Example 7: TREM2 is mainly expressed in mouse TAM.

[0464] Materials and methods

[0465] Tumor tissue was processed using standard methods to isolate a single-cell suspension. Briefly, the tumor was finely minced with a razor blade and digested in RPMI-1640 medium containing enzymes from the Miltenyi MACS Dissociation Kit. The tumor was treated in GentleMACs as recommended by the manufacturer 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 washed with FACS buffer. After centrifugation, the cell clumps were resuspended in FACS buffer and stained with an antibody mixture to identify tumor-associated macrophages and other immune cell populations. 6 TREM2KO cell staining is shown in the shaded image, while wild-type cell staining is shown in the hollow image.

[0466] result

[0467] T cells, B cells, NK cells, and other non-myeloid cell populations, as well as CD45-negative cells, do not exhibit detectable TREM2 expression on their cell surfaces. However, myeloid cell subgroups, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), express TREM2 to varying degrees on their cell surfaces. Among TREM2-positive cell types in the tumor microenvironment, the density of receptor expression on TAMs is significantly higher than in other cell types, regardless of tumor origin (CT26 and MC38 show this density on TAMs). FIG. 6 middle).

[0468] Example 8: Limited TREM2 expression in human peripheral blood leukocytes

[0469] Materials and methods

[0470] Peripheral blood mononuclear cells (PBMCs) and negatively sorted CD14+ mononuclear cells obtained from healthy human volunteers were provided by AllCells Inc. Standard protocols were used. 5 Induces in vitro differentiation of CD14+ monocytes. CD14 +Monocytes were cultured in complete medium consisting of RPMI-1640 supplemented with 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin, and 10% heat-inactivated FBS. To trigger macrophage differentiation, 50 ng / mL M-CSF was added to the medium. Medium was replenished every 2–3 days. After 7 days, macrophages were collected by aspiration, and adherent cells were collected by subsequent trypsin digestion. Cells were then centrifuged and resuspended in RPMI-1640 supplemented with antibiotics, 2% FBS, recombinant human IFN-γ, and 100 ng / mL LPS. These macrophages were surface-stained in parallel with PBMCs using a standard myeloid mixture to assess TREM2 staining in cell subgroups. Cells stained with control mAb are shown in shaded images. Cells stained with anti-TREM2 mAb are shown in hollow images.

[0471] result

[0472] like FIG. 7 As observed, compared to any PBMC-based cell type assessed, ex vivo differentiated macrophages exhibited significantly higher TREM2 cell surface receptor density. Similar to observations reported in the literature, monocytes and some neutrophils expressed lower levels of TREM2.

[0473] Example 9: TREM2 is mainly expressed on human TAM

[0474] Materials and methods

[0475] Human tumor tissues were obtained from the Cooperative Human Tissue Network (CHTN). Fresh human tumor tissues were dissociated into single-cell suspensions using the Miltenyi MACS dissociation kit and the gentleMACS protocol. Surface staining of the single-cell suspensions was performed using a pre-validated multicolor FACS kit. All data were collected on an LSR Fortessa flow cytometer (BD) or an Attune flow cytometer (Thermo Fisher) and analyzed using FlowJo software. Numerical values ​​indicate the staining index for each population, defined as anti-TREM2 staining minus isotype control staining.

[0476] result

[0477] Within the tumor microenvironment, TREM2 expression on TAM cells shows differential expression at high levels compared to other cells. FIG. 8This makes it a translation-related biomarker for TAM. Representative histograms of TREM2 antibody (hollow) or allotype control (shaded) staining are shown in various cell populations of mucinous adenocarcinoma. In summary, this data supports the hypothesis that TREM2 targets will assist in specific TAM reduction with relatively low to no concurrent effects on peripheral cell or other tissue-resident immune subgroups.

[0478] Example 10: Antitumor efficacy of anti-TREM2 antibody in combination with anti-PD-1 antibody in multiple allogeneic tumor models.

[0479] Materials and methods

[0480] 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 were at or below 0.2 EU / mg protein. The amino acid sequence of the anti-mouse PD-1 antibody from clone RMP1-14 was determined by mass spectrometry (LC-MS / MS). The single-point mutation D265A was introduced into the Fc region 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. Both PI-7012 and afuc-PI-7012, in the form of 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 0.1M citrate buffer (pH 3.0) and the buffer was exchanged before use.

[0481] All experimental procedures involving live animals were approved by Murigenics’ Institutional Animal Care and Use Committee. Female BALB / c or C57BL / 6 mice (6–8 weeks old) were purchased from Taconic or The Jackson Laboratory and used after one week of acclimatization at the animal facility. Tumor cells were collected after 3–7 subcultures following thawing from liquid nitrogen stock and then used for in vivo experiments. The right ventral region of the female mice was shaved one day prior to tumor cell inoculation and prepared for injection. On the day of tumor inoculation, cells were collected and used within 30 minutes. To establish subcutaneous tumors, 1 x 102 61 x 10 CT26, EMT6 or Panc-02 cells or 1 x 10 5 4T1 cells were implanted into appropriate strains of mice, and tumor growth in the animals was then monitored. Equal volumes of single-cell suspensions of Py8119 cells were mixed with matrix gel (Corning catalog number 354248 or 354263), and then 2 x 10 cells were implanted into each mouse. 6 Each cell.

[0482] The tumor volume was calculated using the formula (L×W2) / 2, based on the tumor size measurement results obtained using a caliper, where L is the longer measurement result. When the tumor reached an average size of 80-100 cubic millimeters, the mice were randomly assigned to the treatment groups shown in Table 7.

[0483] Tumor volume and body weight were monitored twice a week, and plotted for cohort comparison analysis using univariate ANOVA. Mice were euthanized when tumor volume reached approximately 2,000 cubic millimeters, or when body weight decreased by more than 15% during the study period.

[0484]

[0485] result

[0486] The results are summarized in Table 8. Tumor growth inhibition (TGI%) was determined at the end of the dosing period (t) using the formula: TGI% = (1 - {Tt / T0 / Ct / C0} / 1 - {C0 / Ct}) × 100, where Tt = median tumor volume at time t in the combination therapy recipients, T0 = median tumor volume at time 0 in the combination therapy recipients, Ct = median tumor volume at time t in the isotype control recipients, and C0 = median tumor volume at time 0 in the isotype therapy recipients (before the start of treatment).

[0487]

[0488]

[0489] FIG. 9A-FIG. 9F Anti-TREM2 PI-7012 or afuc-PI7012 in combination with anti-PD-1 antibodies demonstrated antitumor activity in multiple allogeneic mouse tumor models. Anti-TREM2 mAb afuc-PI7012 in combination with an anti-PD-1 mAb resulted in significant antitumor activity in the Panc-02 pancreatic tumor model. FIG. 9A Shows the mean + / - standard deviation of the average tumor volume of 10 mice in each group. FIG. 9B , FIG. 9C , FIG. 9D and FIG. 9E This shows the tumor volume over time for individual animals from each treatment group.FIG. 9F This section presents a statistical analysis of the mean tumor volume at day 32 post-implantation. Differences in tumor volume between groups were assessed using statistical analyses available in Graph Pad Prism software. One-way ANOVA and Sidak's multiple comparison test were performed sequentially on the study data.

[0490] like FIG. 9A and FIG. 9D As observed, subcutaneous Panc-02 tumors did not respond to single-agent PD-1 mAb immune checkpoint blockade therapy or single-agent anti-TREM2 mAb afuc-PI-7012 therapy. However, combination therapy with anti-TREM2 mAb afuc-PI-7012 and anti-PD-1 mAb resulted in significant tumor growth inhibition in animals carrying Panc-02 tumors.

[0491] Combination therapy strategies for reversing CD8 T cell depletion, involving myeloid tuning and immune checkpoint-mediated intervention, were tested in multiple allogeneic tumor models. As shown in Table 8, the combination of anti-TREM2 mAb and anti-PD-1 mAb resulted in significant tumor growth inhibition and complete regression in several tested tumor models. Importantly, these allogeneic models were grown in two distinct mouse strains (prototype Th-1 C57BL / 6 and Th-2 BALB / c strains), which are known to have significantly different compositions of immune infiltrates in tumors grown in vivo in these strains.

[0492] Example 11: Long-term anti-tumor immune memory was induced in mice that responded to combination therapy with anti-TREM2 mAb plus anti-PD-1 mAb.

[0493] Materials and methods

[0494] BALB / c mice without tumors, which had been treated with anti-TREM2 mAb plus anti-PD-1 mAb as described in Example 9 of a previous study, were given 1x10 10 ... 6 CT26 tumor cells were re-excited. Tumor volume was measured 25 days post-implantation. Age-matched, treatment-naïve mice received the same number of CT26 cells, and tumor growth was tracked during the study period. No additional treatment was administered to the mice during the study period.

[0495] result

[0496] Mice whose CT26 tumors were cured after combination treatment with anti-TREM2 mAb afuc-PI-7012 and anti-PD-1 mAb established an effective anti-tumor memory response. FIG. 10The cured mice were able to reject any new tumor growth even in the absence of additional treatment, indicating a long-term immune memory against the original implanted tumor. This form of long-term immune memory utilizes the maintenance of a strong CD8+ effector memory response.

[0497] Example 12: Antitumor efficacy of anti-TREM2 antibody in ID8 ovarian tumor model

[0498] Materials and methods

[0499] All antibodies intended for in vivo use were tested for endotoxin and used at or below 0.2 EU / mg protein. The anti-PD-1 antibody (clone RMP1-14) was purchased from Absolute Antibody Inc. or recombinantly produced at Pionyr in the form of mouse IgG1 D265A. Mouse IgG1 (clone MOPC-21) and mouse IgG2a (clone C1.18.4) isotype controls were purchased from BioXCell or recombinantly prepared at Pionyr in HEK293 cells. The chimeric mouse IgG2a form of the anti-TREM2 antibody (PI-7012) was produced at Pionyr in HEK293 cells, and its monodispersity and purity were assessed by SEC and CE-SDS, and endotoxin was also tested.

[0500] All experimental procedures involving live animals were approved by the Institutional 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 week of acclimatization at the animal facility. Mouse ovarian surface epithelial cells overexpressing a firefly luciferase called ID8-Luc (AJES Life Sciences LLC, Stony Brook, NY) were collected after thawing from liquid nitrogen stock and used for in vivo experiments within 3 to 7 subcultures. Cells were collected on the day of tumor inoculation and used within 45 minutes. To establish intraperitoneal tumors, 5 x 10⁶ cells were used. 6 ID8-Luc cells were injected into the right lower quadrant of the abdomen. Twenty days post-injection, all mice were imaged for luciferase activity to recruit them for the study. Forty tumor-bearing animals were recruited based on mean luminescence readings as a proxy for tumor burden. Four treatment groups of 10 animals each were randomly assigned, resulting in a mean tumor luminescence intensity (p / s / cm² / sr) of 4.7 x 10⁻⁶ per group. 4Animals carrying tumors were treated intraperitoneally every 5 days with the indicated antibody, and luminescence images were captured weekly. Mice were injected intraperitoneally with 0.2 mL of 15 mg / mL D-filamentum luciferin (Promega). Ten minutes after the D-filamentum luciferin injection, the mice were imaged in 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 luminescence (p / s / cm2 / sr) covering the target area of ​​the peritoneum.

[0501] result

[0502] like FIG. 11 As shown, treatment with anti-TREM2 antibody alone resulted in a significant reduction in tumor burden, indicating that TREM2 antibody therapy has monotherapy efficacy. Anti-PD1 antibody therapy resulted in a similar reduction in tumor burden, as did the combination of anti-TREM2 and anti-PD1 antibodies.

[0503] Example 13: TREM2 expression increases with disease severity in various cancers.

[0504] Materials and methods

[0505] Organizational Acquisition

[0506] Tumor microarrays (TMAs) from various tumor indications and disease grades were purchased from Reveal Biosciences (San Diego, CA) and included duplicates of 48 patient cases or >96 single cases, in 2 mm core form. At Reveal Biosciences, TMAs were prepared by acquiring tissue fixed in 10% neutral buffered formalin for 24 hours and treated using the same standard operating procedure (SOP). Sections were picked onto Superfrost Plus or Startfrost adhesive slides, and all TMAs were freshly cut into 4 μm sections upon ordering and stored at 4°C.

[0507] TREM2 IHC staining

[0508] For all TMA staining, the IHC assay used to detect TREM2-positive cells in formalin-fixed paraffin-embedded (FFPE) tissues was standardized. First, slides were baked in a 60°C oven for 45 minutes, followed by deparaffining three times in xylene for 5 minutes each time. The slides were then rehydrated in a series of ethanol gradients from 100% to 70% and finally washed with distilled water. A heat-induced antigen retrieval step was performed in a Biocare decapsulation chamber at 110°C in sodium citrate buffer (Sigma, C9999) at pH 6.0 for 15 minutes. To block endogenous peroxidase activity, blocking solution (Vector Labs) was applied to the slides for 15 minutes, followed by rinsing in PBS buffer containing Tween (Alfa Aesar-J63596). Nonspecific binding was blocked by incubating tissue sections overnight at 4°C with blocking solution (Vector Labs) containing goat serum. PIT2D, also known as the 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. The slides were then washed twice with PBS-Tween for 5 minutes each time, followed by incubation for 20 minutes in HRP-polymer-conjugated anti-rabbit secondary antibody (MP-7500 assay kit from Vector Labs) at a 1:500 dilution. The slides were washed twice with PBS-Tween for 5 minutes each time, and then the assay procedure was performed. The DAB substrate was prepared according to the manufacturer's instructions (Abcam, ab64238) and applied to the slides for 3 minutes, followed by thorough rinsing in distilled water. Hematoxylin was used to counterstain the slides for 30 seconds, followed by rinsing with running water, dehydration in a series of ethanol gradients from 70% to 100% ethanol, and drying in xylene. Finally, the stained slides were mounted in media and covered with coverslips for overnight drying.

[0509] Imaging and scoring

[0510] TMA slides were imaged using a bright-field setting on a Vectra microscope (Perkin Elmer-Akoya Biosciences). Following a full slide scan at 10x, TREM2+ cell quantification was assessed using the following IHC scoring system: 0: no staining in the core-internal matrix region; 1: approximately 25% positive cells in the core-internal matrix region; 2: 50% positive cells in the core-internal matrix region; 3: 75% positive cells in the core-internal matrix region. The percentage of positive cells falling between these groups was scored as 0.5 (approximately 12%), 1.5 (approximately 37.5%), 2.5 (approximately 62.5%), and 3.5% (90%). TREM2 staining intensity was determined as low (score 1), moderate (score 2), and strong (score 3). Cores with missing cores or areas where more than half of the core was distorted and folded were removed from the analysis and were not scored. Each core was examined simultaneously by two operators, and the scores for each patient case (with duplicate cores averaged when available) were plotted in a prism file. The analysis removes the missing core or areas where more than half of the region is a distorted folded core, and does not score them.

[0511] The H score for each core is calculated as follows: TREM2 + cell % x intensity score. For example, if a core has an IHC score of 2 (50%) and moderate staining intensity (2), then the calculated H score would be: 50 x 2 = 100.

[0512] result

[0513] FIG. 12A Using IHC staining and scoring methods, TREM2 expression was increased in ovarian cancer at grades II and III. FIG. 12B Using H-score staining and scoring methods, increased TREM2 expression was observed in ovarian cancer at grades II and III. 16 / 30 (53.3%) of ovarian cancers were TREM2 positive. FIG. 12C TREM2 expression was found to be increased in gastric cancer, in grades I, II, and III, as well as in signet ring cell carcinoma and undifferentiated gastric carcinoma. FIG. 12D TREM2 expression was increased in hepatocellular carcinoma (HCC) at grades I, II, and III, as well as in cholangiocarcinoma. FIG. 12E The results showed that TREM2 expression was increased in prostate cancer, specifically in grades II and III. FIG. 12F TREM2 expression is increased in ductal adenocarcinoma, a type of pancreatic cancer. FIG. 12G The results showed that TREM2 expression was increased in bladder cancer, specifically in grades I-II. FIG. 12H Significant TREM2 expression was observed in lung cancer. FIG. 12I Significant TREM2 expression was observed in colon cancer.FIG. 12J Significant TREM2 expression was observed in renal cell carcinoma. FIG. 12K Increased TREM2 expression was observed in breast cancers of all grades. FIG. 12L TREM2 expression was observed in TNBC cancer. FIG. 12M Significant TREM2 expression was observed in endometrial cancer, across all grades, with approximately 85% of tested TMA samples showing a positive result. FIG. 12N As observed, lung adenocarcinoma samples expressed slightly higher levels of TREM2 compared to lung squamous cell carcinoma samples. Therefore, 77 additional lung adenocarcinoma samples were tested to improve the confidence of this test. FIG. 12O Significant TREM2 expression was observed in lung adenocarcinoma and additional lung cancer subtypes. Table 9 provides an overview of TREM2 expression positively correlated with disease grade and negatively correlated with survival. TREM2 expression was determined in 48–150 subjects according to indication using internal IHC scoring.

[0514]

[0515] Example 14: Anti-TREM2 antibody reduces M2-like TAM

[0516] Materials and methods

[0517] Subcutaneous tumors were established in female BALB / c mice by injecting 1 x 10⁶ EMT6 cells as described in Example 11. Once the average tumor volume reached >200 mm³, mice were administered 30 mg / kg of the allotype antibody or afuc-PI-7012 twice, five days apart. Tumor volume was calculated as previously described. Tumors were collected two days after the second dose. Fatty and fibrous material was removed from the tumors by dissection. The tumors were then weighed and processed by a combination of mechanical and enzymatic dissociation to obtain a single-cell suspension. After tissue fragmentation, the tumors were enzymatically digested in a gentleMACS C tube (Miltenyi Biotec, 130-093-235) in a gentleMACS Octo dissociator (Miltenyi Biotec, 130-095-937) using an optimized enzyme mixture (Miltenyi Biotec, Tumor Dissociation Kit, Mouse 130-096-830). After dissociation, the sample was applied to a filter to remove any remaining larger particles. A suite of antibodies was used to surface-stain single-cell suspensions of tumor tissue for flow cytometry. The antibody suite used to assess the myeloid subgroup included antibodies specific to CD45, XCR1, F4 / 80, CD64, CD11c, Ly6C, CD11b, Ly6G, CD24, class II MHC, and lineage markers in the discard channel (CD45R, CD90.2, CD3e, NKp46, CD19, Siglec F). The antibody suite used to assess the lymphoid subgroup included antibodies specific to 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.

[0518] result

[0519] like FIG. 13A As shown, anti-TREM2 antibody treatment resulted in a reduction in tumor size in vivo. Furthermore, compared to allotype antibodies alone, anti-TREM2 antibody therapy reduced MHC II levels. 低 The number of TAMs was increased, and MHCII was added. 高 The number of TAMs ( FIG. 13B Compared to allotype antibodies alone, anti-TREM2 antibody therapy also increased the number of CD8+ T cells and NKp46+ NK cells. FIG. 13C ).

[0520] MHCII 低TAMs, also known as "M2-like" TAMs, possess immunosuppressive activities, such as blocking CD8+ T cell infiltration and expressing and secreting immunosuppressive factors IL-10 and TGFβ. MHCII 高 TAM is also known as “M1-like” TAM. Therefore, as shown in this example, treatment with the TREM2 antibody leads to a reduction in total immunosuppressive M2-like TAM and cells associated with antitumor immunity, such as CD8+ T cells, NKp46+ NK cells, and M1-like MHCII. 高 The increase of TAM.

[0521] Example 15: Cytokine and CD8 assays using anti-TREM2 and anti-PD1 antibodies + T cell stimulation

[0522] As described in Example 11, using 1x10 6Subcutaneous tumors were established by injecting CT26 cells into female BALB / c mice. Once the tumor reached approximately 1000 mm³, mice were administered two doses five days apart with either an allotype antibody, anti-PD1 antibody (5 mg / kg), afuc-PI-7012 (15 mg / kg), or anti-PD1 antibody and anti-TREM2 antibody. Tumors were collected two days after the second dose. Fatty and fibrous material was removed from the tumors by dissection. The tumors were then weighed and processed to obtain a single-cell suspension by a combination of mechanical and enzymatic dissociation. After tissue fragmentation, the tumors were enzymatically digested using a gentleMACS C-tube (Miltenyi Biotec, 130-093-235) in a gentleMACS Octo dissociator (Miltenyi Biotec, 130-095-937) with an optimized enzyme mixture (Miltenyi Biotec, Tumor Dissociation Kit, Mouse 130-096-830). After dissociation, the sample was gently rotated to aggregate the cells, and the clarified supernatant was then collected and treated with a protease inhibitor (Thermo Fisher Scientific, 100X Halt Protease Inhibitor Mixture, 78429) to inactivate tumor dissociation enzymes. After supernatant collection, the aggregated sample was applied to a filter to remove any remaining larger particles. For intratumoral cytokine levels, the cytokine composition of the supernatant was then analyzed using the Meso Scale Discovery Analytical Platform (Meso Scale Discovery, V-PLEX Mouse Cytokine 19-Way Kit, K-15255D-1). For lymphocyte cytokine analysis, single-cell suspensions were stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P8139), iomycin (Thermo Fisher Scientific, I24222), and brefeldin A (BFA) (Sigma-Aldrich, B7651-5MG). Next, the stimulated cells were surface-stained using a suite of antibodies, including those specific to CD45, CD90.2, CD44, CD8α, CD4, and CD44, for flow cytometry. The cells were then fixed and permeable (Thermo Fisher Scientific, Foxp3 / transcription factor staining buffer kit, 00-5523-00) and stained with antibodies against granzyme B (BioLegend, 515406), TNF-α (BioLegend, 506328), IFN-γ (BioLegend, 50584), and FOXP3 (BioLegend, 126406).Cells were analyzed by flow cytometry on an Attune flow cytometer and analyzed using FlowJo software.

[0523] like FIG. 14A As shown, combination therapy with anti-TREM2 and anti-PD1 antibodies induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, and CXCL1. Although anti-TREM2 antibody treatment alone did not induce pro-inflammatory cytokines in this experiment, the combination of TREM2 and PD1 antibodies resulted in greater cytokine production compared to PD-1 antibody alone.

[0524] Furthermore, combination therapy with anti-TREM2 and anti-PD1 antibodies enhanced anti-tumor CD8+ T cells in mice. FIG. 14B As shown, anti-TREM2 antibody and anti-PD-1 antibody alone have minimal effect on the number of CD8+ T cells expressing granzyme B (GrzB), TNF-α, or IFN-γ. However, combination therapy with both anti-TREM2 antibody and anti-PD-1 antibody increases the number of CD8+ T cells expressing granzyme B (GrzB), TNF-α, or IFN-γ.

[0525] Example 16: TREM2 antibody induces CXCL10 secretion

[0526] C57BL / 6 bone marrow mononuclear cells were collected and incubated with 25 ng / ml CSF-1 for 6 days to differentiate into bone marrow-derived macrophages (BMDM). BMDM was then incubated for 24 hours with increasing concentrations of PI-7012, afuc-PI-7012, or an allotype control antibody. Following incubation, the cytokine composition of the cell supernatant was analyzed using the Meso Scale Discovery analyte assay platform (MesoScale Discovery, V-PLEX Mouse Cytokine 19-way kit, K-15255D-1).

[0527] like FIG. 15 As shown, incubation with anti-TREM2 antibodies PI-7012 and afuc-PI-7012 induced a dose-dependent increase in CXCL10 secretion mediated by BMDM. The trehalosylated TREM2 antibody exhibited the largest CXCL10 response. Therefore, TREM2 antibodies induce increased levels of chemokines secreted by BMDM.

[0528] Example 17: TREM2 expression in gastric cancer

[0529] Using firehose_get, we downloaded Level 2 RNAseq data from the Cancer Genome Atlas cohort of gastric cancer samples from the Broad Institute, converted the RSEM values ​​of TREM2 from both tumor and adjacent normal samples to log 2 per million counts, and plotted them in R.

[0530] Prenormalized TREM2 expression profiles and related clinical data from 192 gastric cancer patients were downloaded from the NCBI GEO website (accession number GSE15459). Expression profiles were divided into two groups based on median TREM2 levels. Kaplan-Meier survival curves were plotted for each group, and relevant log-rank tests were performed using the survival and survminer packages in R.

[0531] like FIG. 16 As shown in the left-hand panel, gastric cancer patients exhibit increased TREM2 expression compared to normal tissue samples. Furthermore, TREM2 expression levels in gastric cancer are inversely correlated with patient survival probability. FIG. 16 (See right-hand panel). Therefore, TREM2 expression is elevated in gastric cancer patients, and higher TREM2 expression leads to worse survival outcomes.

[0532] Example 18: TREM2 expression in ovarian cancer

[0533] Materials and methods

[0534] Normalized TREM2 expression profiles and related clinical data from 285 ovarian cancer patients were downloaded from the NCBI GEO website (accession number GSE9899). Expression profiles were divided into three groups based on median, tertile, and quartile levels of TREM2 expression. Kaplan-Mayer survival curves were plotted for each group, and relevant log-rank tests were performed using the survival and survminer packages in R. The tertile group compared the top 33% and bottom 33% of TREM2 expression, and the quartile group compared the top 25% and bottom 25% of TREM2 expression.

[0535] Normalized TREM2 expression and clinical data from the ovarian dataset were also used to compare TREM2 expression and malignancy potential. Tumors were divided into two groups based on pathological examination of tumor phenotype and malignancy potential. TREM2 expression levels between the two groups were compared using the Wilcoxon rank-sum test, implemented in the R statistical programming language.

[0536] result

[0537] Regarding TREM2 低 and TREM 高 Patients who express [these symptoms] FIG. 17A Display the survival curve for the median group. FIG. 17B Display the survival curve of the tertile group, and FIG. 17C The quartile survival curves are shown. TREM2 expression levels in ovarian cancer are inversely correlated with patient survival probability. Furthermore, higher TREM2 expression leads to worse survival outcomes, such as those observed in patients with significantly lower TREM2 expression compared to the quartile split groups (top 33% TREM2 expression, p = 0.0007). FIG. 17B ) or quartile splitting group (first 25% TREM2 expression, p = 0.00049, FIG. 17C ), median split group (top 50% TREM2 expression, p = 0.0041, FIG. 17A The survival probability is shown.

[0538] Furthermore, TREM2 expression is associated with malignancy in ovarian tumors. FIG. 18 Lower TREM2 expression was associated with tumors of low malignancy potential (LMP), while tumors of higher TREM2 expression were more likely to be malignant (MAL), p = 0.0018.

[0539] Example 19: TREM2 is mainly expressed in ovarian tumor TAM.

[0540] Dissociated tumor cells (DTCs) from a single ovarian cancer patient were purchased from Discovery Life Sciences. CD45-positive cells were sorted by flow cytometry to enrich immune cells and encapsulated for single-cell RNA sequencing using the Chromium Controller from 10X Genomics. The resulting raw data were processed sequentially using the CellRanger program in 10X and the Seurat module in the R programming language. Cell populations in t-distributed random nearest neighbor embeddings (tSNE) dimensionality reduction were manually annotated using cell type-specific marker genes, and TREM2 expression was plotted. TREM2+ cells were defined as any cell expressing TREM2.

[0541] Analysis of single-cell sequencing results from CD45+ immune infiltrates sorted from human ovarian tumor cells showed that TREM2 was mainly expressed on tumor-associated macrophages (TAMs). FIG. 20 (The percentage of TREM2+ cells is shown in dark gray).

[0542] Example 20: Soluble TREM2 Levels in Ovarian Cancer

[0543] Materials and methods

[0544] The plasma concentration of soluble human TREM2 in ovarian cancer samples was determined as described below.

[0545] Human soluble TREM2 (hsTREM2) in plasma samples obtained from commercial suppliers was quantified using a sandwich immunoassay developed on the MSD platform. A biotinylated mouse monoclonal antibody against human TREM2 (clone 7245, an internally developed antibody) was used as a trapping agent and bound to a pre-coated streptavidin plate. After washing, plasma samples from ovarian cancer patients (n=28) or non-cancer patients (n=32) were added to the plate. The soluble TREM2 protein in the samples bound to the trapping antibody and was detected by a sulfonated mouse monoclonal antibody against hTREM2 (clone 7222, an internally developed antibody). The bound soluble TREM2 was then quantified by electrochemiluminescence (ECL).

[0546] The MSD 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.

[0547] result

[0548] Increased levels of soluble human TREM2 were detected in plasma samples from ovarian cancer patients. FIG. 19 This study compares the amount (ng / ml) of soluble TREM2 in plasma samples from ovarian cancer patients and normal non-cancer plasma samples. Patients with ovarian cancer had higher levels of soluble TREM2 in their serum. The mean plasma concentration of TREM2 in ovarian cancer samples was 11.11 ng / ml, while the mean plasma concentration of TREM2 in non-cancer samples was 4.8 ng / ml (p < 0.0001).

[0549] Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0550] All references, patents and patent applications cited in the body of this specification are incorporated herein by reference in their entirety for all purposes.

[0551] References

[0552] 1. Nimmerjahn, F. & Ravetch, J. V. Divergent Immunoglobulin G Subclass Activity Through Selective Fc Receptor Binding Science(80-). 310, 1510 LP-1512 (2005).

[0553] 2. Ford, J. W. & McViear, D. W. TREM and TREM-like receptors in inflamation and disease. Curr. Opin. Immunol. 21, 38 - 46 (2009).

[0554] 3. Colonna, M. TREMs in the immune system and beyond. Nat Rev. Immunol. 3, 445 (2003).

[0555] 4. Takahashi, K., Rochford, C. D. P. & Neumann, H. Clearance of apoptotic neurons without inflamation by microglial triggering receptor expressed on myeloid cells-2. J. Exp. Med. 201, 647 LP-657 (2005).

[0556] 5. Piccio, L. et al. Blockade of TREM-2 exacerbates experimental autoimmune encephalomyelitis. Eur. J. Immunol. 37, 1290 - 1301 (2007).

[0557] 6. Broz, M. L. et al. Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell Immunity. Cancer Cell 26, 638 - 652 (2017).

[0558] Sequence

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

Claims

1. Use of isolated humanized antibodies against human TREM2 in the preparation of medicaments for the treatment of ovarian cancer in subjects of need. The antibody comprises: a. CDR-H1 as described in SEQ ID NO: 9, b. CDR-H2 as described in SEQ ID NO:10, c. CDR-H3 as described in SEQ ID NO:11 d. CDR-L1 as described in SEQ ID NO: 12, e. CDR-L2 as described in SEQ ID NO: 13, and f. CDR-L3 as described in SEQ ID NO: 14, or The antibody comprises: a. CDR-H1 as described in SEQ ID NO: 39, b. CDR-H2 as described in SEQ ID NO: 40, c. CDR-H3 as described in SEQ ID NO: 41, d. CDR-L1 as described in SEQ ID NO: 42, e. CDR-L2 as described in SEQ ID NO: 43, and f. CDR-L3 as described in SEQ ID NO:

44.

2. The use as claimed in claim 1, wherein the antibody is non-trehalosylated and comprises the VH sequence shown in SEQ ID NO: 1; the VL sequence shown in SEQ ID NO: 2; and the active human IgG1 Fc region.

3. The use as claimed in claim 1, wherein the antibody comprises an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a VH sequence comprising a K to R substitution at position 98 of the sequence shown in SEQ ID NO:

7.

4. The use as claimed in claim 1, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3 or 5.

5. The use as claimed in claim 1, wherein the antibody comprises the VL sequence shown in SEQ ID NO: 2, 4 or 6.

6. The use as claimed in claim 1, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3 or 5 and the VL sequence shown in SEQ ID NO: 2, 4 or 6.

7. The use as claimed in claim 1, wherein the antibody comprises the VH sequence shown in SEQ ID NO:

1.

8. The use as claimed in claim 1, wherein the antibody comprises the VL sequence shown in SEQ ID NO:

2.

9. The use as claimed in claim 1, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown in SEQ ID NO:

2.

10. The use as claimed in claim 1, wherein the antibody comprises the heavy chain sequence shown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO:

26.

11. The use according to any one of claims 1-10, wherein, according to surface plasmon resonance (SPR) determination, the antibody has a value of less than or equal to 5 x 10⁻⁶. -9 M of K D Combined with human TREM2.

12. The use according to any one of claims 1-10, wherein the antibody is capable of specifically killing, reducing, or rendering TREM2+ myeloid cells incapable.

13. The use as described in claim 12, wherein the TREM2+ myeloid cells are non-stimulated myeloid cells.

14. The use as claimed in claim 12, wherein the TREM2+ myeloid cells are intratumoral myeloid cells.

15. The use according to any one of claims 1-10, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

16. The use according to any one of claims 1-10, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity.

17. The use according to any one of claims 1-10, wherein the antibody has complement-dependent cytotoxic (CDC) activity.

18. The use according to any one of claims 1-10, wherein the antibody is at least one of: monoclonal antibody, IgG1 antibody, IgG3 antibody, trehalosyl-free antibody, human antibody, chimeric antibody, full-length antibody, and antigen-binding fragments thereof.

19. The use as described in any one of claims 1-10, wherein the antibody is a monoclonal antibody.

20. The use according to any one of claims 1-10, wherein the antibody is untrehaloxylated.

21. The use according to any one of claims 1-10, wherein the antibody is an antigen-binding fragment thereof, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a linear antibody, or a V-domain antibody.

22. The use as claimed in any one of claims 1-10, wherein the antibody comprises a backbone.

23. The use as claimed in claim 22, wherein the skeleton is an Fc or a human Fc.

24. The use according to any one of claims 1-10, wherein the antibody comprises a heavy chain constant region selected from the classes of IgG, IgA, IgD, IgE and IgM.

25. The use according to any one of claims 1-10, wherein the antibody comprises a heavy chain constant region of IgG class and subclasses selected from IgG1, IgG2, IgG3 and IgG4.

26. The use as claimed in claim 25, wherein the antibody comprises the heavy chain constant region of IgG1.

27. The use as claimed in claim 23, wherein the Fc comprises one or more modifications, wherein the one or more modifications result in an increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more modifications.

28. The use as claimed in claim 23, wherein the Fc binds to an Fcγ receptor selected from the group consisting of: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

29. The use according to any one of claims 1-10, wherein the antibody binds to the extracellular domain of TREM2 on TREM2+ myeloid cells.

30. The use according to any one of claims 1-10, wherein the antibody binds to the extracellular domain of TREM2 on myeloid cells, wherein the myeloid cells are CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3- non-stimulated myeloid cells, wherein the antibody kills the non-stimulated myeloid cells via ADCC, CDC, and / or ADCP, rendering the non-stimulated myeloid cells incapacitated or reduced to a level lower than the level of non-stimulated myeloid cells present in the ovarian cancer before the non-stimulated myeloid cells come into contact with the antibody, wherein the non-stimulated myeloid cells are present in an immune cell population comprising CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+ stimulated myeloid cells and the non-stimulated myeloid cells, and wherein killing the non-stimulated myeloid cells, rendering the non-stimulated myeloid cells incapacitated or reduced, treats the ovarian cancer.

31. The use as claimed in claim 30, wherein the contact enhances the immune response in the subject.

32. The use as described in claim 31, wherein the enhanced immune response is an adaptive immune response.

33. The use as described in claim 31, wherein the enhanced immune response is an innate immune response.

34. The use as described in any one of claims 1-10, wherein the subject has previously received, is receiving concurrently with, or will subsequently receive immunotherapy.

35. The use as described in claim 34, wherein the immunotherapy is a checkpoint inhibitor.

36. The use as described in claim 35, wherein the immunotherapy is a T-cell checkpoint inhibitor.

37. The use as described in claim 34, wherein the immunotherapy is selected from the group consisting of: anti-PD1 antibody, anti-PDL1 antibody; or anti-CTLA4 antibody.

38. The use as described in claim 34, wherein the immunotherapy is adoptive T-cell therapy.

39. The use as described in claim 38, wherein the immunotherapy is CAR-T cell therapy.

40. The use as described in claim 34, wherein the immunotherapy is an antigen-binding protein that binds both T cells and antigen-presenting cells or a BiTE dual antigen-binding protein.

41. The use as described in claim 34, wherein the immunotherapy is an immunomodulator.

42. The use as described in claim 34, wherein the immunotherapy is a Toll-like receptor ligand or a cytokine.

43. The use as described in claim 34, wherein the immunotherapy is a cytotoxic therapy.

44. The use as described in claim 34, wherein the immunotherapy is chemotherapy or radiotherapy.

45. The use as described in claim 34, wherein the immunotherapy is a small molecule inhibitor or a small molecule agonist.

46. ​​The use as described in claim 34, wherein the immunotherapy is an epigenetic regulator.

47. Use of isolated humanized antibodies conjugated to human TREM2 in the preparation of medicaments for the treatment of gastric cancer in subjects of need. The antibody comprises: a. CDR-H1 as described in SEQ ID NO: 9, b. CDR-H2 as described in SEQ ID NO:10, c. CDR-H3 as described in SEQ ID NO:11 d. CDR-L1 as described in SEQ ID NO: 12, e. CDR-L2 as described in SEQ ID NO: 13, and f. CDR-L3 as described in SEQ ID NO: 14 or The antibody comprises: a. CDR-H1 as described in SEQ ID NO: 39, b. CDR-H2 as described in SEQ ID NO: 40, c. CDR-H3 as described in SEQ ID NO: 41, d. CDR-L1 as described in SEQ ID NO: 42, e. CDR-L2 as described in SEQ ID NO: 43, and f. CDR-L3 as described in SEQ ID NO:

44.

48. The use as claimed in claim 47, wherein the antibody is non-trehalosylated and comprises the VH sequence shown in SEQ ID NO: 1; the VL sequence shown in SEQ ID NO: 2; and the active human IgG1 Fc region.

49. The use as claimed in claim 47, wherein the antibody comprises an A to T substitution at position 97 of the sequence shown in SEQ ID NO:7; and a VH sequence comprising a K to R substitution at position 98 of the sequence shown in SEQ ID NO:

7.

50. The use as claimed in claim 47, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3 or 5.

51. The use as claimed in claim 47, wherein the antibody comprises the VL sequence shown in SEQ ID NO: 2, 4 or 6.

52. The use as claimed in claim 47, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1, 3 or 5 and the VL sequence shown in SEQ ID NO: 2, 4 or 6.

53. The use as claimed in claim 47, wherein the antibody comprises the VH sequence shown in SEQ ID NO:

1.

54. The use as claimed in claim 47, wherein the antibody comprises the VL sequence shown in SEQ ID NO:

2.

55. The use as claimed in claim 47, wherein the antibody comprises the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown in SEQ ID NO:

2.

56. The use as claimed in claim 47, wherein the antibody comprises the heavy chain sequence shown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO:

26.

57. The use according to any one of claims 47-56, wherein, according to surface plasmon resonance (SPR) determination, the antibody has a value of less than or equal to 5 x 10⁻⁶. -9 M of K D Combined with human TREM2.

58. The use as described in any one of claims 47-56, wherein the antibody is capable of specifically killing, reducing, or rendering TREM2+ myeloid cells incapable.

59. The use as claimed in claim 58, wherein the TREM2+ myeloid cells are non-stimulated myeloid cells.

60. The use as claimed in claim 58, wherein the TREM2+ myeloid cells are intratumoral myeloid cells.

61. The use according to any one of claims 47-56, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

62. The use according to any one of claims 47-56, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity.

63. The use according to any one of claims 47-56, wherein the antibody has complement-dependent cytotoxic (CDC) activity.

64. The use according to any one of claims 47-56, wherein the antibody is at least one of: monoclonal antibody, IgG1 antibody, IgG3 antibody, trehalosyl-free antibody, human antibody, chimeric antibody, full-length antibody, and antigen-binding fragments thereof.

65. The use as described in any one of claims 47-56, wherein the antibody is a monoclonal antibody.

66. The use as described in any one of claims 47-56, wherein the antibody is non-trehalosylated.

67. The use as described in any one of claims 47-56, wherein the antibody is an antigen-binding fragment thereof, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a linear antibody, or a V-domain antibody.

68. The use as claimed in any one of claims 47-56, wherein the antibody comprises a backbone.

69. The use as claimed in claim 68, wherein the skeleton is an Fc or a human Fc.

70. The use according to any one of claims 47-56, wherein the antibody comprises a heavy chain constant region selected from the classes of IgG, IgA, IgD, IgE and IgM.

71. The use according to any one of claims 47-56, wherein the antibody comprises an IgG class and a heavy chain constant region selected from subclasses of IgG1, IgG2, IgG3 and IgG4.

72. The use according to any one of claims 47-56, wherein the antibody comprises the heavy chain constant region of IgG1.

73. The use as claimed in claim 69, wherein the Fc comprises one or more modifications, wherein the one or more modifications result in an increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more modifications.

74. The use as claimed in claim 69, wherein the Fc binds to an Fcγ receptor selected from the group consisting of: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

75. The use as described in any one of claims 47-56, wherein the antibody binds to the extracellular domain of TREM2 on TREM2+ myeloid cells.

76. The use as claimed in claim 75, wherein the TREM2+ myeloid cells are within the tumor.

77. The use according to any one of claims 47-56, wherein the antibody binds to the extracellular domain of TREM2 on myeloid cells, wherein the myeloid cells are CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3- non-stimulated myeloid cells, wherein the antibody kills the non-stimulated myeloid cells via ADCC, CDC, and / or ADCP, rendering the non-stimulated myeloid cells incapacitated or reduced to a level lower than the level of non-stimulated myeloid cells present in the gastric cancer before the non-stimulated myeloid cells come into contact with the antibody, wherein the non-stimulated myeloid cells are present in an immune cell population comprising CD45+, HLA-DR+, CD14-, CD11c+, BDCA1-, and BDCA3+ stimulated myeloid cells and the non-stimulated myeloid cells, and wherein killing the non-stimulated myeloid cells, rendering the non-stimulated myeloid cells incapacitated or reduced, treats the gastric cancer.

78. The use as claimed in claim 77, wherein the contact enhances the immune response in the subject.

79. The use as described in claim 78, wherein the enhanced immune response is an adaptive immune response.

80. The use as described in claim 78, wherein the enhanced immune response is an innate immune response.

81. The use as described in any one of claims 47-56, wherein the subject has previously received, is receiving concurrently with, or will subsequently receive immunotherapy.

82. The use as described in claim 81, wherein the immunotherapy is a checkpoint inhibitor.

83. The use as described in claim 82, wherein the immunotherapy is a T-cell checkpoint inhibitor.

84. The use as claimed in claim 81, wherein the immunotherapy is selected from the group consisting of: anti-PD1 antibody, anti-PDL1 antibody; or anti-CTLA4 antibody.

85. The use as described in claim 81, wherein the immunotherapy is adoptive T-cell therapy.

86. The use as described in claim 85, wherein the immunotherapy is CAR-T cell therapy.

87. The use as described in claim 81, wherein the immunotherapy is an antigen-binding protein that binds both T cells and antigen-presenting cells or a BiTE dual antigen-binding protein.

88. The use as described in claim 81, wherein the immunotherapy is an immunomodulator.

89. The use as described in claim 81, wherein the immunotherapy is a Toll-like receptor ligand or a cytokine.

90. The use as described in claim 81, wherein the immunotherapy is a cytotoxic therapy.

91. The use as described in claim 81, wherein the immunotherapy is chemotherapy or radiotherapy.

92. The use as described in claim 81, wherein the immunotherapy is a small molecule inhibitor or a small molecule agonist.

93. The use as described in claim 81, wherein the immunotherapy is an epigenetic regulator.

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