Anti-TREM1 Antibodies and Related Methods

Abstract

To provide antibodies and pharmaceutical compositions for use in enhancing an immune response and / or in treating an immune-related condition in an individual, e.g., cancer.SOLUTION: The present invention provides an isolated antibody that binds to human TREM 1, where the isolated antibody comprises a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, consisting of specific amino acid sequences, respectively.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 802,161, filed February 6, 2019, and U.S. Provisional Patent Application No. 62 / 889,994, filed August 21, 2019, which are incorporated by reference herein in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been transmitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on February 17, 2020, is named PII-010WO_SL.txt and is 92,256 bytes in size. [Background technology]

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

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

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

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

[0007] Triggering receptor expressed on myeloid cells 1 (TREM1, also known as CD354, HGNC:17760, Entrez Gene:54210, UniProtKB:Q9NP99) belongs to the Ig superfamily of receptors and is highly expressed on subsets of myeloid cells, including neutrophils, monocytes, and macrophages. TREM1 lacks signaling motifs; instead, receptor activation is mediated through the adaptor DAP12 (DNAX-activating protein 12), leading to an amplification of the inflammatory response (Bouchon, et al. (2000) J. Immunol. 164(10):4991-4995). Specifically, cross-linking of TREM1 induces the expression of IL-8, myeloperoxidase, TNFα, and MCP-1. TREM1 expression is upregulated on myeloid cells in response to Toll-like receptor stimulation (bacterial and fungal stimuli) and has been shown to contribute to and amplify acute inflammatory responses during septic shock and infection (Cohen, (2001) Lancet. 358:776-778 (Non-Patent Document 7)). The ligand for TREM1 remains unidentified, but PGLYRP1 (peptidoglycan recognition protein 1) has recently been identified as a potent ligand for TREM1 (Read et al., (2015) J of Immunol. 194:1417-1421 (Non-Patent Document 8)). Mice have five activating TREM receptors, including TREM1, 2, 3, 4, and 5, and soluble TREM1 (sTREM1) is released during infection. Mouse TREM1 and its human homolog TREM1 share a relatively low sequence identity of 46% (Radaev, et al. (2003) Structure 11:1527-1535 (Non-Patent Document 9)). Structurally, TREM1 consists of a single immunoglobulin V (Ig)-like domain of approximately 108 amino acids (Ig-V), followed by a 70-amino acid stalk region. In addition to the role that TREM1 plays in sepsis, TREM1 has also been implicated in inflammatory bowel disease. However, little is known about the role of TREM1 in the tumor microenvironment (Schenk, et al. (2007) JCI. 117:3097-3106 (Non-Patent Document 10)).

[0008] There is an unmet need for novel cancer treatment approaches that involve selectively reducing the amount of cells that are ineffective in stimulating T cell responses or repolarizing such cells, thereby enhancing the immune response within the tumor microenvironment.

[0009] Related patent applications include PCT / US2018 / 045680, filed August 7, 2018, which is incorporated by reference in its entirety for all purposes. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] PCT / US2018 / 045680 [Non-patent literature]

[0011] [Non-Patent Document 1] de Visser,Cancer Immunol Immunother,2008;57:1531-9 [Non-patent document 2] Hanada et al.,Int J Urol 2000;7:263-9 [Non-patent document 3] Yao et al.Clin Cancer Res,520,2001;7:4021-6 [Non-patent document 4] Ruffell et al.,PNAS,523 2012;109:2796-801 [Non-Patent Document 5] Bell et al., J Exp Med 555, 1999;190:1417-26 [Non-patent document 6] Bouchon, et al (2000) J. Immunol. 164(10):4991-4995 [Non-Patent Document 7] Cohen,(2001)Lancet.358:776-778 [Non-patent document 8] Read et al,(2015)J of Immunol.194:1417-1421 [Non-Patent Document 9] Radaev, et al. (2003) Structure 11:1527-1535 [Non-Patent Document 10] Schenk,et al(2007)JCI.117:3097-3106 Summary of the Invention

[0012] overview In one aspect, provided herein is an isolated antibody that binds to human TREM1 (SEQ ID NO: 1), wherein the antibody i) binds within residues 21-34 (SEQ ID NO: 42), 103-109 (SEQ ID NO: 43), and 128-136 (SEQ ID NO: 44) of human TREM1 (SEQ ID NO: 1), and comprises a human Fc region.

[0013] In one aspect, provided herein is an isolated humanized antibody that binds to human TREM1 (SEQ ID NO: 1), wherein the antibody i) binds within residues 21-34 (SEQ ID NO: 42), 103-109 (SEQ ID NO: 43), and 128-136 (SEQ ID NO: 44) of human TREM1 (SEQ ID NO: 1), and ii) optionally comprises a human Fc region.

[0014]

[0014] In one aspect, provided herein is an isolated antibody(ies) that binds to human TREM1 (SEQ ID NO: 1), wherein the isolated antibody comprises a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, CDR-H2 comprises the sequence set forth in SEQ ID NO: 24, and CDR-H3 comprises the sequence set forth in SEQ ID NO: 29, wherein X is leucine (L), glutamine (Q), methionine (M), isoleucine (I), or glutamic acid (E), wherein CDR-L1 comprises the sequence set forth in SEQ ID NO: 26, CDR-L2 comprises the sequence set forth in SEQ ID NO: 27, and CDR-L3 comprises the sequence set forth in SEQ ID NO: 28.

[0014] In one aspect, provided herein is an isolated antibody(ies) that binds to human TREM1 (SEQ ID NO: 1), the isolated antibody comprising a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, CDR-H2 comprises the sequence set forth in SEQ ID NO: 24, and CDR-H3 comprises the sequence set forth in SEQ ID NO: 29, wherein X is leucine (L), glutamine (Q), methionine (M), isoleucine (I), or glutamic acid (E), wherein CDR-L1 comprises the sequence set forth in SEQ ID NO: 26, CDR-L2 comprises the sequence set forth in SEQ ID NO: 27, and CDR-L3 comprises the sequence set forth in SEQ ID NO: 28.

[0015] In some embodiments, the antibody comprises a CDR-H3 comprising the sequence RXAAMDY (SEQ ID NO:29), where X is leucine (L), glutamine (Q), methionine (M), isoleucine (I), or glutamic acid (E). In some embodiments, the antibody further comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO:23 and a CDR-H2 comprising the sequence set forth in SEQ ID NO:24. In some embodiments, the CDR-H3 comprises the sequence set forth in SEQ ID NO:33, and the antibody further comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO:23 and a CDR-H2 comprising the sequence set forth in SEQ ID NO:24.

[0016] In some embodiments, the antibody further comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 26, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 27, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 28.

[0017] In some embodiments, the antibody comprises a VH sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 17.

[0018] In some embodiments, the antibody comprises a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22. In some embodiments, the antibody comprises the VL sequence set forth in SEQ ID NO: 20.

[0019] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO:17 and the VL sequence set forth in SEQ ID NO:20.

[0020] In some embodiments, the antibody is an scFv. In some embodiments, the antibody is an scFv and comprises the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20.

[0021] In some embodiments, the antibody is an scFv and comprises a VH sequence selected from the sequence set forth in SEQ ID NO: 16, 17, or 18 and a VL sequence selected from the sequence set forth in SEQ ID NO: 20, 21, or 22. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20, and the human Fc region comprises wild-type human IgG1 Fc.

[0022] In some embodiments, the antibody comprises the heavy chain sequence set forth in SEQ ID NO:34 and the light chain sequence set forth in SEQ ID NO:35.

[0023] In some embodiments, the antibody comprises a VH sequence selected from the sequences set forth in SEQ ID NO:4, 5, or 6.

[0024] In some embodiments, the antibody comprises a VL sequence selected from the sequences set forth in SEQ ID NOs: 20, 21, or 22.

[0025] In some embodiments, the antibody comprises a VH sequence selected from the sequences set forth in SEQ ID NO:8, 9, or 10.

[0026] In some embodiments, the CDR-H3 comprises the sequence set forth in SEQ ID NO: 32, and the antibody further comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 23 and a CDR-H2 comprising the sequence set forth in SEQ ID NO: 24.

[0027] In some embodiments, the antibody comprises a VH sequence selected from the sequences set forth in SEQ ID NO: 12, 13, or 14. In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 13.

[0028] In some embodiments, the antibody comprises a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22. In some embodiments, the antibody comprises the VL sequence set forth in SEQ ID NO: 20.

[0029] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody is an scFv and comprises the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 20.

[0030] In some embodiments, the antibody is an scFv and comprises a VH sequence selected from the sequence set forth in SEQ ID NO: 112, 13, or 14 and a VL sequence selected from the sequence set forth in SEQ ID NO: 20, 21, or 22.

[0031] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 20, and the human Fc region comprises wild-type human IgG1 Fc.

[0032] In some embodiments, the antibody comprises the heavy chain sequence set forth in SEQ ID NO:36 and the light chain sequence set forth in SEQ ID NO:37.

[0033] In some embodiments, the antibody consists of the VH sequence set forth in SEQ ID NO: 17, 13, or 9, and the VL sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody consists of the VH sequence set forth in SEQ ID NO: 17, and the VL sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody consists of the VH sequence set forth in SEQ ID NO: 13, and the VL sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody consists of the VH sequence set forth in SEQ ID NO: 9, and the VL sequence set forth in SEQ ID NO: 20.

[0034] In some embodiments, the antibody consists of the heavy chain sequence set forth in SEQ ID NO:34 and the light chain sequence set forth in SEQ ID NO:35.

[0035] In some embodiments, the antibody consists of the heavy chain sequence set forth in SEQ ID NO:36 and the light chain sequence set forth in SEQ ID NO:37.

[0036] In some embodiments, the antibody is afucosylated.

[0037] In another aspect, provided herein is an isolated antibody that binds to human TREM1 (SEQ ID NO: 1), wherein the antibody is afucosylated, and the antibody comprises a VH sequence set forth in SEQ ID NO: 17, 13, or 9, and a VL sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 17 and a VL sequence set forth in SEQ ID NO: 20.

[0038] In some embodiments, the antibody comprises the heavy chain sequence set forth in SEQ ID NO:34 and the light chain sequence set forth in SEQ ID NO:35.

[0039] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO:13 and the VL sequence set forth in SEQ ID NO:20.

[0040] In some embodiments, the antibody is afucosylated, and the antibody comprises the heavy chain sequence set forth in SEQ ID NO:36 and the light chain sequence set forth in SEQ ID NO:37.

[0041] In some embodiments, the antibody comprises the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:20.

[0042] In some embodiments, the antibody is afucosylated, and the antibody comprises the heavy chain sequence set forth in SEQ ID NO:38 and the light chain sequence set forth in SEQ ID NO:39.

[0043] In some embodiments, the antibody is a humanized antibody, a human antibody, or a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody comprises a human heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human Fc region comprises wild-type human IgG1 Fc.

[0044] In some embodiments, the antibody consists of the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20, and the human Fc region comprises wild-type human IgG1 Fc.

[0045] In some embodiments, the Fc region comprises one or more amino acid substitutions, which result in increased antibody half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more substitutions. In some embodiments, the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

[0046] In some embodiments, the antibody is a monoclonal antibody.

[0047] In some embodiments, the antibody has a molecular weight of about 0.5, 1, 2, 3, 4, 5, 6, or 7 x 10 as measured by a surface plasmon resonance (SPR) assay. -9 In some embodiments, the antibody binds to human TREM1 with a KD of about 7 nM or less as measured by surface plasmon resonance (SPR) assay.

[0048] In some embodiments, the antibody is an agonist antibody.

[0049] In some embodiments, the antibody induces increased expression of at least one cytokine or chemokine in the cells compared to an isotype control antibody.

[0050] In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB). In some embodiments, the cytokine or chemokine is CXCL10 or IFN-γ.

[0051] In some embodiments, the antibody induces increased expression of at least one myeloid costimulatory protein in the cells compared to an isotype control antibody.

[0052] In some embodiments, the myeloid costimulatory protein is HLA-DR, CD40, CD80, or CD86 on the cells.

[0053] In some embodiments, the antibody induces increased activation of the ERK and / or STAT3 intracellular signaling pathways in cells compared to an isotype control antibody.

[0054] In some embodiments, the antibody induces an anti-tumor memory response compared to an isotype control antibody.

[0055] In some embodiments, the antibody is capable of competing with human TREM-26 antibody for binding to human TREM1, binding to human TREM1, binding to cynomolgus monkey TREM1, stimulating TREM1 signaling, inducing immune signaling pathways, inducing cytokine or chemokine secretion, inducing expression of costimulatory molecules, killing, neutralizing, or depleting myeloid cells, or any combination of a-h.

[0056] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody has antibody-mediated phagocytosis (ADCP) activity. In some embodiments, the antibody has complement-dependent cytotoxicity (CDC) activity.

[0057] In some embodiments, the cells are TREM1+ cells.

[0058] In some embodiments, the TREM1+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs). In some embodiments, the TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils.

[0059] In some embodiments, the antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells.

[0060] In some embodiments, the isolated antibody is for use as a medicament. In some embodiments, the isolated antibody is for use in the treatment of cancer or an infectious disease. In some embodiments, the isolated antibody is for use in the treatment of cancer, wherein the cancer is selected from a solid tumor and a liquid tumor.

[0061] In another aspect, provided herein is an isolated polynucleotide or set of polynucleotides encoding an antibody, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof, optionally a cDNA, as described herein.

[0062] In another aspect, provided herein is a vector or set of vectors comprising a polynucleotide or set of polynucleotides described herein.

[0063] In another aspect, provided herein is a host cell comprising a polynucleotide or set of polynucleotides or a vector or set of vectors described herein.

[0064] In another aspect, provided herein is a method of producing an antibody, comprising expressing the antibody in a host cell and isolating the expressed antibody.

[0065] In another aspect, provided herein is a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable excipient.

[0066] In another aspect, provided herein is a kit comprising an antibody or pharmaceutical composition described herein and instructions for use.

[0067] In another aspect, provided herein is a method of increasing an immune response comprising administering to a subject a composition comprising an anti-TREM1 antibody or antigen-binding fragment thereof.

[0068] In some embodiments, the composition comprises an antibody or pharmaceutical composition described herein.

[0069] In some embodiments, the antibody has receptor-ligand blocking activity, agonist activity, or antagonist activity.

[0070] In some embodiments, the antibody has agonist activity.

[0071] In some embodiments, the antibody induces increased expression of at least one cytokine or chemokine in the cells compared to an isotype control antibody.

[0072] In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB).

[0073] In some embodiments, the cytokine or chemokine is CXCL10 or IFN-γ.

[0074] In some embodiments, the antibody induces increased expression of at least one myeloid costimulatory protein compared to an isotype control antibody.

[0075] In some embodiments, the myeloid costimulatory protein is HLA-DR, CD40, CD80, or CD86 on the cells.

[0076] In some embodiments, the antibody induces increased activation of the ERK and / or STAT3 intracellular signaling pathways in cells compared to an isotype control antibody.

[0077] In some embodiments, the antibody induces a memory immune response.

[0078] In some embodiments, the cells are TREM1+ cells.

[0079] In some embodiments, the TREM1+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs).

[0080] In some embodiments, the TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils.

[0081] In some embodiments, the antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells.

[0082] In some embodiments, the subject is a human.

[0083] In another aspect, provided herein is a method of treating cancer comprising administering to a subject a composition comprising an anti-TREM1 antibody or antigen-binding fragment thereof.

[0084] In some embodiments, the composition comprises an antibody or pharmaceutical composition described herein.

[0085] In some embodiments, the antibody has receptor-ligand blocking activity, agonist activity, or antagonist activity.

[0086] In some embodiments, the antibody has agonist activity.

[0087] In some embodiments, the antibody induces increased expression of at least one cytokine or chemokine in the cells compared to an isotype control antibody.

[0088] In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB).

[0089] In some embodiments, the cytokine or chemokine is CXCL10 or IFN-γ.

[0090] In some embodiments, the antibody induces increased expression of at least one myeloid costimulatory protein compared to an isotype control antibody.

[0091] In some embodiments, the myeloid costimulatory protein is HLA-DR, CD40, CD80, or CD86 on the cells.

[0092] In some embodiments, the antibody induces increased activation of the ERK and / or STAT3 intracellular signaling pathways in cells compared to an isotype control antibody.

[0093] In some embodiments, the antibody induces an anti-tumor memory response compared to an isotype control antibody.

[0094] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

[0095] In some embodiments, the antibody has complement dependent cytotoxicity (CDC) activity.

[0096] In some embodiments, the antibody has antibody-mediated phagocytosis (ADCP) activity.

[0097] In some embodiments, the cells are TREM1+ cells.

[0098] In some embodiments, the TREM1+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs).

[0099] In some embodiments, the TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils.

[0100] In some embodiments, the antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells.

[0101] In some embodiments, the subject is a human.

[0102] In some embodiments, the cancer is a solid tumor.

[0103] In some embodiments, the cancer is a liquid cancer.

[0104] In some embodiments, the cancer is selected from the group consisting of melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, urothelial cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, renal cancer, endometrial cancer, cervical cancer, testicular cancer, and mesothelioma.

[0105] In some embodiments, the cancer is gastric cancer, ovarian cancer, colon cancer, or breast cancer.

[0106] In some embodiments, the contacting enhances the subject's immune response.

[0107] In some embodiments, the enhanced immune response is an adaptive immune response.

[0108] In some embodiments, the enhanced immune response is an innate immune response.

[0109] In some embodiments, the subject has previously received, is concurrently receiving, or will subsequently receive immunotherapy.

[0110] In some embodiments, the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive cell therapy; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a STING agonist; a monocyte vaccine; a Bacillus Calmette-Guerin vaccine; an antigen binding protein that binds to both T cells and antigen presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulatory agent; an oncolytic virus; and an epigenetic modulatory agent.

[0111] 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.

[0112] In another aspect, provided herein is a method of killing, neutralizing, or depleting myeloid cells that express triggering receptor expressed on myeloid cells 1 (TREM1) on the cell surface, comprising contacting the myeloid cells with an antibody or pharmaceutical composition described herein.

[0113] In some embodiments, the antibody kills, neutralizes, or depletes bone marrow cells by at least one of ADCC, CDC, and ADCP; optionally, the antibody kills, neutralizes, or depletes bone marrow cells by ADCC; optionally, the antibody kills, neutralizes, or depletes bone marrow cells by CDC; and optionally, the antibody kills, neutralizes, or depletes bone marrow cells by ADCP.

[0114] In some embodiments, the antibody kills bone marrow cells by at least one of ADCC, CDC, and ADCP.

[0115] In some embodiments, the antibody neutralizes the myeloid cells through at least one of ADCC, CDC, and ADCP.

[0116] In some embodiments, the antibody depletes bone marrow cells by at least one of ADCC, CDC, and ADCP.

[0117] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

[0118] In some embodiments, the antibody has complement dependent cytotoxicity (CDC) activity.

[0119] In some embodiments, the antibody has antibody-mediated phagocytosis (ADCP) activity.

[0120] In some embodiments, the antibody has receptor-ligand blocking activity, agonist activity, or antagonist activity.

[0121] In some embodiments, the bone marrow cells are stimulator bone marrow cells.

[0122] In some embodiments, the bone marrow cells are unstimulated bone marrow cells.

[0123] In some embodiments, the myeloid cells comprise at least one of dendritic cells, tumor-associated macrophages (TAMs), neutrophils, monocytes, or myeloid-derived suppressor cells.

[0124] In some embodiments, the bone marrow cells are neutrophils or tumor-associated neutrophils.

[0125] In some embodiments, the myeloid cells are tumor-associated macrophages.

[0126] In some embodiments, the myeloid cells are monocytic myeloid-derived suppressor cells.

[0127] In some embodiments, the bone marrow cells are intratumor.

[0128] In some embodiments, the bone marrow cells are in a population of immune cells that includes stimulated and unstimulated bone marrow cells.

[0129] In some embodiments, the contacting is in vitro or in vivo.

[0130] In some embodiments, the contacting occurs in vivo in a subject, and optionally, the subject has cancer.

[0131] In some embodiments, the subject is a human.

[0132] In some embodiments, the cancer is a solid tumor.

[0133] In some embodiments, the cancer is a liquid cancer.

[0134] In some embodiments, the cancer is selected from the group consisting of melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, urothelial cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, renal cancer, endometrial cancer, cervical cancer, testicular cancer, and mesothelioma.

[0135] In some embodiments, the cancer is gastric cancer, ovarian cancer, colon cancer, or breast cancer.

[0136] In some embodiments, the contacting enhances the subject's immune response.

[0137] In some embodiments, the enhanced immune response is an adaptive immune response.

[0138] In some embodiments, the enhanced immune response is an innate immune response.

[0139] In some embodiments, the enhanced immune response comprises expression of at least one cytokine or chemokine.

[0140] In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB).

[0141] In some embodiments, the at least one cytokine or chemokine is CXCL10 or IFN-γ.

[0142] In some embodiments, the subject has previously received, is concurrently receiving, or will subsequently receive immunotherapy.

[0143] In some embodiments, the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive cell therapy; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a STING agonist; a monocyte vaccine; a Bacillus Calmette-Guerin vaccine; an antigen binding protein that binds to both T cells and antigen presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulatory agent; an oncolytic virus; and an epigenetic modulatory agent.

[0144] 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.

[0145] [The present invention 1001] 1. An isolated antibody that binds to human TREM1 (SEQ ID NO: 1), said antibody comprising: i) binds within residues 21-34 (SEQ ID NO: 42), 103-109 (SEQ ID NO: 43), and 128-136 (SEQ ID NO: 44) of human TREM1 (SEQ ID NO: 1); ii) optionally comprising a human Fc region; The isolated antibody. [The present invention 1002] 1. An isolated antibody that binds to human TREM1 (SEQ ID NO: 1), comprising: a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3; and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 24; c. CDR-H3 comprises the sequence set forth in SEQ ID NO:29, and X is leucine (L), glutamine (Q), methionine (M), isoleucine (I), or glutamic acid (E); d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 26; e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 27; f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 28; The isolated antibody. [The present invention 1003] 1001. An isolated antibody of the present invention, wherein said antibody comprises a CDR-H3 comprising the sequence RXAAMDY (SEQ ID NO: 29), wherein X is leucine (L), glutamine (Q), methionine (M), isoleucine (I), or glutamic acid (E). [The present invention 1004] The isolated antibody of claim 1001 or 1003, further comprising a CDR-H1 comprising the sequence set forth in SEQ ID NO:23 and a CDR-H2 comprising the sequence set forth in SEQ ID NO:24. [The present invention 1005] 33. The isolated antibody of any of the preceding inventions, wherein the CDR-H3 comprises the sequence set forth in SEQ ID NO: 33, and the antibody further comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 23 and a CDR-H2 comprising the sequence set forth in SEQ ID NO: 24. [The present invention 1006] 20. The isolated antibody of any of the preceding inventions, further comprising a CDR-L1 comprising the sequence set forth in SEQ ID NO: 26, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 27, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 28. [The present invention 1007] 16. The isolated antibody of any of the preceding inventions, comprising a VH sequence selected from the sequence set forth in SEQ ID NO: 16, 17, or 18. [The present invention 1008] 1007. An isolated antibody of the present invention, comprising a VH sequence set forth in SEQ ID NO: 17. [The present invention 1009] 20. The isolated antibody of any of the preceding inventions, comprising a VL sequence selected from the sequence set forth in SEQ ID NO: 20, 21, or 22. [The present invention 1010] 20. Any of the preceding isolated antibodies of the present invention comprising the VL sequence set forth in SEQ ID NO:20. [The present invention 1011] 10. Any of the preceding isolated antibodies of the invention, comprising the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20. [The present invention 1012] Any of the isolated antibodies of the preceding invention which is an scFv. [The present invention 1013] The isolated antibody of any of claims 1001 to 1011 of the present invention, which is an scFv and comprises the VH sequence shown in SEQ ID NO: 17 and the VL sequence shown in SEQ ID NO: 20. [The present invention 1014] An isolated antibody according to any one of claims 1001 to 1011 of the present invention, which is an scFv and comprises a VH sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18 and a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22. [The present invention 1015] The isolated antibody of any one of claims 1001 to 1011, wherein the antibody comprises the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20, and the human Fc region comprises wild-type human IgG1 Fc. [The present invention 1016] An isolated antibody according to any one of claims 1001 to 1011 or 1015 of the present invention, comprising a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35. [The present invention 1017] 1002. An isolated antibody of the present invention comprising a VH sequence selected from the sequences set forth in SEQ ID NO: 4, 5, or 6. [The present invention 1018] 1017. An isolated antibody of the invention, comprising a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22. [The present invention 1019] 1017 or 1018. The isolated antibody of the invention, comprising a VH sequence selected from the sequences set forth in SEQ ID NO: 8, 9, or 10. [The present invention 1020] 1002. An isolated antibody of the present invention, wherein the CDR-H3 comprises the sequence set forth in SEQ ID NO: 32, and the antibody further comprises CDR-H1 comprising the sequence set forth in SEQ ID NO: 23 and CDR-H2 comprising the sequence set forth in SEQ ID NO: 24. [The present invention 1021] 1020. An isolated antibody of the invention comprising a VH sequence selected from the sequences set forth in SEQ ID NO: 12, 13, or 14. [The present invention 1022] 1021. An isolated antibody of the present invention comprising a VH sequence set forth in SEQ ID NO: 13. [The present invention 1023] 1023. The isolated antibody of any of claims 1020 to 1022, comprising a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22. [The present invention 1024] 1023. An isolated antibody of the present invention, comprising a VL sequence set forth in SEQ ID NO: 20. [The present invention 1025] The isolated antibody of any one of claims 1001 to 1024 of the present invention, which comprises the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 20. [The present invention 1026] The isolated antibody of any one of 1020 to 1025 of the present invention, which is an scFv and comprises the VH sequence shown in SEQ ID NO: 13 and the VL sequence shown in SEQ ID NO: 20. [The present invention 1027] An isolated antibody according to any one of claims 1001 to 1011 of the present invention, which is an scFv and comprises a VH sequence selected from the sequences set forth in SEQ ID NO: 112, 13, or 14 and a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22. [The present invention 1028] The isolated antibody of any of claims 1020 to 1025, wherein the antibody comprises the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 20, and the human Fc region comprises wild-type human IgG1 Fc. [The present invention 1029] An isolated antibody according to any one of claims 1020 to 1028 of the present invention, comprising a heavy chain sequence set forth in SEQ ID NO: 36 and a light chain sequence set forth in SEQ ID NO: 37. [The present invention 1030] An isolated antibody according to any one of claims 1001 to 1004 or 1006 of the present invention, which consists of a VH sequence set forth in SEQ ID NO: 17, 13, or 9 and a VL sequence set forth in SEQ ID NO: 20. [The present invention 1031] An isolated antibody according to any one of claims 1001 to 1006 of the present invention, which consists of the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20. [The present invention 1032] An isolated antibody according to any one of claims 1001 to 1004 and 1006 of the present invention, which consists of the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 20. [The present invention 1033] An isolated antibody according to any one of claims 1001 to 1004 and 1006 of the present invention, which consists of the VH sequence set forth in SEQ ID NO: 9 and the VL sequence set forth in SEQ ID NO: 20. [The present invention 1034] An isolated antibody according to any one of claims 1001 to 1006 of the present invention, which consists of a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35. [This invention 1035] An isolated antibody according to any one of claims 1001 to 1004 and 1006 of the present invention, which consists of a heavy chain sequence set forth in SEQ ID NO: 36 and a light chain sequence set forth in SEQ ID NO: 37. [The present invention 1036] Any of the preceding isolated antibodies of the invention, which is afucosylated. [This invention 1037] An isolated antibody that binds to human TREM1 (SEQ ID NO: 1), which is afucosylated and comprises a VH sequence set forth in SEQ ID NO: 17, 13, or 9 and a VL sequence set forth in SEQ ID NO: 20. [The present invention 1038] 1037. An isolated antibody of the present invention, comprising a VH sequence set forth in SEQ ID NO: 17 and a VL sequence set forth in SEQ ID NO: 20. [This invention 1039] 1038. An isolated antibody of the present invention, comprising a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35. [The present invention 1040] 1037. An isolated antibody of the present invention, comprising a VH sequence set forth in SEQ ID NO: 13 and a VL sequence set forth in SEQ ID NO: 20. [This invention 1041] 1040. The isolated antibody of the present invention, which is afucosylated and comprises a heavy chain sequence set forth in SEQ ID NO: 36 and a light chain sequence set forth in SEQ ID NO: 37. [The present invention 1042] 1037. An isolated antibody of the present invention, comprising a VH sequence set forth in SEQ ID NO: 9 and a VL sequence set forth in SEQ ID NO: 20. [This invention 1043] 1042. An isolated antibody of the present invention, which is afucosylated and comprises a heavy chain sequence set forth in SEQ ID NO: 38 and a light chain sequence set forth in SEQ ID NO: 39. [This invention 1044] Any of the isolated antibodies of the preceding invention which is a humanized antibody, a human antibody, or a chimeric antibody. [This invention 1045] The isolated antibody of the present invention 1044, which is a humanized antibody. [The present invention 1046] Any of the preceding isolated antibodies of the invention, comprising a heavy chain human constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. [This invention 1047] Any of the preceding isolated antibodies of the invention comprising an Fc region. [This invention 1048] Any of the isolated antibodies of the preceding invention, wherein said Fc region is a human Fc region. [This invention 1049] 10. The isolated antibody of any of the preceding invention, wherein said human Fc region comprises a human heavy chain constant region of the class IgG, and of a subclass selected from IgG1, IgG2, IgG3, and IgG4. [The present invention 1050] 1049. The isolated antibody of the present invention, wherein said human Fc region comprises wild-type human IgG1 Fc. [This invention 1051] 10. Any of the isolated antibodies of the preceding invention, wherein the antibody consists of the VH sequence set forth in SEQ ID NO: 17 and the VL sequence set forth in SEQ ID NO: 20, and the human Fc region comprises wild-type human IgG1 Fc. [This invention 1052] Any of the isolated antibodies of the preceding invention, wherein the Fc region comprises one or more amino acid substitutions, which confers increased antibody half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity, relative to an Fc without said one or more substitutions. [This invention 1053] Any of the isolated antibodies of the preceding invention, wherein the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. [This invention 1054] Any isolated antibody of the preceding invention that is a monoclonal antibody. [This invention 1055] Approximately 0.5, 1, 2, 3, 4, 5, 6, or 7 × 10 as measured by surface plasmon resonance (SPR) assay -9M or lower K D 2. Any of the preceding isolated antibodies of the invention that bind to human TREM1 at [The present invention 1056] K of approximately 7 nM or less as measured by surface plasmon resonance (SPR) assay D 2. Any of the preceding isolated antibodies of the invention that bind to human TREM1 at [This invention 1057] Any of the isolated antibodies of the preceding invention that is an agonist antibody. [This invention 1058] Any of the preceding isolated antibodies of the invention that induces an increase in the expression of at least one cytokine or chemokine in a cell compared to an isotype control antibody. [This invention 1059] 1058. The isolated antibody of the present invention, wherein said at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB). [The present invention 1060] The isolated antibody of the present invention 1059, wherein said cytokine or chemokine is CXCL10 or IFN-γ. [The present invention 1061] Any of the preceding isolated antibodies of the invention that induces increased expression of at least one myeloid co-stimulatory protein in a cell compared to an isotype control antibody. [The present invention 1062] 1061. The isolated antibody of the present invention, wherein said myeloid costimulatory protein is HLA-DR, CD40, CD80, or CD86 on a cell. [The present invention 1063] Any of the preceding isolated antibodies of the invention that induces increased activation of the ERK and / or STAT3 intracellular signaling pathways in a cell compared to an isotype control antibody. [The present invention 1064] Any of the isolated antibodies of the preceding invention that induces an anti-tumor memory response compared to an isotype control antibody. [This invention 1065] The antibody a. competes with human TREM-26 antibody for binding to human TREM1; b. binds to human TREM1; C. binds to cynomolgus monkey TREM1; d. Stimulate TREM1 signaling; e. Inducing immune signaling pathways; f. Inducing cytokine or chemokine secretion; g. Inducing the expression of costimulatory molecules; h. Killing, disabling, or depleting bone marrow cells; or Possessing the ability to perform any combination of ia to h Any of the preceding isolated antibodies of the invention. [The present invention 1066] Any of the preceding isolated antibodies of the invention, which have antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [This invention 1067] Any of the preceding isolated antibodies of the invention, which have antibody-mediated cellular phagocytosis (ADCP) activity. [The present invention 1068] Any of the isolated antibodies of the preceding invention, which have complement dependent cytotoxicity (CDC) activity. [The present invention 1069] The isolated antibody of any one of claims 1058 to 1068, wherein the cell is a TREM1+ cell. [The present invention 1070] 1069. The isolated antibody of the present invention, wherein said TREM1+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs). [This invention 1071] The isolated antibody of the present invention 1070, wherein said TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils. [This invention 1072] 10. The isolated antibody of any of claims 1001 to 1071, wherein said antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells. [This invention 1073] Any of the isolated antibodies of the preceding invention for use as a medicament. [This invention 1074] Any of the isolated antibodies of the preceding invention for use in the treatment of cancer or an infectious disease. [This invention 1075] The isolated antibody of any of the preceding inventions for use in the treatment of cancer selected from solid tumors and liquid tumors. [This invention 1076] An isolated polynucleotide or set of polynucleotides encoding any of the preceding antibodies of the invention, its VH, its VL, its light chain, its heavy chain, or antigen-binding portion thereof, optionally a cDNA. [This invention 1077] A vector or set of vectors comprising a polynucleotide or set of polynucleotides of the present invention 1076. [This invention 1078] A host cell comprising a polynucleotide or set of polynucleotides of the invention 1076, or a vector or set of vectors of the invention 1077. [This invention 1079] A method for producing an antibody comprising expressing the antibody in a host cell of the invention 1078 and isolating the expressed antibody. [The present invention 1080] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1075 and a pharmaceutically acceptable excipient. [This invention 1081] A kit comprising the antibody of any one of the present inventions 1001 to 1075 or the pharmaceutical composition of the present invention 1080 and instructions for use. [This invention 1082] A method for increasing an immune response comprising administering to a subject a composition comprising an anti-TREM1 antibody or an antigen-binding fragment thereof. [This invention 1083] The method of the present invention 1082, wherein the composition comprises the antibody of any one of the present inventions 1001 to 1075 or the pharmaceutical composition of the present invention 1080. [This invention 1084] The method of claim 1082, wherein said antibody has receptor-ligand blocking activity, agonist activity, or antagonist activity. [This invention 1085] 1084. The method of claim 1084, wherein said antibody has agonist activity. [This invention 1086] 1086. The method of any of claims 1082 to 1085, wherein said antibody induces increased expression of at least one cytokine or chemokine in the cell compared to an isotype control antibody. [This invention 1087] 1086. The method of claim 1086, wherein said at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB). [This invention 1088] 1087. The method of claim 1077, wherein said cytokine or chemokine is CXCL10 or IFN-γ. [This invention 1089] 1089. The method of any of claims 1082 to 1088, wherein said antibody induces increased expression of at least one myeloid costimulatory protein compared to an isotype control antibody. [The present invention 1090] 1089. The method of claim 1089, wherein said myeloid co-stimulatory protein is HLA-DR, CD40, CD80, or CD86 on a cell. [This invention 1091] The method of any of claims 1082 to 1090, wherein said antibody induces increased activation of the ERK and / or STAT3 intracellular signaling pathways in cells compared to an isotype control antibody. [This invention 1092] 1092. The method of any one of claims 1082 to 1091, wherein the antibody induces a memory immune response. [This invention 1093] The method of any one of claims 1082 to 1092, wherein the cells are TREM1+ cells. [This invention 1094] 1093. The method of claim 1093, wherein said TREM1+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs). [This invention 1095] 1094. The method of claim 1094, wherein said TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils. [This invention 1096] The method of any of claims 1082 to 1095, wherein said antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells. [This invention 1097] The method of any one of claims 1082 to 1096, wherein the subject is a human. [This invention 1098] A method of treating cancer comprising administering to a subject a composition comprising an anti-TREM1 antibody or an antigen-binding fragment thereof. [This invention 1099] The method of the present invention 1098, wherein the composition comprises the antibody of any one of the present inventions 1001 to 1075 or the pharmaceutical composition of the present invention 1080. [The present invention 1100] The method of claim 1098, wherein said antibody has receptor-ligand blocking activity, agonist activity, or antagonist activity. [The present invention 1101] The method of claim 1098, wherein the antibody has agonist activity. [The present invention 1102] 1102. The method of any of claims 1098 to 1101, wherein said antibody induces an increase in the expression of at least one cytokine or chemokine in the cell compared to an isotype control antibody. [The present invention 1103] 1102. The method of claim 1102, wherein said at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB). [The present invention 1104] 1104. The method of claim 1103, wherein said cytokine or chemokine is CXCL10 or IFN-γ. [This invention 1105] The method of any of claims 1098 to 1104, wherein said antibody induces increased expression of at least one myeloid costimulatory protein compared to an isotype control antibody. [The present invention 1106] 1105. The method of claim 1105, wherein said myeloid co-stimulatory protein is HLA-DR, CD40, CD80, or CD86 on a cell. [This invention 1107] The method of any of claims 1098 to 1106, wherein said antibody induces increased activation of the ERK and / or STAT3 intracellular signaling pathways in cells compared to an isotype control antibody. [This invention 1108] 8. The method of any of claims 1098 to 1107, wherein said antibody induces an anti-tumor memory response compared to an isotype control antibody. [This invention 1109] 1098. The method of claim 1098, wherein said antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [The present invention 1110] The method of claim 1098, wherein the antibody has complement-dependent cytotoxicity (CDC) activity. [The present invention 1111] 1098. The method of claim 1098, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity. [The present invention 1112] The method of any one of claims 1098 to 1108, wherein the cells are TREM1+ cells. [The present invention 1113] 1112. The method of claim 1112, wherein said TREM1+ cells are selected from the group consisting of dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs). [This invention 1114] 1113. The method of claim 1113, wherein said TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils. [This invention 1115] The method of any of claims 1098 to 1114, wherein said antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells. [The present invention 1116] The method of any one of claims 1098 to 1115, wherein the subject is a human. [This invention 1117] The method of any one of claims 1098 to 1116, wherein the cancer is a solid cancer. [This invention 1118] The method of any one of claims 1098 to 1117, wherein the cancer is a liquid cancer. [This invention 1119] 1098-1118. The method of any of claims 1098 to 1118, wherein said cancer is selected from the group consisting of melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, urothelial cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, kidney cancer, endometrial cancer, cervical cancer, testicular cancer, and mesothelioma. [The present invention 1120] 1119. The method of claim 1119, wherein said cancer is gastric cancer, ovarian cancer, colon cancer, or breast cancer. [This invention 1121] The method of any of claims 1098 to 1120, wherein said contacting enhances an immune response in said subject. [This invention 1122] The method of claim 1121, wherein the enhanced immune response is an adaptive immune response. [This invention 1123] The method of claim 1122, wherein the enhanced immune response is an innate immune response. [This invention 1124] The method of any of claims 1098 to 1123, wherein said subject has previously undergone, is concurrently undergoing, or will subsequently undergo immunotherapy. [This invention 1125] The method of claim 1124, wherein the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive cell therapy; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a STING agonist; a monocyte vaccine; a Bacillus Calmette-Guerin vaccine; an antigen binding protein that binds to both T cells and antigen-presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulatory agent; an oncolytic virus; and an epigenetic modulator. [Invention 1126] 1125. The method of claim 1125, wherein said immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody. [This invention 1127] A method for killing, neutralizing, or depleting bone marrow cells that express triggering receptor expressed on bone marrow cells 1 (TREM1) on their cell surface, the method comprising contacting the bone marrow cells with any of the antibodies of the present inventions 1001 to 1075 or the pharmaceutical composition of the present invention 1080. [This invention 1128] The method of claim 1127, wherein the antibody kills, neutralizes, or depletes the bone marrow cells by at least one of ADCC, CDC, and ADCP, optionally wherein the antibody kills, neutralizes, or depletes the bone marrow cells by ADCC, optionally wherein the antibody kills, neutralizes, or depletes the bone marrow cells by CDC, and optionally wherein the antibody kills, neutralizes, or depletes the bone marrow cells by ADCP. [This invention 1129] 1129. The method of claim 1127 or 1128, wherein the antibody kills the bone marrow cells by at least one of ADCC, CDC, and ADCP. [The present invention 1130] The method of any one of claims 1127 to 1129, wherein said antibody neutralizes said bone marrow cells by at least one of ADCC, CDC, and ADCP. [This invention 1131] 1129. The method of any of claims 1127 to 1129, wherein said antibody depletes said bone marrow cells by at least one of ADCC, CDC, and ADCP. [This invention 1132] The method according to any one of claims 1127 to 1131, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [This invention 1133] The method according to any one of claims 1127 to 1131, wherein the antibody has complement-dependent cytotoxicity (CDC) activity. [This invention 1134] The method of any one of claims 1127 to 1131, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity. [This invention 1135] The method of any one of claims 1127 to 1134, wherein said antibody has receptor-ligand blocking activity, agonist activity, or antagonist activity. [This invention 1136] The method according to any one of claims 1127 to 1135, wherein said bone marrow cells are stimulator bone marrow cells. [This invention 1137] The method according to any one of claims 1127 to 1135, wherein said bone marrow cells are unstimulated bone marrow cells. [This invention 1138] 1138. The method of any of claims 1127 to 1137, wherein said bone marrow cells comprise at least one of dendritic cells, tumor-associated macrophages (TAMs), neutrophils, monocytes, or myeloid-derived suppressor cells. [This invention 1139] 1138. The method of claim 1138, wherein said bone marrow cells are neutrophils or tumor-associated neutrophils. [This invention 1140] 1139. The method of claim 1139, wherein said bone marrow cells are tumor-associated macrophages. [This invention 1141] 1138. The method of claim 1138, wherein said bone marrow cells are monocytic myeloid-derived suppressor cells. [This invention 1142] The method of any one of claims 1127 to 1141, wherein the bone marrow cells are present in a tumor. [This invention 1143] The method of any of claims 1127 to 1142, wherein said bone marrow cells are in a population of immune cells comprising stimulated and non-stimulated bone marrow cells. [This invention 1144] The method of any one of claims 1127 to 1143, wherein said contacting is in vitro or in vivo. [Invention 1145] The method of any of claims 1127 to 1144, wherein said contacting occurs in vivo in a subject, and optionally said subject has cancer. [Invention 1146] The method of any one of claims 1127 to 1145, wherein the subject is a human. [This invention 1147] The method according to any one of claims 1127 to 1146, wherein the cancer is a solid cancer. [Invention 1148] The method of any one of claims 1127 to 1146, wherein the cancer is a liquid cancer. [This invention 1149] 1146. The method of claim 1146, wherein said cancer is selected from the group consisting of melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, urothelial cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, renal cancer, endometrial cancer, cervical cancer, testicular cancer, and mesothelioma. [This invention 1150] 1149. The method of claim 1149, wherein said cancer is gastric cancer, ovarian cancer, colon cancer, or breast cancer. [This invention 1151] The method of claim 1150, wherein said contacting enhances an immune response in said subject. [This invention 1152] The method of claim 1151, wherein the enhanced immune response is an adaptive immune response. [This invention 1153] The method of claim 1151, wherein the enhanced immune response is an innate immune response. [This invention 1154] The method of claim 1151, wherein the enhanced immune response comprises expression of at least one cytokine or chemokine. [Invention 1155] 1154. The method of claim 1154, wherein said at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB). [Invention 1156] 1155. The method of claim 1155, wherein said at least one cytokine or chemokine is CXCL10 or IFN-γ. [This invention 1157] The method of any of claims 1127 to 1156, wherein said subject has previously undergone, is concurrently undergoing, or will subsequently undergo immunotherapy. [This invention 1158] The method of claim 1157, wherein the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; adoptive cell therapy; adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; a STING agonist; a monocyte vaccine; a Bacillus Calmette-Guerin vaccine; an antigen binding protein that binds to both T cells and antigen-presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulatory agent; an oncolytic virus; and an epigenetic modulator. [This invention 1159] 1158. The method of claim 1158, wherein said immunotherapy is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody. These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief explanation of the drawings]

[0146] [Figure 1] 1 shows the SPR binding kinetics of PI-4026-5. [Figure 2] A shows the binding of PI-4026-5 to HEK293 control cells. B shows the binding of PI-4026-5 to HEK293 cells overexpressing human TREM1. [Figure 3] A shows the binding of PI-4026-5 to neutrophils in human peripheral blood. B shows the binding of PI-4026-5 to monocytes in human peripheral blood. [Figure 4] A shows binding of PI-4026-5 to cells expressing cynomolgus monkey TREM1, and B shows no binding of PI-4026-5 to cells expressing mouse TREM1. [Figure 5] A shows FcγR signaling induced by PI-4026-5 (using a hCD16 reporter assay system), and B shows FcγR signaling induced by PI-4026-5 (using a hCD32 reporter assay system). [Figure 6] (A) PI-4026-5 does not induce ADCP of parental expi293 cells by primary human macrophages. (B) PI-4026-5 induces ADCP of hTREM1-expressing expi293 cells by primary human macrophages. [Figure 7A] The interactions of residues within CDRH1 of PI-4026 Fab and the TREM1 IgV domain are shown. [Figure 7B] The interactions of residues within CDRH2 of PI-4026 Fab and the TREM1 IgV domain are shown. [Figure 7C] The interactions of residues within CDRH3 of PI-4026 Fab and the TREM1 IgV domain are shown. [Figure 7D] The interactions of residues within CDRL1 of PI-4026 Fab and the TREM1 IgV domain are shown. [Figure 7E]The interactions of residues within CDRL3 of PI-4026 Fab and the TREM1 IgV domain are shown. [Figure 8] 1 shows the SEC profile of PI-4026-5 after exposure to different concentrations of H2O2. [Figure 9] A. Fucosylated PI-4026-5 antibody binds to human peripheral monocytes. B. Afucosylated PI-4026-5 antibody binds to human peripheral monocytes. [Figure 10] A. Fucosylated PI-4026-5 antibody binds to peripheral monocytes of cynomolgus monkeys. B. Afucosylated PI-4026-5 antibody binds to peripheral monocytes of cynomolgus monkeys. [Figure 11] A shows FcγR signaling induced by fucosylated PI-4026-5 (using the hCD16 reporter assay system), and B shows FcγR signaling induced by afucosylated PI-4026-5 antibody (using the hCD16 reporter assay system). [Figure 12] A shows the mean radius (determined by DLS) of the PI64052 (PI-4026-5-M100L) and PI64062 (PI-4026-5-M100Q) antibodies in response to heat stress. B shows the % polydispersity (determined by DLS) of the PI64052 and PI64062 antibodies in response to heat stress. These are stability kinetic measurements. [Figure 13] A shows the % monomer (determined by SEC) of PI64052 and PI64062 antibodies in response to heat stress. B shows the % aggregate (determined by SEC) of PI64052 and PI64062 antibodies in response to heat stress. These are stability kinetic measurements. [Figure 14] A shows the % major species of PI64052 and PI64062 antibodies after 24 hours of exposure to different concentrations of HO by hydrophobic interaction chromatography. B shows the % hydrophilic species of PI64052 and PI64062 antibodies after 24 hours of exposure to different concentrations of HO by hydrophobic interaction chromatography. These are measures of oxidative stress susceptibility. [Figure 15] A shows the % major species of PI64052 and PI64062 antibodies after 14 days of exposure to different concentrations of HO by hydrophobic interaction chromatography. B shows the % hydrophilic species of PI64052 and PI64062 antibodies after 14 days of exposure to different concentrations of HO by hydrophobic interaction chromatography. These are measures of oxidative stress susceptibility. [Figure 16] A shows the % major species of PI64052 and PI64062 antibodies after 28 days of exposure to different concentrations of H2O2 by hydrophobic interaction chromatography. B shows the % hydrophilic species of PI64052 and PI64062 antibodies after 28 days of exposure to different concentrations of H2O2 by hydrophobic interaction chromatography. [Figure 17] A shows the results of a deamidation assay of the PI64052 and PI64062 antibodies. B shows the results of a deamidation assay of the PI64052 and PI64062 antibodies. C shows the results of a deamidation assay of the PI64052 and PI64062 antibodies. D shows the results of a deamidation assay of the PI64052 and PI64062 antibodies. [Figure 18] A shows no background binding of PI64052 and PI64062 to HEK293 cells. B shows no background binding of PI64052 and PI64062 to HEK293 cells. C shows no background binding of PI64052 and PI64062 to HEK293 cells. [Figure 19] A indicates no binding of PI64052 and PI64062 to lymphocytes (T cells). B indicates no binding of PI64052 and PI64062 to lymphocytes (T cells). C indicates no binding of PI64052 and PI64062 to lymphocytes (T cells). D indicates no binding of PI64052 and PI64062 to lymphocytes (B cells). E indicates no binding of PI64052 and PI64062 to lymphocytes (B cells). F indicates no binding of PI64052 and PI64062 to lymphocytes (B cells). [Figure 20]Figure 1 shows the binding of PI64052 and PI64062 antibodies to HEK293 cells overexpressing human TREM1 (A), (B), and (C) of PI64052 and PI64062 antibodies to HEK293 cells overexpressing human TREM1. [Figure 21] Figure 1 shows the binding of PI64052 and PI64062 antibodies to HEK293 cells overexpressing cynomolgus monkey TREM1 (A), (B), and (C) the binding of PI64052 and PI64062 antibodies to HEK293 cells overexpressing cynomolgus monkey TREM1 (C). [Figure 22] A. Binding of PI64052 and PI64062 to human monocytes. B. Binding of PI64052 and PI64062 to human monocytes. C. Binding of PI64052 and PI64062 to human monocytes. [Figure 23] A. Binding of PI64052 and PI64062 to cynomolgus monkey monocytes. B. Binding of PI64052 and PI64062 to cynomolgus monkey monocytes. [Figure 24] FcγR signaling induced by PI64052 and PI64062 (using the hCD16 reporter assay system) is shown and is representative of three separate experiments. [Figure 25] A shows that PI64052 induces ADCP of hTREM1-expressing expi cells by primary human macrophages, but not the parental expi cells. B shows that PI64062 induces ADCP of hTREM1-expressing expi cells by primary human macrophages, but not the parental expi cells. C shows that PI64052 induces ADCP of hTREM1-expressing expi cells by primary human macrophages, but not the parental expi cells. D shows that PI64062 induces ADCP of hTREM1-expressing expi cells by primary human macrophages, but not the parental expi cells. [Figure 26-1] A shows the binding of PI64052 and PI64062 and their fucosylated parental forms (PI-4026-5-M100L and PI-4026-5-M100Q, respectively) to hFcγRI. B shows the binding of PI64052 and PI64062 and their fucosylated parental forms to hFcγRIIα. C shows the binding of PI64052 and PI64062 and their fucosylated parental forms to hFcγRIIβ. D shows the binding of PI64052 and PI64062 and their fucosylated parental forms to hFcγRIIIα. E shows the binding of PI64052 and PI64062 and their fucosylated parental forms to hFcγRIIIα. F shows the binding of PI64052 and PI64062 and their fucosylated parental forms to hFcγRIIIβ. [Figure 26-2] See description of Figure 26-1. [Figure 27] (A) Dose-dependent receptor occupancy of PI-4026-5-M100Q and afucosylated PI-4026-5-M100Q on peripheral blood-derived monocytes. (B) Dose-dependent receptor occupancy of PI-4026-5-M100Q and afucosylated PI-4026-5-M100Q on peripheral blood-derived neutrophils. [Figure 28] (A) Afucosylated PI-4026-5-M100Q induces a more potent dose-dependent cytokine release of IFN-γ from human peripheral blood leukocytes than PI-4026-5-M100Q. (B) Afucosylated PI-4026-5-M100Q induces a more potent dose-dependent cytokine release of IL-8 from human peripheral blood leukocytes than PI-4026-5-M100Q. (C) Afucosylated PI-4026-5-M100Q induces a more potent dose-dependent cytokine release of IL-2 from human peripheral blood leukocytes than PI-4026-5-M100Q. D shows that afucosylated PI-4026-5-M100Q induces a more potent dose-dependent cytokine release of IP-10 from human peripheral blood leukocytes than PI-4026-5-M100Q. [Figure 29](A) Combination therapy with afucosylated PI-4928 anti-TREM1 and anti-PD-1 antibodies induces a peripheral cytokine signature in vivo. (B) Combination therapy with afucosylated PI-4928 anti-TREM1 and anti-PD-1 antibodies induces a peripheral cytokine signature in vivo. [Figure 30] 1 shows that the afucosylated PI-9067 anti-TREM1 antibody has monotherapy activity in the Panc02 tumor model. [Figure 31] A provides the growth curves for each mouse from the isotype group of the Panc02 tumor model. B provides the growth curves for each mouse from the anti-PD-1 group of the Panc02 tumor model. C provides the growth curves for each mouse from the anti-TREM1 group of the Panc02 tumor model. D provides the growth curves for each mouse from the anti-TREM1 and anti-PD-1 groups of the Panc02 tumor model. [Figure 32] Tumor volumes at day 28 for each mouse from each indicated group in the Panc02 tumor model are provided. [Figure 33] We show that human TREM1 is expressed across a variety of tumor indications and is restricted to myeloid cells. [Figure 34] A shows the fold change in cytokines induced by afucosylated PI64062 in human blood cells. B shows the upregulation of HLA-DR surface expression after treatment with afucosylated PI64062. C shows the upregulation of CD40 surface expression after treatment with afucosylated PI64062. [Figure 35] Figure 1 shows an expression matrix of 1000 genes with the highest variance across blood samples, sorted according to cell type. Gene expression clustering correlated well with the sorted immune cell populations. [Figure 36]A shows HLA-DR gene expression in the indicated cell types after isotype antibody treatment (ISO) or anti-TREM1 antibody (afcosylated PI64062). B shows CD40 gene expression in the indicated cell types after isotype antibody treatment (ISO) or anti-TREM1 antibody (afcosylated PI64062). C shows CD80 gene expression in the indicated cell types after isotype antibody treatment (ISO) or anti-TREM1 antibody (afcosylated PI64062). D shows CD86 gene expression in the indicated cell types after isotype antibody treatment (ISO) or anti-TREM1 antibody (afcosylated PI64062). [Figure 37] A shows a representative histogram overlay of cell surface HLA-DR expression on monocytes after treatment with an anti-TREM1 antibody (afcosylated PI64062). B shows a representative histogram overlay of cell surface CD40 expression on monocytes after treatment with an anti-TREM1 antibody (afcosylated PI64062). C shows a representative histogram overlay of cell surface CD80 expression on monocytes after treatment with an anti-TREM1 antibody (afcosylated PI64062). D shows a representative histogram overlay of cell surface CD86 expression on monocytes after treatment with an anti-TREM1 antibody (afcosylated PI64062). [Figure 38A] The % of pERK and pSTAT3 in neutrophils, monocytes, or T cells after treatment with isotype control or afucosylated PI64062 antibody is shown. [Figure 38B] (*<0.05, **<0.001) Results of RNAseq analysis of the ERK and STAT pathways are shown, showing that genes associated with the pathways are significantly enriched in monocytes but not in neutrophils or NK cells. [Figure 39] Chemokines and cytokines upregulated by anti-TREM1 antibodies in mouse or human blood cells are shown. [Figure 40] 1 shows that the afucosylated PI-9067L anti-TREM1 antibody has monotherapy activity in the ID8 ovarian tumor model. [Figure 41]1 shows that afucosylated PI-9067L antibody induces immunological memory in rechallenged mice in the CT26 tumor model. [Figure 42] A shows TREM1 expression in colorectal cancer. B shows survival probability and TREM1 expression in colorectal cancer patients. [Figure 43] (A) Afucosylated PI-4928 anti-TREM1 antibody exhibits dose-dependent pharmacokinetics. (B) Soluble mouse TREM1 in serum after treatment with afucosylated PI-4928 anti-TREM1 antibody. [Figure 44] A shows the blood counts of mice on day 7. B shows the blood counts of mice on day 14. C shows the red blood cell parameters of mice on day 7. D shows the red blood cell parameter values ​​of mice on day 14. [Figure 45] (A) Antitumor efficacy of anti-TREM1, anti-PD-1, or a combination of anti-TREM1 and anti-PD-1 treatment in female BALB / c mice bearing small subcutaneous EMT6 tumors. (B) Antitumor efficacy of anti-TREM1, anti-PD-1, or a combination of anti-TREM1 and anti-PD-1 treatment in female BALB / c mice bearing large subcutaneous EMT6 tumors. DETAILED DESCRIPTION OF THE INVENTION

[0147] Detailed Description definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not be construed as representing a difference beyond that commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those of ordinary skill in the art using conventional techniques, such as the widely used molecular cloning methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-specified protocols and conditions unless otherwise noted.

[0148] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0149] Aspects and embodiments of the invention described herein are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0150] For all compositions and methods of using the compositions described herein, the composition may include any recited components or steps or may "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" recited components, the composition contains the recited components and may contain other components other than the explicitly recited components that do not substantially affect the condition being treated, but does not contain any other components that substantially affect the condition being treated, or if the composition contains additional components other than the recited components that substantially affect the condition being treated, the composition does not contain the additional components in concentrations or amounts sufficient to substantially affect the condition being treated. When a method is described as "consisting essentially of" recited steps, the method contains the recited steps and may contain other steps that do not substantially affect the condition being treated, but the method does not contain any other steps other than those explicitly recited that substantially affect the condition being treated. As a specific, non-limiting example, when a composition is described as "consisting essentially of" a component, the composition may further contain any amount of a pharmaceutically acceptable carrier, vehicle, or diluent, and such other components that do not substantially affect the condition being treated.

[0151] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0152] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include "transformants" (or "transformed cells") and "transfectants" (or "transfected cells"), which include the primary transformed or transfected cell, respectively, and their derived progeny. Such progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations.

[0153] As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic compound, such as an anti-TREM1 antibody, administered to an individual as a single dose or as part of a series, which is effective to produce or contribute to a desired therapeutic effect, either alone or in combination with another therapeutic modality. Examples of desired therapeutic effects are an enhanced immune response; slowing or delaying tumor progression; stabilizing disease; or amelioration of one or more symptoms. An effective amount may be given in one or more administrations.

[0154] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can occur during the course of clinical disease. Desirable effects of treatment include prevention of disease recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0155] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate an immune response in a subject.

[0156] As used herein, the term "subject" or "individual" means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with an antibody provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0157] The term "in vitro" refers to processes that occur in living cells grown isolated from an organism, such as growth in tissue culture.

[0158] The term "in vivo" refers to a process that occurs within a living organism.

[0159] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic or diagnostic product (e.g., a kit) that contain information regarding the indications, uses, dosages, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic product.

[0160] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0161] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth.

[0162] The term "cytostatic agent" refers to a compound or composition that inhibits cell growth either in vitro or in vivo. In some embodiments, a cytostatic agent is an agent that reduces the percentage of cells in S phase. In some embodiments, a cytostatic agent reduces the percentage of cells in S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.

[0163] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematological malignancy.

[0164] The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredients contained therein to be effective in treating a subject, and which does not contain additional components that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0165] The terms "co-administration," "co-administering," and "in combination with" include the administration of two or more therapeutic agents simultaneously, in parallel, or sequentially, without specific time limitations. In one embodiment, the agents are present in a cell or in a subject's body at the same time or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered before the administration of a second therapeutic agent.

[0166] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0167] The terms "increase" and "activate" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater increase in the recited variable.

[0168] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater decrease in the recited variable.

[0169] The term "about" refers to and encompasses the indicated value and the range above and below that value. In certain embodiments, the term "about" refers to ±10%, ±5%, or ±1% of the given value. In certain embodiments, where applicable, the term "about" refers to the given value(s) ±1 standard deviation of that value(s).

[0170] The term "stimulate" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and stimulates a receptor.

[0171] The term "antagonize" refers to the inhibition of receptor signaling to inhibit the biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0172] For any of the structural and functional properties described herein, methods for determining these properties are known in the art.

[0173] The term "optionally," when used sequentially, means to include from one to all of the listed combinations, and contemplates all subcombinations.

[0174] The term "amino acid" refers to the 20 common naturally occurring amino acids, including alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0175] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., using publicly available computer software, such as BLAST, BLASTP, BLASTN, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software, or other algorithms available to those of skill in the art) or by visual inspection. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the entire length of the sequences being compared. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., a functional domain, or, alternatively, over the entire length of the two sequences to be compared.

[0176] For sequence comparison, one sequence to which a test sequence is compared usually serves as a reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0177] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0178] Ranges recited herein are understood to be shorthand for all of the values ​​within the range, inclusive of the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0179] TREM1 antibody structure The present application provides antibodies and compositions, including antibodies that bind to TREM1 protein, including antibodies that neutralize unstimulated bone marrow cells.

[0180] The term "antibody" is used herein in the broadest sense to include a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0181] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0182] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminal domain of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. An IgG1 heavy chain is composed of, from N- to C-terminus, VH, CH1, CH2, and CH3 domains, respectively. The light chain is composed of, from N- to C-terminus, VL and CL domains. An IgG1 heavy chain contains a hinge between the CH1 and CH2 domains. In certain embodiments, an immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are derived from or are derived from immunoglobulin-based constructs such as diabodies or nanobodies. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain derived from a heavy chain antibody, such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain derived from a mammalian antibody, such as a bovine antibody, a human antibody, a camelid antibody, a murine antibody, or any chimeric antibody.

[0183] In some embodiments, an antibody provided herein comprises a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0184] In some embodiments, the antibody is an IgG1 antibody.

[0185] In some embodiments, the antibody is an IgG3 antibody.

[0186] In some embodiments, the antibody is an IgG2 antibody.

[0187] In some embodiments, the antibody is an IgG4 antibody.

[0188] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, a naturally occurring four-chain antibody comprises six HVRs: three VH (H1, H2, H3) and three VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or complementarity-determining regions (CDRs), the latter of which are of the highest sequence variability and / or are involved in antigen recognition. With the exception of CDR1 of VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen-binding region. This particular region is described by Kabat et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), and the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody.

[0189] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0190] Table A provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0191] CDRs may be assigned using antibody numbering software such as Abnum, for example, as described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, available at www.bioinf.org.uk / abs / abnum / and incorporated by reference in its entirety.

[0192] Table A: CDR residues according to the Kabat and Chothia numbering scheme TIFF0007759996000001.tif43145 * The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the CDR.

[0193] The "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0194] The term "antigen-binding domain" refers to a portion of an antibody capable of specifically binding to an antigen or epitope. An example of an antigen-binding domain is the antigen-binding domain formed by the VH-VL dimer of an antibody. Another example of an antigen-binding domain is the antigen-binding domain formed by diversifying a specific loop from the tenth fibronectin type III domain of an Adnectin. The antigen-binding domain may comprise CDR1, 2, and 3, in that order, from the heavy chain, and CDR1, 2, and 3, in that order, from the light chain.

[0195] The term "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter can be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for determining epitopes, such as, for example, testing antibody binding to TREM1 variants with different point mutations or chimeric TREM1 variants.

[0196] To screen for antibodies that bind to the epitope on the target antigen to which the antibody of interest (e.g., TREM1) binds, routine cross-blocking assays can be performed, such as those described in *Antibodies, A Laboratory Manual*, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, or additionally, epitope mapping can be performed by methods known in the art.

[0197] 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.

[0198] A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0199] A "humanized antibody" has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody is less likely to induce an immune response and / or induces a relatively mild immune response when administered to a human subject, compared to the non-human species antibody. In one embodiment, specific amino acids within the framework and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In another embodiment, constant domain(s) from a human antibody are fused to variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the immunogenic potential of the non-human antibody when administered to a human subject, either because none of the altered amino acid residues are important for immunospecific binding of the antibody to an antigen, or because the changes made to the amino acid sequence are conservative changes, such that binding of the humanized antibody to the antigen is significantly lower than binding of the non-human antibody to the antigen. Examples of methods for making humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596 (each of which is incorporated by reference in its entirety).

[0200] 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 may be epitopes on the same antigen (e.g., a single TREM1 molecule expressed by a cell) or different antigens (e.g., different TREM1 molecules, or a TREM1 molecule and a non-TREM1 molecule expressed by the same cell). In some embodiments, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some embodiments, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody").

[0201] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is a naturally occurring IgG molecule that is bivalent (i.e., has two antigen-binding domains), but recognizes the same epitope in each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.

[0202] The term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope(s), excluding variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present only in minor amounts. Monoclonal antibodies are typically obtained by a process that includes selection of a single antibody from a plurality of antibodies. For example, the selection process can be selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve production in cell culture, and / or to reduce immunogenicity in a subject.

[0203] The term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain such embodiments, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain of the single-chain Fab molecule. As described in more detail herein, an scFv has a variable domain of the light chain (VL) linked by a polypeptide chain from the C-terminus to the N-terminus of the variable domain of the heavy chain (VH). Alternatively, an scFv is composed of a polypeptide chain in which the C-terminus of the VH is linked by a polypeptide chain to the N-terminus of the VL.

[0204] A "Fab fragment" (also called fragment antigen binding) contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), along with the variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain the complementarity-determining loops (CDRs, also called hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0205] An "F(ab')2" fragment contains two Fab' fragments linked near the hinge region by a disulfide bond. F(ab')2 fragments may be produced, for example, by recombinant methods or by pepsin digestion of intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0206] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0207] A "single-chain Fv" or "sFv" or "scFv" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plückthun 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.

[0208] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. The Fc domain may follow a VH or VL, depending on the orientation of the variable domains within the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0209] The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. sdAbs are fairly stable and amenable to expression as fusion partners with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Appl. Microbiol. Biotechnol. 77(1):13-22).

[0210] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain that includes an Fc region. For example, when used to refer to an IgG molecule, a "full length antibody" is an antibody that includes two heavy chains and two light chains.

[0211] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0212] Anti-TREM1 antibodies may include those described herein, such as the clones listed in the tables. In some embodiments, the antibody comprises an alternative scaffold. In some embodiments, the antibody consists of an alternative scaffold. In some embodiments, the antibody consists essentially of an alternative scaffold. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the antibody consists of an antibody fragment. In some embodiments, the antibody consists essentially of an antibody fragment. A "TREM1 antibody," "anti-TREM1 antibody," or "TREM1-specific antibody" is an antibody as provided herein that specifically binds to the antigen TREM1. In some embodiments, the antibody binds to the extracellular domain of TREM1. In certain embodiments, the TREM1 antibodies provided herein bind to an epitope of TREM1 that is conserved between or among TREM1 proteins from different species. An anti-TREM1 antibody may include the TREM-26 clone (BioLegend; Cat. No. 314907; Li J, et al. 2011. Dev. Comp. Immunol. epub.).

[0213] In some embodiments, the antibody is a monoclonal antibody.

[0214] In some embodiments, the antibody is a polyclonal antibody.

[0215] In some embodiments, the antibody is produced by a hybridoma, hi other embodiments, the antibody is produced by a recombinant cell that has been engineered to express the desired variable and constant domains.

[0216] In some embodiments, the antibody may be a single chain antibody or other antibody derivative that retains the antigen specificity and a lower hinge region or variant thereof.

[0217] In some embodiments, the antibody may be a multifunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment, or a variant thereof. In some embodiments, the antibody is a monoclonal antibody, a neutral antibody, an antagonist antibody, an agonist antibody, a polyclonal antibody, an afucosylated antibody, a bispecific antibody, a human antibody, a chimeric antibody, a full-length antibody, or an antigen-binding fragment thereof. In certain embodiments, the antibody fragment or derivative thereof is selected from a Fab fragment, a Fab'2 fragment, a CDR, and an ScFv. In some embodiments, the antibody is an antigen-binding fragment thereof, a Fab, a Fab', a F(ab')2, an 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.

[0218] In some embodiments, the antibody is capable of forming an immune complex, for example, an immune complex may be a tumor cell coated with the antibody.

[0219] In some embodiments, the TREM1 antibody competes for binding to human TREM1 (SEQ ID NO: 1) with a reference antibody.

[0220] TREM1 antibody sequence V H domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:3. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:4. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:5. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:6. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:7. HIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 8. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 11. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 13. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 14. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 15. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 16. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 17. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 18. H Contains arrays.

[0221] In some embodiments, the antibodies provided herein comprise a V nucleotide sequence as provided in SEQ ID NOs: 4, 5, 6, 8, 9, 10, 12, 13, 14, 16, 17, and 18. H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H In some embodiments, the antibodies provided herein comprise the V sequences provided in SEQ ID NOs: 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. HIn some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0222] V L domain In some embodiments, the antibodies provided herein comprise a VL sequence selected from SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, the antibodies provided herein comprise the VL sequence of SEQ ID NO: 19. In some embodiments, the antibodies provided herein comprise the VL sequence of SEQ ID NO: 20. In some embodiments, the antibodies provided herein comprise the VL sequence of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise the VL sequence of SEQ ID NO: 22.

[0223] In some embodiments, the antibodies provided herein comprise VL sequences having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary VL sequences provided in SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, the antibodies provided herein comprise VL sequences provided in SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 19, 3, 20, 5, 21, 7, 22, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0224] VH-VL combinations In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. H and V selected from SEQ ID NOs: 19, 20, 21, and 22. L Contains arrays.

[0225] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 17. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 17. H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 17. H Sequence and V of SEQ ID NO: 22 L Contains arrays.

[0226] In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 16. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 16. H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 16. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 16. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 18. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 18. H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 18. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 18. H Sequence and V of SEQ ID NO: 22 L Contains arrays.

[0227] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 13. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 13. H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 13. H Sequence and V of SEQ ID NO: 22 L Contains arrays.

[0228] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 14. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 14. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 14. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 14. H Sequence and V of SEQ ID NO: 20 L Contains arrays.

[0229] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H Sequence and V of SEQ ID NO: 22 L Contains arrays.

[0230] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 8. H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 8. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 8. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H Sequence and V of SEQ ID NO: 20 L Contains arrays.

[0231] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:5. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:5. H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:5. H Sequence and V of SEQ ID NO: 22 L Contains arrays.

[0232] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:4. H Sequence and V of SEQ ID NO: 20 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:4. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:4. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:6. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:6. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:6. H Sequence and V of SEQ ID NO: 22 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO:6. H Sequence and V of SEQ ID NO: 20 L Contains arrays.

[0233] In certain aspects, any of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 can be combined with any of SEQ ID NOs: 19, 20, 21, and 22. For example, SEQ ID NO: 9 can be combined with any of SEQ ID NOs: 19, 20, 21, and 22. As another example, SEQ ID NO: 17 can be combined with any of SEQ ID NOs: 19, 20, 21, and 22.

[0234] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H VL sequences having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary VL sequences provided in SEQ ID NOs: 19, 20, 21, and 22. L In some embodiments, the antibodies provided herein comprise VH sequences as provided in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions, and VL sequences as provided in SEQ ID NOs: 19, 20, 21, and 22, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0235] CDR In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. HIn some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. H In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. 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.

[0236] In some embodiments, the CDRs are CDRs that have at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. In some embodiments, the CDR-H1 is the CDR-H1 of a VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is the CDR-H2 of a VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 is the CDR-H3 of a VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0237] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VL domain selected from SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VL domain selected from SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, the antibodies provided herein comprise three CDRs of a VL domain selected from SEQ ID NOs: 19, 20, 21, and 22. 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.

[0238] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, CDR-L1 is CDR-L1 of a VL domain selected from SEQ ID NOs: 2, 4, 21, and 22 with up to 1, 19, 3, 20, or 5 amino acid substitutions. In some embodiments, CDR-L19 is CDR-L2 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 2, 3, 20, 5, 21, 7, or 22 amino acid substitutions. In some embodiments, CDR-L3 is CDR-L3 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8 with up to 1, 19, 3, 20, 5, 21, 7, or 22 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0239] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and one to three CDRs of a VL domain selected from SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and two to three CDRs of a VL domain selected from SEQ ID NOs: 19, 20, 21, and 22. In some embodiments, the antibodies provided herein comprise three CDRs of the VH domain selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and three CDRs of the VL domain selected from SEQ ID NOs: 19, 20, 21, and 22. 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.

[0240] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NOs: 25, 29, 30, 31, 32, and 33. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NOs: 25, 29, 30, 31, 32, and 33. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NOs: 25, 29, 30, 31, 32, and 33 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0241] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NO: 33. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 33. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NO: 33 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0242] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NO: 32. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 32. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NO: 32 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0243] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NO: 25. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 25. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0244] In some embodiments, the antibodies provided herein comprise a CDR-H2 of SEQ ID NO: 24. In some aspects, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24. In some embodiments, the CDR-H2 is a CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0245] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23. In some aspects, the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H1 is a CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0246] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29 and a CDR-H2 of SEQ ID NO: 24. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 24, and a CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 29, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 29 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions.

[0247] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25 and a CDR-H2 of SEQ ID NO: 24. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 24, and a CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 25, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions.

[0248] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 33 and a CDR-H2 of SEQ ID NO: 24. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 33, a CDR-H2 of SEQ ID NO: 24, and a CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 33, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 33 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions.

[0249] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 32 and a CDR-H2 of SEQ ID NO: 24. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 32, a CDR-H2 of SEQ ID NO: 24, and a CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 32, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 32 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0250] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30 and a CDR-H2 of SEQ ID NO: 24. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30, a CDR-H2 of SEQ ID NO: 24, and a CDR-H1 of SEQ ID NO: 23. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 31 and a CDR-H2 of SEQ ID NO: 24. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 24, and a CDR-H1 of SEQ ID NO: 23.

[0251] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 28. In some aspects, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L3 of SEQ ID NO: 28. In some embodiments, the CDR-L3 is a CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0252] In some embodiments, the antibodies provided herein comprise a CDR-L2 of SEQ ID NO: 27. In some aspects, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L2 of SEQ ID NO: 27. In some embodiments, the CDR-L2 is a CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0253] In some embodiments, the antibodies provided herein comprise a CDR-L1 of SEQ ID NO: 26. In some aspects, the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-L1 is a CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0254] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 28 and a CDR-L2 of SEQ ID NO: 27. In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 27, and a CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L3 of SEQ ID NO: 28, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L2 of SEQ ID NO: 27, and the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-L3 is the CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is the CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is the CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods for obtaining antibodies provided herein.

[0255] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 33, a CDR-H2 of SEQ ID NO: 24, a CDR-H1 of SEQ ID NO: 23, a CDR-L3 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 27, and a CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 33, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. wherein CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 28; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 27; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 33 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0256] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 32, a CDR-H2 of SEQ ID NO: 24, a CDR-H1 of SEQ ID NO: 23, a CDR-L3 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 27, and a CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 32, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. wherein CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 28; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 27; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 32 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0257] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 24, a CDR-H1 of SEQ ID NO: 23, a CDR-L3 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 27, and a CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 29, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. wherein CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 28; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 27; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 29 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0258] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 24, a CDR-H1 of SEQ ID NO: 23, a CDR-L3 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 27, and a CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 25, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. wherein CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 28; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 27; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0259] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30 or 31, a CDR-H2 of SEQ ID NO: 24, a CDR-H1 of SEQ ID NO: 23, a CDR-L3 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 27, and a CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 30 or 31, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 28, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 27, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 30 or 31 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 27 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants."In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., according to the methods provided herein for obtaining antibodies.

[0260] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23, a CDR-H2 of SEQ ID NO: 24, a CDR-H3 of SEQ ID NO: 33, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 27, and a CDR-L3 of SEQ ID NO: 28.

[0261] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23, a CDR-H2 of SEQ ID NO: 24, a CDR-H3 of SEQ ID NO: 25, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 27, and a CDR-L3 of SEQ ID NO: 28.

[0262] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23, a CDR-H2 of SEQ ID NO: 24, a CDR-H3 of SEQ ID NO: 29, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 27, and a CDR-L3 of SEQ ID NO: 28.

[0263] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23, a CDR-H2 of SEQ ID NO: 24, a CDR-H3 of SEQ ID NO: 32, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 27, and a CDR-L3 of SEQ ID NO: 28.

[0264] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23, a CDR-H2 of SEQ ID NO: 24, a CDR-H3 of SEQ ID NO: 30, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 27, and a CDR-L3 of SEQ ID NO: 28.

[0265] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 23, a CDR-H2 of SEQ ID NO: 24, a CDR-H3 of SEQ ID NO: 31, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 27, and a CDR-L3 of SEQ ID NO: 28.

[0266] Fc area The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The structures of various immunoglobulin Fc regions and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or an Fc region that has been modified as described in the art or elsewhere in this disclosure.

[0267] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc polypeptide of a dimeric IgGFc comprises the IgG CH2 and IgG CH3 constant domain sequences. The Fc may be of the classes IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0268] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, an FcR may be a native-sequence human FcR. Generally, FcRs bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes can also bind to specific FcRs (see, e.g., Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0269] Modification of the CH2 domain can affect the binding of FcR to Fc. Many amino acid modifications in the Fc region are known in the art to selectively alter the affinity of Fc to different Fc gamma receptors. In some embodiments, the Fc contains one or more modifications that promote selective binding to Fc-gamma receptors.

[0270] Exemplary mutations that alter binding of FcR to Fc are listed below: S298A / E333A / K334A, S298A / E333A / K334A / K326A(Lu Y, Vernes JM, Chiang N, et al.J Immunol Methods.2011 Feb 28;365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L(Stavenhagen JB,Gorlatov S,Tuaillon N,et al.Cancer Res.2007 Sep 15;67(18):8882-90;Nordstrom JL, Gorlatov S, Zhang W, et al.Breast Cancer Res.2011 Nov 30;13(6):R123); F243L(Stewart R,Thom G,Levens M,et al.Protein Eng Des Sel.2011 Sep;24(9):671-8.),S298A / E333A / K334A(Shields RL,Namenuk AK,Hong K,et al.J Biol Chem.2001 Mar 2;276(9):6591-604); S239D / I332E / A330L, S239D / I332E(Lazar GA,Dang W,Karki S,et al.Proc Natl Acad Sci US A.2006 Mar 14;103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S, et al.Mol Immunol.2008 Sep;45(15):3926-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D and other mutations listed in WO2011 / 120134 and WO2011 / 120135 (incorporated herein by reference). Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012) lists the mutations on page 283.

[0271] In some embodiments, the antibodies described herein comprise modifications that improve their ability to mediate effector function. Such modifications are known in the art and include afucosylation or engineering the affinity of Fc for activating receptors, primarily FCGR3a for ADCC and C1q for CDC. Table B below summarizes various designs reported in the literature for effector function engineering.

[0272] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn297 EU numbering) without modifying the amino acid sequence are well known in the art. GlymaxX® technology (ProBioGen AG) is based on the introduction of a gene encoding an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the antibody-producing cells from adding the sugar "fucose" to N-linked antibody carbohydrate moieties (von Horsten et al. (2010) Glycobiology. 2010 December;20(12):1607-18). Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Pat. No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to produce reduced levels of antibody fucosylation. Antibodies can be fully afucosylated, meaning they contain no detectable fucose, or they can be partially afucosylated, meaning that the isolated antibody contains less than 95%, 85%, 75%, 65%, 55%, 45%, 35%, 25%, 15%, or 5% of the amount of fucose typically found in a similar antibody produced in a mammalian expression system.

[0273] Thus, in one embodiment, the antibodies described herein may comprise a dimeric Fc containing one or more amino acid modifications that confer improved effector function, as described in Table B. In another embodiment, the antibodies may be afucosylated to improve effector function.

[0274] (Table B) CH2 domain and effector function engineering TIFF0007759996000002.tif85149

[0275] Fc modifications that reduce FcγR and / or complement binding and / or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curr Opin Biotech 20:685-691, and Strohl, WR and Strohl LM, "Antibody Fc engineering for optimal antibody performance," In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp. 225-249). These strategies include reducing effector function by modifying glycosylation, using an IgG2 / IgG4 scaffold, or introducing mutations into the hinge or CH2 region of the Fc. For example, U.S. Patent Publication No. 2011 / 0212087 (Strohl), International Patent Publication No. WO2006 / 105338 (Xencor), U.S. Patent Publication No. 2012 / 0225058 (Xencor), U.S. Patent Publication No. 2012 / 0251531 (Genentech), and Strop et al ((2012) J. Mol. Biol. 420:204-219) describe certain modifications that reduce FcγR or complement binding to Fc.

[0276] Specific, non-limiting examples of known amino acid modifications that reduce FcγR or complement binding to Fc include those identified in Table C below.

[0277] Table C: Modifications that reduce FcγR or complement binding to Fc TIFF0007759996000003.tif110128

[0278] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn297EU numbering) without modifying the amino acid sequence are well known in the art. GlymaxX® technology (ProBioGen AG) is based on the introduction of a gene encoding an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the addition of the sugar "fucose" to N-linked antibody carbohydrate moieties by antibody-producing cells (von Horsten et al. (2010) Glycobiology. 2010 Dec;20(12):1607-18). Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), which inhibits protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Publication No. 2003 / 0157108; WO2004 / 056312, each of which is incorporated by reference in its entirety), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al., al., Biotech. Bioeng., 2004, 87:614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO2003 / 085107 (each of which is incorporated by reference in its entirety). Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Pat. No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to produce reduced levels of antibody fucosylation.

[0279] Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Publication No. 2003 / 0157108; WO2004 / 056312, each of which is incorporated by reference in its entirety), which are deficient in protein fucosylation and have stable overexpression of the bacterial GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), as well as knockout cell lines such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al., J. Immunol. 2004, 20:1607-1618). al., Biotech. Bioeng., 2004, 87:614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107 (each of which is incorporated by reference in its entirety).

[0280] Antibodies can be fully afucosylated, meaning they contain no detectable fucose, or they can be partially afucosylated, meaning that the isolated antibody contains less than 95%, 85%, 75%, 65%, 55%, 45%, 35%, 25%, 15%, or 5% of the amount of fucose typically found in a similar antibody produced in a mammalian expression system.

[0281] In some embodiments, the antibodies provided herein comprise an IgG1 domain with a reduced fucose content at Asn297 compared to naturally occurring IgG1 domains. Such Fc domains are known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, which is incorporated by reference in its entirety. In some embodiments, such antibodies do not comprise any fucose at Asn297. The amount of fucose may be determined using any suitable method, such as, for example, that described in WO2008 / 077546, which is incorporated by reference in its entirety.

[0282] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at one or more of the Fc region positions 298, 333, and 334. In some embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions at positions 239, 332, and 330 as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103:4005-4010, which is incorporated by reference in its entirety.

[0283] Other exemplary glycosylation variants that can be incorporated into the antibodies provided herein are described in, e.g., U.S. Patent Publication Nos. 2003 / 0157108, 2004 / 0093621, 2003 / 0157108, 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0132140, 2004 / 01 10704, 2004 / 0110282, 2004 / 0109865; International Patent Publication Nos. 2000 / 61739, 2001 / 29246, 2003 / 085119, 2003 / 084570, 2005 / 035586, 2005 / 035778, 2005 / 053742, 2002 / 031140, Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249, and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87:614-622, each of which is incorporated by reference in its entirety.

[0284] In some embodiments, the antibodies provided herein comprise an Fc region having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764, each of which is incorporated by reference in its entirety.

[0285] In some embodiments, the antibodies provided herein contain one or more modifications that improve or decrease C1q binding and / or CDC. See U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 2000, 164:4178-4184 (each of which is incorporated by reference in its entirety).

[0286] In some embodiments, the antibody comprises a human heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human Fc region comprises a wild-type human IgG1 Fc.

[0287] join "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise specified, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of molecule X for partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0288] With respect to antibody binding to a target molecule, the terms "binds with," "specifically binding with," "specifically binds to," "specific for," "selectively binds to," and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to the non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Specific binding is then indicated if binding of the antibody to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 50% of its affinity for TREM1. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 40% of its affinity for TREM1. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 30% of its affinity for TREM1. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 20% of its affinity for TREM1. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 10% of its affinity for TREM1. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 1% of its affinity for TREM1. In some embodiments, the affinity of the TREM1 antibody for the non-target molecule is less than about 0.1% of its affinity for TREM1.

[0289] As used herein, "k" d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is off Also called value.

[0290] As used herein, "k" a " (M -1 ×sec -1 The term k ) refers to the association rate constant for a particular antibody-antigen interaction. This value is onAlso called value.

[0291] As used herein, "K" D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. D =k d / k a In some embodiments, the affinity of an antibody is determined by the K D For clarity, as known in the art, the smaller K D values ​​indicate a high affinity interaction, but a large K D Values ​​indicate low affinity interactions.

[0292] As used herein, "K" A " (M -1 The term K ) refers to the association equilibrium constant of a particular antibody-antigen interaction. A =k a / k d .

[0293] As used herein in the context of two or more antibodies, the terms "compete with" or "cross-compete with" indicate that the two or more antibodies compete for binding to an antigen (e.g., TREM1). In one exemplary assay, TREM1 is coated on a surface and contacted with a first TREM1 antibody, after which a second TREM1 antibody is added. In another exemplary assay, a first TREM1 antibody is coated on a surface and contacted with TREM1, after which a second TREM1 antibody is added. In either assay, antibodies compete with each other if the presence of the first TREM1 antibody reduces binding of the second TREM1 antibody. The term "compete with" also includes antibody combinations in which one antibody reduces binding of another antibody, but no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit each other's binding regardless of the order in which they are added. In some embodiments, one antibody reduces the binding of another antibody to an antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (as measured in a competitive binding assay). One skilled in the art can select the concentration of antibody to use in a competition assay based on the affinity of the antibody for TREM1 and the valency of the antibody. The assays described in this definition are exemplary, and one skilled in the art can utilize any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358, each of which is incorporated by reference in its entirety.

[0294] A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits or blocks binding of the reference antibody, e.g., by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (as measured in a competitive binding assay). Antibodies identified by a competitive assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody so that steric hindrance occurs. For example, a second competing antibody can be identified that competes with a first antibody described herein for binding to TREM1. In certain cases, the second antibody can block or inhibit binding of the first antibody, e.g., by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (as measured in a competitive binding assay). In certain cases, the second antibody can replace the first antibody by more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0295] In some embodiments, the anti-TREM1 antibody does not substantially bind to myeloid cells present outside the cancer tissue, hi some embodiments, the anti-TREM1 antibody does not substantially bind to stimulatory myeloid cells present in the cancer tissue.

[0296] In some embodiments, the anti-TREM1 antibody binds to residues 21-34 (SEQ ID NO: 42), 103-109 (SEQ ID NO: 43), and 128-136 (SEQ ID NO: 44) of human TREM1. The binding epitope includes residues within a numerical range (e.g., residues 22-33 of TREM1), the starting residue of each range (e.g., residues 21-33 of TREM1), and the ending residue of each range (e.g., residues 22-34 of TREM1), or any combination thereof. In some embodiments, the anti-TREM1 antibody binds to residues 22-33, 104-108, and 129-135 of human TREM1. In some embodiments, the anti-TREM1 antibody binds to residues 21-33, 103-108, and 128-135 of human TREM1. In some embodiments, the anti-TREM1 antibody binds to residues 22-34, 104-109, and 129-136 of human TREM1. In some embodiments, the anti-TREM1 antibody binds to residues selected from residues 21-34, 22-33, 21-33, or 22-34; 103-109, 104-108, 103-108, or 104-109; and 128-136, 129-135, 128-135, or 129-136.

[0297] In some embodiments, the antibodies provided herein have a concentration of about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 8, 9, or 10 x 10 -9 K below M D In some embodiments, the K of the antibodies provided herein binds to human TREM1 (as measured by Biacore assay). Dis 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, 5~6, 5~5.5, 5.5~6, 5~7, 6~7, 6~6.5, 6.5~7, 6~8, 7~9, 7~10, or 5~10 x 10 -9 M (measured by Biacore assay).

[0298] In some embodiments, the antibodies provided herein are at a concentration of about 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 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, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, or 0.1 x 10 -9 M or lower, or K D In some embodiments, the antibodies provided herein bind to human TREM1 at a concentration of 5 to 3, 4 to 2, 3 to 1, 1.9 to 1.8, 1.8 to 1.7, 1.7 to 1.6, 1.6 to 1.5, 1.9 to 1.5, 1.5 to 1, 1 to 0.8, 1 to 0.5, 0.9 to 0.6, 0.7 to 0.4, 0.6 to 0.2, 0.5 to 0.3, 0.3 to 0.2, or 0.2 to 0.1 x 10 -9 K of M D In some embodiments, the antibodies provided herein bind to human TREM1 at a binding affinity of about 10, 9.56, 9.5, 9.0, 8.88, 8.84, 8.5, 8, 7.5, 7.32, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 x 10 (as measured by Biacore assay). -4 (1 / s) or less K dIn some embodiments, the antibodies provided herein bind to human TREM1 at 7-10, 7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, or 9.5-10 x 10 -4 (1 / s)K d In some embodiments, the antibodies provided herein bind to human TREM1 with a ribozyme activity of about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 45, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 7, 8, 9, or 10 x 10 (as measured by Biacore assay). 5 K greater than (1 / Ms) a In some embodiments, the antibodies provided herein bind to human TREM1 at a ribonucleotide concentration of 4-7, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10 x 10 (as measured by Biacore assay). 5 (1 / Ms)K a Binds to human TREM1 (as measured by Biacore assay).

[0299] In some embodiments, the antibody binds to human monocytes with an EC50 (measured by flow cytometry) of about 0.1, 0.15, 0.2, 0.22, 0.25, 0.27, 0.3, 0.32, 0.35, 0.37, 0.4, 0.05, 0.5, 0.55, 0.6, 0.65, 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, or 2 nM. In some embodiments, the antibodies provided herein bind to human monocytes with an EC50 (as measured by flow cytometry) of 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM or less. In some embodiments, the antibodies bind to human monocytes with an EC50 (as measured by flow cytometry) of about 0.2-1.4, 0.2-0.3, 0.3-0.5, 0.5-0.7, 0.7-1, 1-1.2, or 1.2-1.4 nM.

[0300] In some embodiments, the antibody binds to human neutrophils with an EC50 (measured by flow cytometry) of about 0.1, 0.15, 0.2, 0.22, 0.25, 0.27, 0.3, 0.32, 0.35, 0.37, 0.4, 0.05, 0.5, 0.55, 0.6, 0.65, 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, or 2 nM. In some embodiments, the antibodies provided herein bind to neutrophils with an EC50 (measured by flow cytometry) of about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM or less. In some embodiments, the antibodies bind to human neutrophils with an EC50 (measured by flow cytometry) of about 0.15-1, 0.15-0.3, 0.3-0.5, 0.5-0.7, 0.7-1, 1-1.5, or 1.5-2 nM.

[0301] In some embodiments, the antibodies provided herein do not bind to human neutrophils with an EC50 (measured by flow cytometry) of 3 nM or greater.

[0302] function "Effector function" refers to a biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP), receptor-ligand blockade, agonism, or antagonism.

[0303] In some embodiments, the antibody has antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC can occur when an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell. Effector cells bearing Fc gamma receptors (FcγR or FCGR) on their cell surface, including cytotoxic T cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, dendritic cells, or monocytes, recognize and bind the Fc region of an antibody bound to a target cell. Such binding can cause activation of intracellular signaling pathways leading to cell death. In certain embodiments, the immunoglobulin Fc region subtype (isotype) of the antibody comprises human IgG1 and IgG3. As used herein, ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize antibody bound to a target cell and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0304] In some embodiments, the antibody has complement-dependent cytotoxicity (CDC) activity. Antibody-induced CDC is mediated by proteins of the typical complement cascade and is triggered by the binding of the complement protein C1q to the antibody. The antibody Fc region that binds to C1q can induce activation of the complement cascade. In certain embodiments, the immunoglobulin Fc region subtype (isotype) of the antibody includes human IgG1 and IgG3. As used herein, CDC refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide (e.g., an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0305] In some embodiments, the antibody has antibody-dependent cellular phagocytosis (ADCP) activity. ADCP can occur when an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell. Phagocytes, including monocytes and macrophages, which have Fc receptors on their cell surface, recognize and bind the Fc region of antibodies bound to target cells. Binding of the Fc receptor to the antibody-bound target cell can initiate phagocytosis of the target cell. ADCP can be considered a form of ADCC.

[0306] In some embodiments, the TREM1 antibody induces reprogramming of unstimulated myeloid cells into stimulated myeloid cells. In some embodiments, the TREM1 antibody induces expression of a proinflammatory cytokine or chemokine and / or induces expression of a costimulatory molecule. In some embodiments, the costimulatory molecule is CD40 or HLA-DR. In some embodiments, the proinflammatory cytokine or chemokine is IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB).

[0307] 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 TREM1 protein expressed on a cell. An agonist antibody can bind to and activate NSM, causing changes in cell proliferation or modifying antigen-presenting ability. An agonist antibody can bind to and activate NSM, triggering intracellular signaling pathways that lead to modifications of cell growth or apoptosis. An agonist antibody can bind to TREM1 and induce signaling downstream of TREM1. In some embodiments, TREM1 signaling increases an immune response in cells. In some embodiments, the immune response is activation, cytokine or chemokine secretion, or expression of myeloid costimulatory proteins.

[0308] In some embodiments, the antibody is an antagonist antibody. An antagonist antibody can block (e.g., reduce) one or more activities or functions of NSM after the antibody binds to the TREM1 protein expressed on a cell. For example, an antagonist antibody can bind to and block ligands that bind to one or more NSM proteins, preventing cell differentiation and proliferation or modifying antigen-presenting ability. An antagonist antibody can bind to and prevent activation of the TREM1 protein by ligands, preventing binding and activation and modifying intracellular signaling pathways that contribute to cell growth and survival.

[0309] In some embodiments, the antibody is a depleting antibody. A depleting antibody kills unstimulated bone marrow cells upon contact via antibody interaction with other immune cells. For example, when the antibody binds to cells bearing the TREM1 protein, it can capture complement proteins and induce complement-dependent cell lysis. When the antibody binds to cells bearing the TREM1 protein, it can also induce nearby cells bearing Fc receptors to kill them by antibody-dependent cellular cytotoxicity (ADCC).

[0310] In some embodiments, the antibody is a neutralizing antibody, and the antibody neutralizes one or more biological activities of the NSM. In some embodiments, the TREM1 protein is expressed on the surface of unstimulated bone marrow cells, and the antibody recognizes the extracellular domain of the TREM1 protein.

[0311] In some embodiments, the antibody is selective for NSM (binds preferentially to TREM1). In certain embodiments, an antibody that selectively binds to NSM has a dissociation constant (Kd) in the range of 0.0001 nM to 1 μM. In certain embodiments, the antibody specifically binds to an epitope on the TREM1 protein that is conserved among proteins from different species. In other embodiments, selective binding includes, but does not require, exclusive binding.

[0312] In one embodiment, an anti-TREM1 antibody bound to a target causes in vivo depletion of unstimulated bone marrow cells to which it binds. In some embodiments, effector proteins induced by clustered antibodies can elicit various responses, including the release of inflammatory cytokines, control of antigen production, endocytosis, or cell killing. In one embodiment, the antibody can recruit and activate complement, mediate antibody-dependent cellular cytotoxicity (ADCC) in vivo, or mediate phagocytosis by binding Fc receptors in vivo. The antibody can also deplete unstimulated bone marrow cells by inducing apoptosis or necrosis of unstimulated bone marrow cells upon binding.

[0313] In some embodiments, the antibody is capable of forming an immune complex, for example, an immune complex may be a tumor cell coated with the antibody.

[0314] In some embodiments, the anti-TREM1 antibody does not substantially bind to myeloid cells present outside the cancer tissue, hi some embodiments, the anti-TREM1 antibody does not substantially bind to stimulatory myeloid cells present in the cancer tissue.

[0315] In some embodiments, deactivation of unstimulated bone marrow cells is in vitro and is achieved by a) killing the unstimulated bone marrow cells, b) magnetic bead depletion of the unstimulated bone marrow cells; or c) fluorescence-activated cell sorting (FACS) sorting of the unstimulated bone marrow cells.

[0316] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), such as an effector molecule, or a therapeutic (eg, cytokine) or diagnostic agent.

[0317] In certain embodiments, the antibody is conjugated to a drug, such as a toxin, a chemotherapeutic agent, an immunomodulator, or a radioisotope. Several methods for preparing ADCs (antibody-drug conjugates) are known in the art, and are described, for example, in U.S. Patent Nos. 8,624,003 (pot method), 8,163,888 (one-step method), and 5,208,020 (two-step method). The antibody or its antigen-binding fragment can be conjugated to at least one agent, including an antigen-binding radionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a prodrug, a toxin, an enzyme, an immunomodulator, an antiangiogenic agent, a proapoptotic agent, a cytokine, a hormone, an oligonucleotide, an antisense molecule, an siRNA, a second antibody, and a second antibody fragment.

[0318] Non-stimulated bone marrow cells (NSM) Described herein are methods and compositions for neutralizing and / or detecting non-stimulated myeloid cells (NSM), including the use of anti-TREM1 antibodies. Also provided herein are methods and compositions for targeting and / or detecting non-stimulated myeloid cells that express NSM proteins.

[0319] Also provided herein are methods and compositions for neutralizing and / or detecting unstimulated bone marrow cells in the non-human individual, comprising the use of antibodies directed against non-human homologs of human NSM proteins.

[0320] As used herein, unstimulated bone marrow cells are bone marrow cells that are not sufficiently effective at stimulating an immune response (e.g., not as effective at stimulating an anti-tumor response in the tumor microenvironment as stimulated bone marrow cells). In some embodiments, unstimulated bone marrow cells are not as effective at presenting antigens (e.g., tumor antigens) to T cells or at stimulating tumor-specific T cell responses as stimulated bone marrow cells. In some embodiments, unstimulated bone marrow cells may exhibit reduced ability to uptake, process, and / or present tumor-associated antigens to T cells as compared to stimulated bone marrow cells. Unstimulated bone marrow cells may contain reduced or no ability to reprime cytotoxic T lymphocytes, or in some cases, may not stimulate effective tumor cell killing. Unstimulated bone marrow cells may exhibit reduced expression of genes and cell surface markers involved in antigen processing, antigen presentation, and / or antigen costimulation, including, but not limited to, CD80, CD86, MHC1, and MHCII, as compared to stimulated bone marrow cells.

[0321] Unstimulated bone marrow cells may exhibit decreased expression of genes associated with cross-presentation, costimulation, and / or stimulatory cytokines, including, but not limited to, any one or more of TAP1, TAP2, PSMB8, PSMB9, TAPBP, PSME2, ​​CD24a, CD274, BTLA, CD40, CD244, ICOSL, ICAM1, TIM3, PDL2, RANK, FLT3, CSF2RB, CSF2RB2, CSF2RA, IL12b, XCR1, CCR7, CCR2, CCL22, CXCL9, and CCL5, and increased expression of the anti-inflammatory cytokine IL-10, when compared to stimulated bone marrow cells. In some embodiments, unstimulated bone marrow cells depend on the transcription factor IRF4 and the cytokines GM-CSF or CSF-1 for differentiation and survival. In some embodiments, unstimulated bone marrow cells can contribute to tumor angiogenesis by secreting vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS), and support tumor growth by secreting epidermal growth factor (EGF).

[0322] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages (TAMs) or dendritic cells (DCs). In some embodiments, the unstimulated bone marrow cells are not dendritic cells (DCs).

[0323] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages (TAMs). TAMs are macrophages that reside near or within cancerous tumors and are derived from circulating monocytes or resident tissue macrophages.

[0324] In some embodiments, the unstimulated bone marrow cells are tumor-associated neutrophils (TANs). TANs are neutrophils that reside near or within cancerous tumors.

[0325] In some embodiments, unstimulated and stimulated bone marrow cells are distinguished based on the markers they express or preferentially express. Expression of a cell surface marker can be described as "+" or "positive." Absence of a cell surface marker can be described as "-" or "negative." Expression of a cell surface marker can be further described as "high" (cells expressing high levels of the marker) or "low" (cells expressing low levels of the marker), indicating the relative expression of each marker on the cell surface. Marker levels may be determined by various methods known in the art, such as immunostaining and FACS analysis, or gel electrophoresis and Western blotting.

[0326] In some embodiments, the non-stimulatory bone marrow cells are dendritic cells (DCs). In some embodiments, dendritic cells can be distinguished by the morphology of their processes or dendrites. In one embodiment, the non-stimulatory dendritic cells are at least CD45+, HLA-DR+, CD14-, CD11c+, and BDCA1+ (also referred to as DC1 cells). In one embodiment, the non-stimulatory dendritic cells are not CD45+, HLA-DR+, CD14-, CD11c+, and BDCA3+ (also referred to as DC2 cells). In one embodiment, dendritic cells that are CD45+, HLA-DR+, CD14-, CD11c+, and BDCA3+ are stimulatory bone marrow cells.

[0327] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages (TAMs). In some embodiments, for example, in humans, the unstimulated tumor-associated macrophages are at least CD45+, HLA-DR+, CD14+. In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , CD11c + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11c+ In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated tumor-associated macrophages are at least CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + is.

[0328] In some embodiments, the methods and compositions of the present invention are useful for targeting TAMs and DCs in other mammals, e.g., mice. In such embodiments, mouse TAMs and DCs are contacted with a TREM1 antibody. In one embodiment, for example, in mice, tumor-associated macrophages are at least CD45+, IA / I-E+, CD14+, CD11b high , and CD11c low In one embodiment, for example in a mouse, tumor-associated macrophages are at least CD45+, IA / I-E+, CD14+, CD11b low , and CD11c high (also known as TAM2). high The term "macrophage" refers to a macrophage that expresses high levels of CD11b. low The term "macrophage" refers to CD11b high As used herein, "CD11c" refers to macrophages that express CD11b on their surface at levels substantially lower than those of macrophages. high The term "CD11c" refers to macrophages that express high levels of CD11c. low The term "macrophage" refers to CD11c highIt concerns macrophages that express CD11c on their surface at levels substantially lower than those of macrophages.

[0329] In some embodiments, the unstimulated bone marrow cells of the invention comprise one or more of TAM and DC1 cells.

[0330] In some embodiments, for example in a mouse, the unstimulated bone marrow cells of the invention comprise one or more of TAM1, TAM2, and DC1 cells. In such embodiments, the unstimulated bone marrow cells of the invention are contacted with a TREM1 antibody.

[0331] In some embodiments, the unstimulated myeloid cells are myeloid-derived suppressor cells (MDSCs). In some embodiments, the unstimulated myeloid cells are tumor-associated neutrophils (TANs).

[0332] In some embodiments, unstimulated bone marrow cells localize within the margins of tumor lesions or within transformed tumor ducts, where they are in contact with allogeneic T cells. In one embodiment, the localization of unstimulated bone marrow cells is modified, such that the cells no longer localize to the tumor margin or are no longer in contact with T cells.

[0333] In some embodiments, the unstimulated bone marrow cells are in a population of immune cells that includes stimulated and unstimulated bone marrow cells. In some embodiments, the unstimulated bone marrow cells are in a population of immune cells that includes only unstimulated bone marrow cells. The immune cell populations of the present invention may be pure, homogeneous, heterogeneous, derived from a variety of sources (e.g., diseased tissue, tumor tissue, healthy tissue, cell banks), maintained in primary cell culture, and / or maintained in ex vivo culture.

[0334] In some embodiments, the unstimulated bone marrow cells are tumor-associated macrophages.

[0335] In some embodiments, the unstimulated bone marrow cells are dendritic cells.

[0336] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA1 + The cell of the present invention essentially consists of cells that are

[0337] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - The cell of the present invention essentially consists of cells that are

[0338] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + The cell of the present invention essentially consists of cells that are

[0339] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11c + The cell of the present invention essentially consists of cells that are

[0340] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 -, and CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , and CD11c + The cell of the present invention essentially consists of cells that are

[0341] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + The cell of the present invention essentially consists of cells that are

[0342] In some embodiments, the unstimulated bone marrow cells are CD45+ , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , CD11b + , and CD11c + The cell of the present invention essentially consists of cells that are

[0343] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14 + , BDCA3 - , CD11b + , and CD11c + In some embodiments, the unstimulated bone marrow cells comprise cells that are CD45 + , HLA-DR + , CD14+ , BDCA3 - , CD11b + , and CD11c + The cell of the present invention essentially consists of cells that are

[0344] In some embodiments, the unstimulated bone marrow cells are CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA3 + In some embodiments, the unstimulated bone marrow cells are not CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA3 + Includes cells that are not

[0345] In some embodiments, for example in mice, unstimulated bone marrow cells express CD45 + , IA / IE + , CD14 + , CD11b high , and CD11c low In some embodiments, for example in mice, unstimulated bone marrow cells are CD45 + , IA / IE + , CD14 + , CD11b high , and CD11c low In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , IA / IE + , CD14 + , CD11b high , and CD11c low In some embodiments, for example in mice, the unstimulated bone marrow cells consist essentially of cells that are CD45 + , IA / IE + , CD14 + , CD11b high , and CD11c low In such embodiments, unstimulated mouse bone marrow cells are contacted with the TREM1 antibody.

[0346] In some embodiments, for example in mice, unstimulated bone marrow cells express CD45 + , IA / IE + , CD14 + , CD11b low , and CD11c high In some embodiments, for example in mice, unstimulated bone marrow cells are CD45 + , IA / IE + , CD14 + , CD11b low , and CD11c high In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , IA / IE + , CD14 + , CD11b low , and CD11c high In some embodiments, for example in mice, the unstimulated bone marrow cells comprise cells that are CD45 + , IA / IE + , CD14 + , CD11b low , and CD11c high In such embodiments, unstimulated mouse bone marrow cells are contacted with the TREM1 antibody.

[0347] In some embodiments, the unstimulated bone marrow cells are in cancer tissue.

[0348] In some embodiments, the population of immune cells is in cancer tissue.

[0349] In some embodiments, the unstimulated cells and the stimulated bone marrow cells are in cancer tissue.

[0350] In some embodiments, the biological sample comprises a population of immune cells comprising unstimulated bone marrow cells and stimulated bone marrow cells.

[0351] NSM cells may collectively refer to DC1, TAM1, and TAM2 cells, which are present in tumor tissues and can be distinguished from other cell types by the expression of NSM cell markers. For example, genes and associated proteins that are more abundantly expressed or translated in NSM cells than in SDCs can serve as NSM markers. An exemplary NSM marker is CD11b. Additional exemplary NSM markers are listed in Table A. NSM cells may express TREM1, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and TMEM119 on the cell surface. In some aspects, NSM cells do not express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1.

[0352] In one embodiment, the NSM cells express one or more of the NSM marker genes listed in Table D. In another embodiment, the NSM cells express 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more of the NSM markers listed in Table A. In another embodiment, the NSM cells express most or all of the NSM markers listed in Table A. In another embodiment, the NSM cells are identified as expressing MRC1, MS4A7, C1QC, APOE, C1QB, C1QA, and C5AR1.

[0353] (Table D) TIFF0007759996000004.tif84128

[0354] Stimulating bone marrow cells As used herein, stimulated bone marrow cells (also referred to in certain aspects as SDCs) are bone marrow cells that are effective in stimulating an immune response (e.g., more effective at stimulating an anti-tumor response in a tumor microenvironment compared to unstimulated bone marrow cells). In some embodiments, stimulated bone marrow cells are more effective at presenting antigens (e.g., tumor antigens) to T cells or more effective at stimulating tumor-specific T cell responses compared to unstimulated bone marrow cells. In some embodiments, stimulated bone marrow cells may exhibit increased ability to uptake, process, and / or present tumor-associated antigens to T cells compared to unstimulated bone marrow cells. Stimulated bone marrow cells may have a reduced ability to reprime cytotoxic T lymphocytes or, in some cases, stimulate effective tumor cell killing compared to unstimulated bone marrow cells. Stimulated bone marrow cells may exhibit higher expression of genes and cell surface markers involved in antigen processing, antigen presentation, and / or antigen costimulation, including, but not limited to, CD80, CD86, MHC1, and MHCII, compared to unstimulated bone marrow cells.

[0355] Exemplary stimulatory myeloid cell markers are listed in Table A. For example, in human SDC, expression of Xcr1, Clec9a, and BDCA3 (CD141) are markers of SDC identity. In mice, CD103 can also be used as a strong marker of SDC identity, although it will be noted that it is not expressed in human SDC.

[0356] In one embodiment, SDCs are tumor-infiltrating myeloid cells that have a dendritic cell identity and also express one or more of the SDC markers listed in Table A. In another embodiment, SDCs are tumor-infiltrating myeloid cells that have a dendritic cell identity and also express two, three, four, five, six, seven, eight, nine, or all of the SDC markers listed in Table A. In another embodiment, SDCs are identified as tumor-infiltrating myeloid dendritic cells that express BDCA3, KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, XCR1, and CLEC9A. SDC cells may express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1. In some embodiments, the SDCs do not substantially express TREM1, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1 / CD206, SIGLEC1, STAB1, TMEM37, MERTK, and / or TMEM119 on the cell surface. In some embodiments, the SDCs do not substantially express C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, MERTK, C1QC, TMEM119, MS4A7, APOE, CYP4F18, TREM1, TLR7, and / or LILRB4. Flow cytometry and PCR, among other art-recognized assays, can be used to assess expression of the markers disclosed herein.

[0357] Stimulated bone marrow cells express CD45 + , HLA-DR + , CD14 - , CD11c + , and BDCA3 + The stimulated bone marrow cells may be CD45 + , HLA-DR + , and BDCA3 + The stimulated bone marrow cells may be CD45 + , HLA-DR + , CD14 - , and BDCA3 + The stimulated bone marrow cells may be CD45+ , HLA-DR + , CD11c + , and BDCA3 + It could be.

[0358] Pharmaceutical Composition The present application provides compositions comprising antibodies, including pharmaceutical compositions comprising any one or more of the antibodies described herein, together with one or more pharmaceutically acceptable excipients. In some embodiments, the compositions are sterile. Pharmaceutical compositions generally comprise an effective amount of an antibody.

[0359] These compositions may contain, in addition to one or more of the antibodies disclosed herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other substance may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0360] The pharmaceutical composition for oral administration can be in tablet, capsule, powder, or liquid form.Tablets can contain solid carriers such as gelatin or adjuvants.Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil.Saline, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0361] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0362] Whether it is a polypeptide, antibody (e.g., an anti-TREM1 antibody), nucleic acid, small molecule, or other pharmaceutically useful compound according to the present invention to be given to an individual, the administration is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (in some cases, prophylaxis can be considered treatment, although prophylaxis can be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Treatment formulation, e.g., determining dosage, etc., is within the responsibility of general practitioners and other doctors, and will usually take into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above-mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0363] The compositions may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition to be treated.

[0364] method Preparation method The antibodies described herein can be produced using recombinant methods and compositions, such as those described in US Pat. No. 4,816,567.

[0365] In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or VH of the antibody (e.g., the light and / or heavy chain of the antibody) or an amino acid sequence comprising the VHH of a single-domain antibody. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In one embodiment, the nucleic acid is provided in a multicistronic vector. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antigen-binding polypeptide construct. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a YO, NS, or Sp20 cell). In one embodiment, a method of producing an antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and, optionally, recovering the antibody from the host cell (or host cell medium).

[0366] For recombinant production of antibodies, nucleic acids encoding the antibodies, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0367] The term "substantially purified" refers to constructs described herein, or variants thereof, which may be substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., naturally occurring cells or, in certain embodiments, host cells in the case of recombinantly produced heteromultimers, substantially free of extracellular material, includes preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein in certain embodiments is present at no more than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% of the dry weight of the cell. When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein is present in the culture medium in certain embodiments at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of dry weight of cells. In certain embodiments, the "substantially purified" heteromultimers produced by the methods described herein have a level of purification of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically at least about 75%, 80%, 85%, more specifically at least about 90%, at least about 95%, at least about 99%, or more, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0368] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0369] "Recombinant host cell" or "host cell" refers to a cell containing an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells. The exogenous polynucleotide may be maintained as a non-integrated vector, e.g., a plasmid, or alternatively, may be integrated into the host genome. Host cells may include CHO, CHO derivatives, NS0, Sp20, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0370] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0371] As used herein, the term "prokaryote" refers to prokaryotic organisms. For example, non-eukaryotic organisms may belong to the Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the Archaea (including, but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, e.g., Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

[0372] For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0373] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast lineages whose glycosylation pathways have been "humanized," resulting in the production of antibodies that contain partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0374] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly Spodoptera frugiperda cells for transfection.

[0375] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0376] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC5 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, NS0, and SP2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0377] In one embodiment, an antibody described herein is produced in a stable mammalian cell by a method comprising transfecting at least one stable mammalian cell with nucleic acids encoding the antibody in a predetermined ratio and expressing the nucleic acids in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that results in the highest proportion of antibody in the expression product.

[0378] In some embodiments, there are methods of producing antibodies in stable mammalian cells as described herein, wherein the expression product of at least one stable mammalian cell comprises a greater proportion of the desired glycosylated antibody compared to monomeric heavy or light chain polypeptides or other antibodies.

[0379] In some embodiments, there are methods for producing glycosylated antibodies in stable mammalian cells as described herein, the methods comprising identifying and purifying a desired glycosylated antibody, in some embodiments, by one or both of liquid chromatography and mass spectrometry.

[0380] If necessary, antibodies can be purified or isolated after expression. Proteins may be isolated or purified by a variety of methods known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, size, or gel filtration, and reversed phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins may find use in the present invention for antibody purification. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can often be enabled by specific fusion partners. For example, antibodies may be purified using glutathione resins if a GST fusion is used, Ni+2 affinity chromatography if a His tag is used or immobilized, or anti-Flag antibodies if a Flag tag is used. For general guidance on suitable purification techniques, see, e.g., Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994, which is incorporated by reference in its entirety. The degree of purification required will vary depending on the use of the antibody. In some cases, no purification is necessary.

[0381] In certain embodiments, antibodies are purified using anion exchange chromatography, including but not limited to, chromatography on Q-Sepharose, DEAE Sepharose, Poros HQ, Poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0382] In certain embodiments, the proteins described herein are purified using cation exchange chromatography, including but not limited to SP-Sepharose, CM Sepharose, Poros HS, Poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and derivatives.

[0383] Additionally, the antibodies described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NY, and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids generally include, but are not limited to, the D isomers of the common amino acids 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, designer amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs. Additionally, amino acids can be D (dextrorotatory) or L (levorotatory).

[0384] Methods for immunomodulation of bone marrow cells The administration methods of TREM1 antibodies described herein can result in the induction of proinflammatory molecules, such as cytokines, chemokines, or the expression of myeloid-activating receptors by myeloid cells. Generally, the induced proinflammatory molecules are present at levels higher than those achieved with isotype controls. In some embodiments, the myeloid cells are unstimulated myeloid cells. In some embodiments, the unstimulated myeloid cells are TREM1-expressing (TREM1+) cells. In some embodiments, the myeloid cells are myeloid-derived suppressor cells or tumor-associated neutrophils. In some embodiments, the TREM1+ cells are myeloid-derived suppressor cells or tumor-associated neutrophils. Such proinflammatory molecules then lead to the activation of anti-tumor immunity, including, but not limited to, T cell activation, M1-like macrophage activation, and NK cell activation. Thus, administration of anti-TREM1 antibodies can induce multiple anti-tumor immune mechanisms that lead to tumor destruction.

[0385] In another aspect, the invention provides methods for increasing an immune response in an individual, comprising administering to the individual an effective amount of a composition comprising an anti-TREM1 antibody or antigen-binding fragment thereof. In some embodiments, a method for increasing an immune response in a subject comprises administering to the subject an antibody that competes with a reference antibody for binding to human TREM1 (SEQ ID NO: 1). In some embodiments, a method for increasing an immune response in a subject comprises administering to the subject an antibody that competes for binding to human TREM1 (SEQ ID NO: 1), wherein the antibody binds within residues 21-34 (SEQ ID NO: 42), 103-109 (SEQ ID NO: 43), and 128-136 (SEQ ID NO: 44) of human TREM1 (SEQ ID NO: 1). In some embodiments, a method for increasing an immune response in a subject comprises administering to the subject an antibody that competes for binding to human TREM1 (SEQ ID NO: 1), wherein the antibody i) binds within residues 21-34 (SEQ ID NO: 42), 103-109 (SEQ ID NO: 43), and 128-136 (SEQ ID NO: 44) of human TREM1 (SEQ ID NO: 1), and ii) optionally comprises a human Fc region. In some embodiments, the antibody is present in a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.

[0386] In any and all aspects of increasing an immune response described herein, any increase or decrease or change in property(ies) or function(ies) aspect is compared to cells not contacted with the anti-TREM1 antibody.

[0387] Increasing an immune response can be both enhancing an immune response or inducing an immune response. For example, increasing an immune response includes both initiating or initiating an immune response or increasing or amplifying an ongoing or existing immune response. In some embodiments, the treatment induces an immune response. In some embodiments, the induced immune response is an adaptive immune response. In some embodiments, the induced immune response is an innate immune response. In some embodiments, the treatment enhances an immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the treatment increases an immune response. In some embodiments, the increased immune response is an adaptive immune response. In some embodiments, the increased immune response is an innate immune response. In some embodiments, the immune response is initiated or initiated by administration of an anti-TREM1 antibody. In some embodiments, the immune response is enhanced by administration of an anti-TREM1 antibody.

[0388] In another aspect, the application provides a method of contacting a cell with an anti-TREM1 antibody, which results in modulation of the immune function of the cell. The modulation can be an increase in immune response or reprogramming of unstimulated bone marrow cells to become stimulatory bone marrow cells. In some embodiments, the modulation is an increase in immune function. In some embodiments, the modulation is reprogramming of unstimulated bone marrow cells to become stimulatory bone marrow cells. In some embodiments, the modulation of function results in activation of unstimulated bone marrow cells. In some embodiments, the modulation of function results in reprogramming of TREM1-expressing bone marrow cells.

[0389] In some embodiments, the cells are unstimulated bone marrow cells. In some embodiments, the cells are TREM1-expressing cells (TREM1+ cells). In some embodiments, the unstimulated cells are TREM1+ cells. In some embodiments, the TREM1+ cells are one or more of DC1 cells, TAM1 cells, TAM2 cells, myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated neutrophils (TANs). In some embodiments, the TREM1+ cells are myeloid-derived suppressor cells. In some embodiments, the TREM1+ cells are tumor-associated neutrophils (TANs). In some embodiments, the unstimulated bone marrow cells are induced to become stimulatory myeloid cells (SDCs) by contacting the unstimulated bone marrow cells with a TREM1 antibody.

[0390] In some embodiments, modulating the function of unstimulated bone marrow cells or TREM1+ cells results in an increased ability of the cells to stimulate both naive and activated CD8+ T cells, e.g., by increasing the ability of the unstimulated cells to cross-present tumor antigens on MHC1 molecules to naive CD8+ T cells or by increasing cytokine or chemokine secretion by the unstimulated bone marrow cells. In some embodiments, modulating the function of unstimulated bone marrow cells or TREM1+ cells results in an increased ability of the cells to stimulate both naive and activated CD4+ T cells, e.g., by increasing the ability of the unstimulated bone marrow cells or TREM1+ to cross-present tumor antigens on MHCII molecules to naive CD4+ T cells. In some embodiments, modulating the function enhances or increases the ability of the cells to produce cytokines, chemokines, or costimulatory or activating receptors. In some embodiments, modulation increases a T cell stimulatory function of myeloid cells or TREM1+ cells, including, for example, the ability of the cells to induce T cell receptor (TCR) signaling, T cell proliferation, or T cell cytokine production.

[0391] In some embodiments, the increased immune response is cytokine and chemokine secretion. In some embodiments, the antibody has agonist activity. In some embodiments, the antibody induces increased expression of at least one cytokine or chemokine in cells compared to an isotype control antibody. In some embodiments, the at least one cytokine or chemokine is selected from the group consisting of IFN-γ, IL-1α, IL-12, IL-2, TNFSF9, TNFSF10, CXCL9, CXCL10, CCL17, CXCL1, CXCL5, CXCL8, CXCL11, CXCL15, CCL3, CCL4, CCL2, CCL8, CCL20, IL-6, CCL2, CCL7, CSF-1, CCL13, CCL19, TNFα, GZMH, PD-L1, MMP7, CCL23, CD70, CD8α, FasL, CD274, CRTAM, granzyme A (GzmA), or granzyme B (GzmB). In some embodiments, the cytokine or chemokine is CXCL10. In some embodiments, the cytokine or chemokine is IFN-γ. In some embodiments, cytokine or chemokine secretion is increased by about 1-100 fold (1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 fold) compared to untreated cells or cells treated with an isotype control antibody. In some embodiments, the chemokine is CXCL10 and secretion is increased by about 1-100 fold (1 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70-80 fold, 80-90 fold, or 90-100 fold) compared to untreated cells or cells treated with an isotype control antibody.In some embodiments, the cytokine is IFN-γ and secretion is increased by about 1-100 fold (1 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70-80 fold, 80-90 fold, or 90-100 fold) compared to untreated cells or cells treated with an isotype control antibody.

[0392] In some embodiments, the modulation increases the expression of at least one myeloid costimulatory protein compared to an isotype control antibody. In some embodiments, the myeloid costimulatory protein is HLA-DR, CD40, CD80, or CD86 on cells. HLA-DR is an MHC class II cell surface receptor that functions primarily to present peptide antigens to T cells to elicit an immune response. HLA-DR is a heterodimer of an α unit and a β unit. HLA-DR is expressed by antigen-presenting cells such as macrophages, B cells, and dendritic cells. CD40 is a costimulatory protein found on antigen-presenting cells and required for their activation. CD80 is a receptor expressed primarily by immune cells such as dendritic cells, B cells, monocytes, and antigen-presenting cells, and is closely related to CD86. CD80 interacts with CD28 and CTLA4 on T cells and acts in concert with CD86 to prime T and B cell activation, proliferation, and differentiation, including signaling T cells to differentiate into cytotoxic T cells. CD80 can also stimulate dendritic cells to enhance cytokine production. Stimulatory proteins, along with primary antigen-specific signals, are required for antigen-presenting cells to fully activate T cells. T cell costimulation is required for T cell proliferation, differentiation, and survival. Activated T cells without costimulation can result in T cell unresponsiveness or loss.

[0393] In some embodiments, the treatment increases HLA-DR expression. In some embodiments, the treatment increases CD40 expression. In some embodiments, the treatment increases CD80 expression. In some embodiments, the treatment increases CD86 expression. In some embodiments, costimulatory molecule expression is increased by about 1-100 fold (1 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70-80 fold, 80-90 fold, or 90-100 fold) or more compared to untreated cells or cells treated with an isotype control antibody.

[0394] In some embodiments, the enhanced immune response is the recruitment and activation of anti-tumor immune cells.

[0395] In some embodiments, the antibody induces increased activation of the ERK and / or JAK-STAT intracellular signaling pathway in cells compared to an isotype control antibody. In some embodiments, the antibody induces increased activation of STAT3 in cells compared to an isotype control antibody. In some embodiments, the antibody induces increased activation of the ERK intracellular signaling pathway in cells compared to an isotype control antibody. In some embodiments, the antibody induces increased activation of the JAK-STAT intracellular signaling pathway in cells compared to an isotype control antibody. The ERK pathway is involved in cell proliferation. STAT3 is a transcription factor and a member of the JAK-STAT signaling pathway. STAT3 mediates the transcription of genes involved in cell growth and promotes T cell proliferation. H 17 It is essential for the differentiation of helper T cells. Loss of STAT3 in vivo is also associated with the inability to maintain antibody-based immunity.

[0396] In some embodiments, the antibody induces a memory immune response compared to an isotype control antibody. Generally, a memory immune response is a protective immune response upon subsequent exposure to a pathogen or antigen that the immune system has previously encountered. Exemplary memory immune responses include immune responses following infection or vaccination with an antigen. Generally, memory immune responses are mediated by lymphocytes such as T cells or B cells. In some embodiments, a memory immune response is a protective immune response against cancer, including the growth, proliferation, or metastasis of cancer cells. In some embodiments, a memory immune response inhibits, prevents, or reduces the growth, proliferation, or metastasis of cancer cells.

[0397] In some embodiments, the antibody crosslinks TREM1 to TREM1 on the cell surface of TREM1+ cells. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In certain embodiments, the contacting is in vivo in a human. In some embodiments, the contacting occurs by administering an anti-TREM1 antibody. In some embodiments, the individual to whom the antibody (e.g., a human) is administered has cancer.

[0398] Methods for disabling, killing, or depleting unstimulated bone marrow cells In one aspect, the application provides a method of contacting unstimulated bone marrow cells with an anti-TREM1 antibody, such as a human or humanized antibody, which results in the neutralization of the unstimulated bone marrow cells.

[0399] In another aspect, the application provides a method of contacting unstimulated bone marrow cells with an anti-TREM1 antibody, which results in the neutralization of the unstimulated bone marrow cells.

[0400] In some embodiments, the non-stimulatory cells are one or more of DC1 cells and TAM cells. In some embodiments, the non-stimulatory cells are one or more of TAM cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated neutrophils (TANs).

[0401] In some embodiments, the present application provides a method for neutralizing unstimulated bone marrow cells, comprising contacting the unstimulated bone marrow cells with a TREM1 antibody, thereby killing the unstimulated bone marrow cells. Neutralization refers to rendering the cells partially or completely non-functional. In some embodiments, neutralization of the unstimulated bone marrow cells results in inducing growth arrest in the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in apoptosis of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in lysis of the cells, e.g., by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, neutralization of the unstimulated bone marrow cells results in necrosis of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in inducing growth arrest in the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in inactivation of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in neutralization of the activity of the TREM1 protein in the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in reduced proliferation of the cells. In some embodiments, neutralization of the unstimulated bone marrow cells results in differentiation of the cells. In some embodiments, nullifying the non-stimulated bone marrow cells results in a decrease in the ability of the cells to act as inhibitory antigen-presenting cells or an increase in the ability of the cells to act as activating antigen-presenting cells. In some embodiments, nullifying the non-stimulated bone marrow cells results in mallocalization of the cells within the tumor tissue or tumor microenvironment (TME). In some embodiments, nullifying the non-stimulated bone marrow cells results in altered spatial organization of cells within the tumor tissue or tumor microenvironment. In some embodiments, nullifying the non-stimulated bone marrow cells results in altered temporal expression of cells within the tumor tissue or TME. In some embodiments, the method further comprises removing the non-stimulated bone marrow cells.

[0402] In any and all aspects of neutralizing unstimulated bone marrow cells described herein, any increase or decrease or change in property(ies) or function(ies) aspect is as compared to cells not contacted with the anti-TREM1 antibody.

[0403] In another aspect, the present application provides a method of contacting unstimulated bone marrow cells with an anti-TREM1 antibody, resulting in modulation of the function of the unstimulated bone marrow cells. The modulation can be any one or more of the following: In some embodiments, the unstimulated cells are one or more of DC1 cells, TAM1 cells, TAM2 cells, MDSCs, and TANs. In some embodiments, the modulation of function results in neutralization of the unstimulated bone marrow cells. In some embodiments, the modulation of function of unstimulated bone marrow cells results in an increased ability of the cells to stimulate both naive and activated CD8+ T cells, for example, by increasing the ability of the unstimulated cells to cross-present tumor antigens on MHC1 molecules to naive CD8+ T cells. In some embodiments, the modulation of function of unstimulated bone marrow cells results in an increased ability of the cells to stimulate both naive and activated CD4+ T cells, for example, by increasing the ability of the unstimulated cells to cross-present tumor antigens on MHCII molecules to naive CD4+ T cells. In some embodiments, the modulation increases a T cell stimulatory function of the bone marrow cells, including, for example, the ability of the cells to induce T cell receptor (TCR) signaling, T cell proliferation, or T cell cytokine production. In some embodiments, the modulation of function enhances or increases the ability of the cells to produce cytokines, chemokines, or costimulatory or activating receptors. In one embodiment, the survival of unstimulated cells is decreased or the proliferation of unstimulated cells is decreased. In one embodiment, the ratio of stimulated to unstimulated bone marrow cells is increased.

[0404] In any and all aspects of reducing the function of unstimulated bone marrow cells described herein, any increase or decrease or change in property(ies) or aspect(s) of function(ies) is as compared to cells not contacted with the TREM1 antibody.

[0405] In some embodiments, the present application provides a method of killing unstimulated bone marrow cells (also referred to as inducing cell death), comprising contacting unstimulated bone marrow cells with an anti-TREM1 antibody, thereby killing the unstimulated bone marrow cells. In some embodiments, killing is increased compared to unstimulated bone marrow cells that have not been contacted with the anti-TREM1 antibody. In some embodiments, the contacting induces apoptosis of the unstimulated bone marrow cells. In some embodiments, the unstimulated bone marrow cells are in a population of immune cells that includes unstimulated bone marrow cells and stimulated bone marrow cells. In some embodiments, the method further comprises removing the unstimulated bone marrow cells. In some embodiments, 10% to 100% of the cells are killed. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells are killed.

[0406] In some embodiments, the present application provides a method for increasing the ratio of stimulated to unstimulated bone marrow cells in a population of immune cells, the ratio including stimulated and unstimulated bone marrow cells, comprising contacting the population of immune cells with an anti-TREM1 antibody. In some embodiments, the ratio is increased compared to a population of cells not contacted with the anti-TREM1 antibody. In some embodiments, the ratio of DC2 cells to DC1 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1 cells is increased. In some embodiments, the ratio of DC2 cells to TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1+TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1+DC1 cells is increased. In some embodiments, the ratio of DC2 cells to DC1+TAM2 cells is increased. In some embodiments, the ratio is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0407] In some embodiments, the ratio of stimulated bone marrow cells to unstimulated bone marrow cells before contact ranges from 0.001:1 to 0.1:1. In some embodiments, the ratio of stimulated bone marrow cells to unstimulated bone marrow cells after contact ranges from 0.1:1 to 100:1.

[0408] In some embodiments, the number of unstimulated bone marrow cells is reduced. In some embodiments, the stimulated bone marrow cells are DC2 cells. In some embodiments, the unstimulated bone marrow cells are killed, for example, by necrosis or apoptosis. In some embodiments, the unstimulated bone marrow cells are induced to undergo growth arrest. In some embodiments, the unstimulated bone marrow cells no longer proliferate. In some embodiments, the spatial localization of unstimulated bone marrow cells is altered, and the ratio is increased in specific regions of the TME. In some embodiments, the temporal expression of unstimulated bone marrow cells is altered, and the ratio is increased during specific times during tumor development.

[0409] In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In certain embodiments, the contacting is in vivo in a human. In some embodiments, the contacting occurs by administering an anti-TREM1 antibody. In some embodiments, the individual to whom the antibody (e.g., a human) is administered has cancer.

[0410] Cancer Treatment Methods In another aspect, the invention provides a method of treating an immune-related condition in an individual (e.g., a method of enhancing an immune response or a method of resulting in the neutralization of unstimulated myeloid cells) comprising administering to the individual an effective amount of a composition comprising an anti-TREM1 antibody. In some embodiments, the methods provided herein are useful for treating cancer, such that the anti-TREM1 antibody or the individual being administered the anti-TREM1 antibody has cancer.

[0411] Any suitable cancer can be treated with the antibodies provided herein. The cancer can be, but is not limited to, any carcinoma, adenocarcinoma, soft tissue, sarcoma, teratoma, melanoma, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or brain cancer, or any other cancer known in the medical field. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is immune-evasive. In some embodiments, the cancer is immune-responsive.

[0412] In some embodiments, the cancer is melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, urothelial cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, renal cancer, endometrial cancer, cervical cancer, testicular cancer, melanoma, leukemia, lymphoma, or mesothelioma. In some embodiments, the cancer is colon cancer, pancreatic cancer, or breast cancer.

[0413] In some embodiments, the treatment results in a decrease in the volume or size of the cancer. In some embodiments, the treatment is effective in reducing the volume of the cancer compared to the volume of the cancer before administration of the antibody. In some embodiments, the treatment results in a decrease in the rate of cancer growth. In some embodiments, the treatment is effective in reducing the rate of cancer growth compared to the rate of cancer growth before administration of the antibody. In some embodiments, the treatment is effective in eliminating the cancer.

[0414] In some embodiments, the immune-related condition is an immune-related condition associated with expression of TREM1 protein on unstimulated (human) bone marrow cells or expression of a homologue of the TREM1 protein in a non-human species. In some embodiments, the immune-related condition is an immune-related condition associated with overexpression of TREM1 protein in unstimulated bone marrow cells compared to stimulated bone marrow cells. In some embodiments, overexpression of TREM1 mRNA or TREM1 protein is at least about 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, or 100-fold higher compared to stimulated bone marrow cells.

[0415] In some embodiments, these methods are further provided in combination with other combination therapies, such as PD-1 / PD-L1 / PD-L2 blocking therapy, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, CTLA4 blocking therapy, anti-CTLA-4 antibodies, systemic checkpoint blockade therapy in which inhibitory molecules on T cells are blocked, adoptive T cell therapy, CAR T cell therapy, dendritic cell, or other cell therapy, and conventional chemotherapy.

[0416] In some embodiments, the method further comprises determining the expression level of TREM1 protein in a biological sample from the individual. In some embodiments, the biological sample includes, but is not limited to, body fluids, tissue samples, organ samples, urine, feces, blood, saliva, CSF, and any combination thereof. In some embodiments, the biological sample is derived from tumor tissue. In some embodiments, the expression level comprises the mRNA expression level of mRNA encoding TREM1 protein. In some embodiments, the expression level of TREM1 protein comprises the protein expression level of NSM. In some embodiments, the expression level of TREM1 protein is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques, and FISH, and combinations thereof.

[0417] In another aspect, the present application provides a method for determining the presence or absence of unstimulated bone marrow cells in general, or a method for determining the presence or absence of specific unstimulated bone marrow cells (e.g., DC1 cells, TAM1 cells, and / or TAM2 cells), comprising contacting a population of cells comprising unstimulated bone marrow cells with an anti-TREM1 antibody and quantifying the number of unstimulated bone marrow cells. In another aspect, the present application provides a method for determining the presence or absence of unstimulated bone marrow cells, comprising contacting a population of immune cells comprising unstimulated bone marrow cells and stimulated bone marrow cells with an anti-TREM1 antibody, detecting complexes or moieties indicative of binding of the antibody to the cells, and optionally quantifying the number of unstimulated bone marrow cells in the population. In another aspect, the present application provides a method for determining the relative ratio of unstimulated bone marrow cells to stimulated bone marrow cells, comprising contacting a population of immune cells comprising unstimulated bone marrow cells and stimulated bone marrow cells with an anti-TREM1 antibody; quantifying the number of stimulated bone marrow cells and unstimulated bone marrow cells; and determining the relative ratio of unstimulated bone marrow cells to stimulated bone marrow cells.

[0418] In the embodiments described herein for detection and / or quantification, the anti-TREM1 antibody binds to the TREM1 protein but does not necessarily affect a biological response such as ADCC, although it may have an effect on a biological response.

[0419] In another aspect, the present invention provides methods for identifying an individual who may respond to immunotherapy (e.g., using an anti-TREM1 antibody) for the treatment of an immune-related condition (e.g., cancer), comprising detecting the expression level of TREM1 protein in a biological sample from the individual and determining whether the individual may respond to immunotherapy based on the expression level of TREM1 protein, wherein an elevated level of the individual's TREM1 protein compared to that of a healthy individual indicates that the individual may respond to immunotherapy. In some embodiments, these methods may also be used to diagnose an immune-related condition (e.g., cancer) in an individual, wherein an elevated level of the individual's TREM1 protein compared to that of a healthy individual indicates that the individual has cancer, based on the expression level of TREM1 protein. In some embodiments, the expression level comprises the mRNA expression level of mRNA encoding TREM1 protein. In other embodiments, the expression level of TREM1 protein comprises the protein expression level of TREM1 protein. In some embodiments, the expression level of TREM1 protein is detected in a sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques, and FISH, and combinations thereof. In these embodiments, the anti-TREM1 antibody binds to the TREM1 protein but does not necessarily affect a biological response such as ADCC. In some embodiments, the biological sample is derived from tumor tissue. In some embodiments, the biological sample includes, but is not limited to, body fluids, tissue samples, organ samples, urine, feces, blood, saliva, CSF, and any combination thereof.

[0420] Also disclosed herein is a method for enhancing the immune response of a subject against tumor or the effectiveness of immunotherapy treatment.Generally, treatment that increases the abundance of SDC will improve subject outcomes such as recurrence-free survival time, and will enhance the effectiveness of cancer immunotherapy treatment.Treatment can increase the relative or absolute abundance of SDC cells in the tumor of a subject.Treatment can decrease the relative or absolute abundance of NSM cells in the tumor of a subject.

[0421] An exemplary general treatment strategy involves increasing the number of SDCs by systemic introduction of Flt3L. Another method is to treat a subject's autologous bone marrow or blood cells with Flt3L while simultaneously blocking CSF1. For example, retroviral expression of SDC transcription factors such as IRF8, Mycl1, BATF3, or ZBTB46 in bone marrow or blood progenitor cell populations can also be used to promote the emergence of SDCs. Another treatment strategy involves systematically eliminating NSM cells while selectively sparing SDCs. This can result in an overall favorable change in the ratio of these populations. Elimination of NSM cells can be achieved by any means, including administration (systemically or locally to the tumor) of antibodies against the TREM1 surface protein.

[0422] In some embodiments, the SDC-enhancing treatment is applied as a therapeutic treatment to make the subject's natural immune system more capable of controlling or eradicating cancer. In another embodiment, the SDC-enhancing treatment of the present invention is applied in combination with a therapeutic treatment, such as an immunotherapy treatment (such application occurs before, simultaneously with, or after the immunotherapy treatment), and the SDC-enhancing treatment acts as a supplementary or adjuvant treatment to increase the effectiveness of the therapeutic treatment.

[0423] Administration method In some embodiments, the methods provided herein are useful for treating an immune-related condition in an individual. In one embodiment, the individual is a human and the antibody is a TREM1 antibody. In another embodiment, the individual is a mouse and the antibody is a TREM1 antibody.

[0424] In some embodiments, the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. An effective amount of an anti-TREM1 antibody may be administered to treat cancer. The appropriate dosage of the anti-TREM1 antibody may be determined based on the type of cancer to be treated, the type of anti-TREM1 antibody, the severity and course of the cancer, the individual's clinical condition, the individual's clinical history, and response to treatment, as well as the judgment of the attending physician.

[0425] Combination therapy In some embodiments, the antibodies provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic or immunotherapeutic agent may be administered with the antibodies provided herein. In some embodiments, the immunotherapy is selected from, but is not limited to, checkpoint inhibitors; T cell checkpoint inhibitors; anti-PD1 antibodies; anti-PDL1 antibodies; anti-CTLA4 antibodies; adoptive cell therapy; adoptive T cell therapy; CAR-T cell therapy; dendritic cell vaccines; STING agonists; monocyte vaccines; Bacillus Calmette-Guérin vaccines; antigen binding proteins that bind to both T cells and antigen-presenting cells; BiTE dual antigen binding proteins; Toll-like receptor ligands; cytokines; cytotoxic therapy; chemotherapy; radiation therapy; small molecule inhibitors; small molecule agonists; immunomodulators; oncolytic viruses; and epigenetic modulators, and combinations thereof.

[0426] In some embodiments, the additional therapeutic agent is an antibody. In some embodiments, the additional therapeutic agent is an antibody that binds to a protein(s) on the surface of a tumor cell.

[0427] For the treatment of cancer, anti-TREM1 antibodies may be combined with one or more antibodies that inhibit immune checkpoint proteins. Immune checkpoint proteins displayed on the surface of tumor cells are of particular interest. The most actively investigated immune checkpoint receptors in the context of clinical cancer immunotherapy, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4, also known as CD152) and programmed cell death protein 1 (PD1, also known as CD279), are both inhibitory receptors. The clinical activity of antibodies that block either of these receptors means that antitumor immunity can be enhanced at multiple levels and that combination strategies can be rationally designed, guided by mechanistic considerations and preclinical models.

[0428] The two ligands for PD-1 are PD-1 ligand 1 (PD-L1, also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). PD-L1 is expressed on cancer cells, and through binding to its receptor PD-1 on T cells, it inhibits T cell activation / function. Inhibitors that block the interaction of PD-1 with its cognate ligands PD-L1 and PD-L2 on cancer cells can result in increased T cell activation and function, preventing cancer cells from evading the immune system.

[0429] 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.

[0430] Various PD-1, PD-L1, and PD-L2 antibodies are known in the art. In some embodiments, the additional therapeutic agent is at least one of atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1), nivolumab (PD-1), pembrolizumab (PD-1), cemiplimab (PD-1), ipilimumab (CTLA-4), tremelimumab (CTLA-4), or any combination thereof.

[0431] The additional therapeutic agent can be administered by any suitable means. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in the same pharmaceutical composition. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in different pharmaceutical compositions.

[0432] In embodiments in which an antibody provided herein and an additional therapeutic agent are contained in different pharmaceutical compositions, administration of the antibody may occur prior to, simultaneously with, and / or after administration of the additional therapeutic agent. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one month of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one week of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one day of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about 12 hours of each other. In some embodiments, administration of an antibody provided herein and an additional therapeutic agent occurs within about one hour of each other.

[0433] Kits and Articles of Manufacture The present application provides kits comprising any one or more of the antibody compositions described herein. In some embodiments, the kit further contains a component selected from a secondary antibody, an immunohistochemistry reagent, a pharmaceutically acceptable excipient, and instructions, and any combination thereof. In a specific embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein together with one or more pharmaceutically acceptable excipients.

[0434] The present application also provides an article of manufacture comprising any one of the antibody compositions or kits described herein. Examples of articles of manufacture include vials (including sealed vials).

[0435] Additional Embodiments Below are paragraphs listing specific embodiments.

[0436] 1. An isolated antibody that binds to human TREM1 (SEQ ID NO: 1), comprising: a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3; and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 24; c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 33; d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 26; e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 27; f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 28; The isolated antibody.

[0437] The isolated antibody of claim 1, wherein the VH chain sequence comprises the set of VH sequences set forth in SEQ ID NO: 17 and the VL chain sequence comprises the set of VL sequences set forth in SEQ ID NO: 20.

[0438] The isolated antibody of claim 1, wherein the VH chain sequence comprises a VH sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18, and the VL chain sequence comprises a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22.

[0439] 2. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35.

[0440] 2. The isolated antibody of claim 1, wherein the VH chain sequence consists of the set of VH sequences set forth in SEQ ID NO: 17 and the VL chain sequence consists of the set of VL sequences set forth in SEQ ID NO: 20.

[0441] 2. The isolated antibody of claim 1, wherein the VH chain sequence consists of a VH sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18, and the VL chain sequence consists of a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22.

[0442] 2. The isolated antibody of claim 1, wherein the antibody consists of a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35.

[0443] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated, the VH chain sequence comprises the VH sequence set forth in SEQ ID NO: 17, and the VL chain comprises the VL sequence set forth in SEQ ID NO: 20.

[0444] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated, and the VH chain sequence comprises a VH sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18, and the VL chain sequence comprises a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22.

[0445] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated and comprises a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35.

[0446] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated, the VH chain sequence comprises the VH sequence set forth in SEQ ID NO: 17, and the VL chain sequence consists of the VL sequence set forth in SEQ ID NO: 20.

[0447] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated, the VH chain sequence consists of a VH sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18, and the VL chain sequence comprises a VL sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22.

[0448] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated and consists of a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO: 35.

[0449] The isolated antibody of claim 1, wherein the antibody is afucosylated.

[0450] The isolated antibody of claim 1, wherein the antibody comprises an active human Fc.

[0451] 16. The isolated antibody of claim 15, wherein the human Fc is a wild-type human IgG1 Fc.

[0452] 2. The isolated antibody of claim 1, wherein the antibody is afucosylated and comprises a wild-type human IgG1 Fc, wherein the VH chain sequence comprises the VH sequence set forth in SEQ ID NO: 17 and the VL chain sequence comprises the VL sequence set forth in SEQ ID NO: 20.

[0453] the antibody has a cytoplasmic affinity of about 0.5, 1, 2, 3, 4, 5, 6, or 7 x 10 as measured by a surface plasmon resonance (SPR) assay -9 The isolated antibody of claim 1, which binds to human TREM1 with a KD of M or less.

[0454] The isolated antibody of claim 1, wherein the antibody is humanized.

[0455] 10. A method for producing an antibody comprising expressing the antibody of claim 1 from a host cell and isolating the expressed antibody.

[0456] A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable excipient.

[0457] A kit comprising the antibody of claim 1 and instructions for use.

[0458] 1. An isolated antibody that binds to human TREM1 (SEQ ID NO: 1), comprising: a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3; and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 24; c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 32; d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 26; e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 27; f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 28; The isolated antibody.

[0459] 24. The isolated antibody of claim 23, wherein the VH chain sequence comprises the set of VH sequences set forth in SEQ ID NO: 13 and the VL chain sequence comprises the set of VL sequences set forth in SEQ ID NO: 20.

[0460] 24. The isolated antibody of claim 23, wherein the antibody comprises a heavy chain sequence set forth in SEQ ID NO: 36 and a light chain sequence set forth in SEQ ID NO: 37.

[0461] 24. A pharmaceutical composition comprising the antibody of claim 23 and a pharmaceutically acceptable excipient.

[0462] 1. A method of treating cancer in a subject, comprising administering to the subject an antibody that competes with a reference antibody for binding to human TREM1 (SEQ ID NO: 1), and comprises a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 24; c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 33; d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 26; e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 27; f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 28; The method.

[0463] 28. The method of claim 27, wherein the subject has previously received, is concurrently receiving, or will subsequently receive immunotherapy, and the immunotherapy is at least one of a checkpoint inhibitor; a T cell checkpoint inhibitor; an anti-PD1 antibody; an anti-PDL1 antibody; an anti-CTLA4 antibody; an adoptive T cell therapy; a CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds to both T cells and antigen-presenting cells; a BiTE dual antigen binding protein; a Toll-like receptor ligand; a cytokine; a cytotoxic therapy; chemotherapy; radiation therapy; a small molecule inhibitor; a small molecule agonist; an immunomodulatory agent; and an epigenetic modulatory agent.

[0464] 29. The method of claim 28, wherein the immunotherapy is an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0465] 1. A method of increasing an immune response in a subject, comprising administering to the subject an antibody that competes with a reference antibody for binding to human TREM1 (SEQ ID NO: 1), and comprising a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 24; c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 33; d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 26; e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 27; f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 28; The method. [Example]

[0466] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0467] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature, see, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0468] Example 1: Sequencing and humanization of anti-TREM1 antibodies A monoclonal mouse IgG1 clone, TREM-26, specific for human TREM1 was obtained and used for sequencing and humanization. Briefly, the antibody's disulfide bonds were reduced with dithiothreitol (DTT), and free sulfhydryl groups were alkylated with iodoacetamide. The alkylated antibody was digested with sequencing-grade endoproteinase, purified using a spin column, and sequenced by LC-MS / MS analysis (see below).

[0469] A chimeric antibody (PI-4026) was constructed by grafting the VL and VH regions of a murine antibody onto a human IgG1 constant region. The chimera was further humanized by cloning the antibody CDRs into human variable domain frameworks, generating 10 additional humanized variants with different framework mutations, four VH variants, and four VL variants (PI-4026-1-10).

[0470] The VH and VL sequences were compared to a library of known human germline sequences on the NCBI website (ncbi.nlm.nih.gov / igblast / ; Ye, J. et al., Nucleic Acids Research 41:W34-W40 (2013)). The databases used were the IMGT human VH gene (F+ ORF, 273 germline sequences) and the IMGT human VL kappa gene (F+ ORF, 74 germline sequences).

[0471] For humanized PI-4026VH, human germline IGHV1-46 (allele 1) was selected as the acceptor sequence, and the human heavy chain IGHJ4 (allele 1) joining region (J gene) was selected from human joining region sequences compiled by IMGT®, the international ImMunoGeneTics information system®, www.imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France).

[0472] For humanized PI-4026VL, human germline IGKV1-39 (allele 1) was selected as the acceptor sequence, and the human light chain IGKJ2 (allele 1) joining region (J gene) was selected from human joining region sequences compiled by IMGT®, the international ImMunoGeneTics information system®, www.imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France).

[0473] CDRs were defined according to AbM definitions (see Dr. Andrew C. Martin at www.bioinf.org.uk / abs / for a table comparing CDR definitions). For example, changes in human germline framework (i.e., non-CDR residues of VH and VL) positions to the corresponding parental murine sequences were used to optimize binding of the humanized antibodies.

[0474] Table 1A shows the VH and VL sequences of the humanized versions of mAb PI-4026 that were created. 4026VH-1 and 4026VL-1 were the parent humanized VH and VL clones from which other humanized versions were created with additional framework mutations. Three humanized clones of the VH region (4026VH-2, VH-3, VH-4) and three humanized clones of the VL region (4026VL-2, VL-3, VL-4) were created. Table 1B shows the CDR sequences.

[0475] (Table 1A) TIFF0007759996000005.tif70170

[0476] Table 1B. CDRs of humanized antibodies TIFF0007759996000006.tif58128

[0477] Ten humanized variants of the TREM1 antibody were generated using different framework mutations. Table 1C shows the number of framework mutations and heavy and light chain pairings used in each humanized variant.

[0478] (Table 1C) TIFF0007759996000007.tif74128

[0479] Example 2: Production and characterization of anti-TREM1 antibodies Antibody production and characterization Heavy and light chain expression vectors were transfected into expi293 cells using standard methods. Cells were grown for up to 7 days, after which the supernatant was harvested for antibody purification. In addition to expi293, antibodies were also produced in expi293 cells that had been CRISPR / Cas9-edited (Alexander Weiss, University of Toronto) to delete mammalian α1,6 fucosyltransferase (FUT8). The pH of the supernatant was adjusted with 1 M HEPES pH 7.4, and sodium azide was added to prevent microbial growth. Proteins were captured using KanCap A resin, and after washing with PBS containing 1 M NaCl and PBS, the antibodies were eluted with 50 mM citric acid pH 3.5, 100 mM NaCl. Immediately after elution, the solution was neutralized with 1 M Tris (pH 8) containing 0.5 M arginine. Biophysical characterization of the buffer-exchanged proteins into PBS was performed using standard methods. Protein was quantified at OD280, and the calculated extinction coefficient was used to determine the amount and concentration. Purity and approximate molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris / glycine / SDS, 4-20%) or a Perkin Elmer GXII capillary electrophoresis system. Aggregation status was determined by UHPLC with detection at 280 nm using a Sepax Zenix-C SEC-300, 3 μm, 300 Å, 4.6*150 mm size exclusion column and PBS running buffer.

[0480] Human binding strength K using surface plasmon resonance (SPR) D measurement The affinity of human TREM1 binding to PI-4026 variants was measured by SPR on a BIAcore T200 (GE Healthcare, UK). All data were collected at 25°C. 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) surfactant P20 were used as the transfer buffer and for all dilutions of mAbs. Anti-mouse Fc was immobilized on a CM4 biosensor chip (GE Healthcare) using amine coupling chemistry. Human chimeric TREM1-mIgG Fc was captured on one flow cell, and the other flow cell (used as the reference surface) was left blank. Various concentrations of PI-4026 variants (all hIgG1 isotypes as analytes) were injected through both flow cells in multiple cycles. After each cycle, the surface was regenerated by injecting glycine HCl buffer (10 mM, pH 2.0). Kinetic evaluation was performed using BIAevaluation software and consisted of generating sensorgrams fitted to a 1:1 Langmuir binding model.

[0481] Human monomeric K using surface plasmon resonance (SPR) D measurement The affinity of human TREM1 binding to PI-4026 variants was measured by SPR on a BIAcore T200 (GE Healthcare, UK). All data were collected at 25°C. 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) surfactant P20 were used as the transfer buffer and for all dilutions of mAbs. Anti-human Fc was immobilized onto a CM4 biosensor chip (GE Healthcare) using amine coupling chemistry. PI-4026 variants (all hIgG1 isotype) were captured with anti-human Fc in one flow cell, while another flow cell (used as the reference surface) was left blank. Varying concentrations of His-tagged human TREM1 (as the analyte) were injected through the flow cell containing the captured PI-4026 variants and the reference cell in multiple cycles. After each cycle, the surface was regenerated by injecting 3 M MgCl2. Kinetic evaluation was performed using BIAevaluation software and consisted of generating sensorgrams fitted to a 1:1 Langmuir binding model.

[0482] Differential scanning fluorometry The melting temperatures of PI-4026 variants were determined by nano-differential scanning fluorimetry (DSF). PI-4026 variant samples were run from 20 °C to 100 °C at a ramp rate of 1.5 °C / min. The samples were loaded into nano-DSF-grade capillaries by immersion. To minimize evaporation during thermal scanning, both ends of the capillary were sealed with an inert oil-based liquid rubber sealing paste.

[0483] T is determined from the change in tryptophan fluorescence intensity or the ratio of tryptophan emission at 350 and 330 nm. m This allows us to accurately investigate the conformational changes during protein unfolding as a function of temperature. m is the temperature at which the protein is 50% unfolded.

[0484] cell binding Logarithmic-phase HEK293 control cells were harvested from culture, washed, and diluted to 1 x 10 5 Cells were plated at 1000 cells / well in a 96-well U-bottom plate. The indicated titrations of PI-4026 variants and isotype controls were added to the cells and incubated on ice for 30 minutes. Bound primary antibody was detected using an Alexa 647-conjugated anti-human Fcγ secondary antibody (Jackson Immunoresearch) diluted 1:500. The secondary antibody was incubated with the cells on ice for 30 minutes. Cells were then washed and stained with Zombie NIR (Biolegend) to determine cell viability for 15 minutes at room temperature. Fluorescent signals of bound antibodies were assessed by flow cytometry (ThermoFisher Attune NxT).

[0485] result Table 2 shows the antibody avidity, monomer affinity, cell binding, and DSF results for each of the humanized TREM1 antibodies.

[0486] (Table 2) TIFF0007759996000008.tif125166

[0487] Based on the results shown, PI-4026-5 was selected for further characterization and testing.

[0488] The affinity of human TREM1 binding to PI-4026-5 was then further measured by SPR on a BIAcore T200 (GE Healthcare). All data were collected at 25°C. 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) surfactant P20 were used as the transfer buffer and diluent for PI-4026-5. Anti-mouse Fc was immobilized on a CM4 biosensor chip (GE Healthcare) using amine coupling chemistry. Human chimeric TREM1-mIgG Fc was captured on one flow cell, and the other flow cell, used as the reference surface, was left blank. Various concentrations of PI-4026-5 were injected through both flow cells in multiple cycles. After each cycle, the surface was regenerated by injecting glycine HCl buffer (10 mM, pH 2.0). Kinetic evaluation was performed using BIAevaluation software. Kinetic evaluation consisted of generating sensorgrams fitted to a 1:1 Langmuir binding model.

[0489] The SPR binding kinetics of PI-4026-5 is shown in Figure 1 .

[0490] Example 3: Cell binding of anti-TREM1 antibodies Species specificity of humanized TREM1 antibodies hTREM1-overexpressing cells Logarithmic-phase HEK293 control or human TREM1-overexpressing cells were harvested from culture, washed, and plated at 1 × 10 5Cells were plated at 1000 cells / well in a 96-well U-bottom plate. Unconjugated hIgG1 control or titrations of PI-4026-5 antibody at the indicated concentrations were added to the cells and incubated on ice for 30 minutes. Bound primary antibody was detected using a 1:500 diluted Alexa 647-conjugated anti-human Fcγ secondary antibody (Jackson Immunoresearch). The secondary antibody was incubated with the cells on ice for 30 minutes. Cells were then washed and stained with Zombie NIR (Biolegend) to determine cell viability for 15 minutes at room temperature. The fluorescent signal of the bound antibody was assessed by flow cytometry (ThermoFisher Attune NxT). EC50 values ​​were calculated using Prism software (Graphpad).

[0491] Cynomolgus monkey TREM1 and mTREM1 overexpressing cells Logarithmically growing HEK293 cells overexpressing cynomolgus monkey or mouse TREM1 were harvested from culture, washed, and plated at 1 x 10 5 Cells were plated at 1000 cells / well in a 96-well U-bottom plate. Unconjugated hIgG1 control or titrations of PI-4026-5 antibody at the indicated concentrations were added to the cells and incubated on ice for 30 minutes. Bound primary antibody was detected using a 1:500 diluted Alexa 647-conjugated anti-human Fcγ secondary antibody (Jackson Immunoresearch). The secondary antibody was incubated with the cells on ice for 30 minutes. Cells were then washed and stained with Zombie NIR (Biolegend) to determine cell viability for 15 minutes at room temperature. The fluorescent signal of the bound antibody was assessed by flow cytometry (ThermoFisher Attune NxT). EC50 values ​​were calculated using Prism software (Graphpad).

[0492] cell binding Red blood cells from fresh blood from human donors (Stanford Blood Center) were lysed and washed to enrich for PBMC and granulocyte content. Cells were plated and Fc receptors were blocked with a combination of human serum (Jackson Immunoresearch), human FcX (Biolegend), and a peptide-based FcR blocking solution (Innovex Biosciences). Cells were also stained with Zombie NIR (Biolegend) to determine cell viability. After staining with Zombie NIR, cells were stained with a flow cytometry cocktail containing markers of major immune subsets and titrations of human IgG1 control or PI-4026-5. All antibodies used were directly conjugated. Data were acquired by flow cytometry (ThermoFisher Attune NxT). EC50 values ​​were calculated using Prism software (Graphpad).

[0493] result As shown in Figures 2A and 2B, PI-4026-5 binds with high specificity to human TREM1-overexpressing cells but not to cells that do not overexpress human TREM1. Furthermore, PI-4026-5 binds to neutrophils and monocytes in human peripheral blood (Figures 3A and 3B).

[0494] To confirm the species specificity of the humanized TREM1 antibody, the binding of PI-4026-5 to cynomolgus monkey and mouse TREM1 was assessed.

[0495] As shown in Figures 4A and 4B, PI-4026-5 binds to cynomolgus monkey TREM1-overexpressing cells, but not to mouse TREM1-overexpressing cells.

[0496] Example 4: Induction of ADCC and ADCP by anti-TREM1 antibodies ADCC and ADCP assays HEK293 target cells overexpressing human TREM1 were incubated with titrations of PI-4026-5 or hIgG1 isotype control at the indicated concentrations in flat-bottom white 96-well plates (ThermoFisher). After 15 minutes of incubation at room temperature, hCD16- or hCD32-expressing NFAT-luciferase reporter Jurkat cells (Promega, Madison, WI) were added to the target cell / antibody mixture at a 3:1 ratio. After adding the reporter cells, the assay was incubated for 6 hours at 37°C in a 5% CO2 atmosphere. The amount of luciferase activity was determined by exposure to luciferase substrate (Promega, Madison, WI) and detected with a luminescence reader (EnVision, Perkin Elmer). EC50 values ​​were calculated using Prism software (Graphpad).

[0497] GFP-positive expi293 parent cells or human TREM1-overexpressing cells were incubated with a titration of unconjugated hIgG1 control or PI-4026-5 antibody at room temperature for 15–30 min. Next, without washing, human macrophages polarized from CD14+ monocytes labeled with Cell Trace Violet (ThermoFisher) were added to the expi293 and antibody mixture at a 1:1 ratio. The assay was incubated for 6 h at 37°C in a 5% CO2 atmosphere, after which the assay was evaluated by flow cytometry (ThermoFisher Attune NxT). ADCP was measured by counting the number of GFP-positive and Cell Trace Violet-positive macrophages from the singlet gate. EC50 values ​​were calculated using Prism software (Graphpad).

[0498] result As shown in Figures 5A and 5B, PI-4026-5 induces downstream FcγR-mediated signaling in a dose-dependent, TREM1-specific manner. Figure 5A shows induction of FcγR signaling (using the hCD16 reporter assay system), and Figure 5B shows induction of FcγR signaling (using the hCD32 reporter assay system).

[0499] Next, the ability of humanized TREM1 antibodies to induce ADCP was assessed in primary human macrophages.

[0500] As shown in Figures 6A and 6B, PI-4026-5 induces ADCP in primary human macrophages and in expi293 cells overexpressing hTREM1 (Figure 6B), but not in parental expi293 cells (Figure 6A).

[0501] Example 5: Crystallization of chimeric PI-4026 Fab with human TREM1 IgV Protein crystallization Diffraction-quality crystals of the SEC-purified hTREM1 IgV:chimeric PI-4026 Fab complex were obtained by sitting-drop vapor diffusion at 20°C. Crystals were cryoprotected, and X-ray diffraction data were collected at the European Synchrotron (ESRF) in Grenoble, France. The structure of the hTREM1 IgV:PI-4026 Fab complex was determined by molecular replacement using the program MOLREP in CCP4. The final model was refined to 1.93 Å resolution with a Rwork of 16.4% and a Rfree of 21.6%. Analysis of the final refined structure indicated that one PI-4026 Fab interacts with one TREM1 IgV molecule.

[0502] result Figures 7A-E show the interactions of specific residues in each CDR of the PI-4026 Fab with specific residues in the TREM1 IgV domain. In each panel, the dark gray structure represents the indicated antibody CDR, and the light gray structure represents the TREM1 IgV domain. Interacting residues are represented by single-letter amino acid names followed by their positions in the human TREM1 sequence or the PI-4026 heavy or light chain. Dotted lines indicate hydrogen bonds between CDR residues and TREM1 IgV residues. Numbers accompanying the dotted lines are the distances between residues measured in angstroms (Å). CDRL2 of the PI-4026 Fab was not important for interactions with the TREM1 IgV domain. All diagrams and interactions were generated and modeled using PyMOL software.

[0503] Figure 7A shows the interaction of residues in CDRH1 of PI-4026 Fab with the TREM1 IgV domain. Figure 7B shows the interaction of residues in CDRH2 of PI-4026 Fab with the TREM1 IgV domain. Figure 7C shows the interaction of residues in CDRH3 of PI-4026 Fab with the TREM1 IgV domain. Figure 7D shows the interaction of residues in CDRL1 of PI-4026 Fab with the TREM1 IgV domain. Figure 7E shows the interaction of residues in CDRL3 of PI-4026 Fab with the TREM1 IgV domain.

[0504] The epitope sequence of the chimeric PI-4026 antibody is non-contiguous residues including amino acids 21 to 34 (SEQ ID NO: 42), 103 to 109 (SEQ ID NO: 43), and 128 to 136 (SEQ ID NO: 44) of human TREM1.

[0505] Example 6: CDRH3 Mutations to Reduce Oxidation Risk and Characterization of Variant CDRH3 mAbs H2O2 oxidation evaluation by size exclusion chromatography (SEC) Oxidation analysis was achieved by incubating PI-4026-5 and PI-4026-7 variants at a final antibody concentration of 1 mg / mL in the indicated concentrations of H2O2 for 1 hour at 40°C. Samples were analyzed by SEC at various concentrations and time points using a ThermoFisher analytical UHPLC system with a Superdex 200 Increase 10 / 300 GL column (GE Healthcare). PBS (pH 7.4) was used as the mobile phase buffer, and the flow rate was 0.5 mL / min. Data were analyzed and the AUC of the A280 peak was integrated using Chromeleon7 instrument software (ThermoFisher).

[0506] Cynomolgus monkey binding strength KD The binding affinity of cynomolgus monkey TREM1 to the PI-4026-5 variants was measured by SPR on a BIAcore T200 (GE Healthcare). All data were collected at 25°C. 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) surfactant P20 were used as the running buffer and for all dilutions of mAbs. Anti-mouse Fc was immobilized onto a CM4 biosensor chip (GE Healthcare) using amine coupling chemistry. Chimeric cynomolgus monkey TREM1-mIgG Fc was captured on one flow cell, and another flow cell used as a reference plane was left blank. Various concentrations of PI-4026-5 variants (hIgG1 isotype as the analyte) were injected through the flow cell containing the captured cynomolgus monkey TREM1-mIgG Fc and the reference cell. After each cycle, the surface was regenerated by injecting glycine HCl buffer (10 mM, pH 2.0). Kinetic evaluation was performed using BIAevaluation software. Kinetic evaluation consisted of generating sensorgrams fitted to a 1:1 Langmuir binding model.

[0507] Afucosylated antibody production Afucosylated antibodies were produced by transfecting the antibody heavy and light chain plasmids of interest (e.g., PI-4026-5) into expi293 cells that had been rendered deficient in mammalian α1,6-fucosyltransferase (FUT8) by CRISPR / Cas9 editing (Alexander Weiss, University of Toronto). Once transfected, the cells were expanded for up to 7 days, after which the medium was harvested and the antibody purified using a single Protein A column purification step (HiTrap MabSelect SuRe, GE Healthcare). The antibody was then buffer-exchanged into PBS and assessed for endotoxin (LAL Endotoxin Test, Charles River Laboratories), concentration (NanoDrop, ThermoFisher Scientific), and any aggregation by UHPLC (ThermoFisher Scientific). Depending on aggregation properties, the antibody was SEC-purified to monomeric form (<95% monomeric). Afucosylation is verified by Western blot using an antibody against Lens Culinaris agglutination (LCA) antigen (L-1040-10, Vector Biolabs) or by assessment of the complete glycan profile of the antibody of interest (Bionova Scientific).

[0508] result As shown in Figure 8, PI-4026-5 has an oxidation risk at Met100 of CDRH3 (RMAAMDY (SEQ ID NO: 25)) as assessed by SEC. The percent heterogeneity of the antibody increases and the percent monomer decreases in an HO dose-dependent manner. This is indicated by the double peak observed in the SEC profiles of samples treated with 0.3%, 0.1%, and 0.01% HO. Table 3 provides a quantification of the SEC results.

[0509] (Table 3) TIFF0007759996000009.tif36128

[0510] To address this, the methionine at residue 100 in PI-4026-7 was mutated to isoleucine, and the mutant antibody was tested as described above. As shown in Table 4, the mutation of the methionine residue (RMAAMDY (SEQ ID NO: 25)) to isoleucine (RIAAMDY (SEQ ID NO: 30)) eliminated the risk of oxidation. Incubation of the PI-4026-7M100I antibody with HO did not result in any increase in protein heterogeneity.

[0511] (Table 4) TIFF0007759996000010.tif44139

[0512] PI-4026-5 was then subjected to additional mutations to determine one or more preferred mutation(s) at this residue position for clinical development. M100 was mutated to isoleucine (M100I), glutamic acid (M100E), leucine (M100L), or glutamine (M100Q). These mutations were rationally selected based on analysis of the solved crystal structure of the interaction between PI-4026 Fab and the TREM1 IgV domain (Example 5). Table 5 summarizes the substitutions and sequences of CDR-H3.

[0513] (Table 5) TIFF0007759996000011.tif30157

[0514] Using the hCD16 reporter assay system, we measured the binding affinity to human and cynomolgus monkey TREM1, SECs, and human monocytes, as well as ECs. 50 Each PI-4026-5 CDRH3 variant was characterized for DSF, and FcγR signaling as described above in Examples 2, 3, and 4.

[0515] Additionally, antibody binding to cells overexpressing cynomolgus monkey TREM1 was assessed. Results for the PI-4026-5 CDRH3 variant are summarized in Table 6.

[0516] (Table 6) TIFF0007759996000012.tif73170

[0517] The afucosylated PI-4026-5 CDRH3 mutant antibody was then similarly characterized.

[0518] The results for the afucosylated PI-4026-5 CDRH3 variants are summarized in Table 7.

[0519] (Table 7) TIFF0007759996000013.tif70170

[0520] PI-4026-5-M100I and PI-4026-5-M100E exhibited higher nonspecific binding to non-TREM1-expressing cells and lost the ability to bind to cells expressing cynomolgus monkey TREM1. PI-4026-5-M100L and PI-4026-5-M100Q were selected for further development because they retained the qualities of PI-4026-5-M100 (the parent antibody), such as binding to human and cynomolgus monkey TREM1, specificity, biophysical properties, and functionality. Importantly, afucosylated PI-4026-5-M100L and PI-4026-5-M100Q exhibited similar biophysical and binding properties compared to their fucosylated counterparts, except for significantly enhanced FcγR signaling in the hCD16 reporter assay.

[0521] Example 7: Cell Binding and Induction of ADCC and ADCP Signaling by Fucosylated and Afucosylated PI-4026-5 Variant CDRH3 Anti...

Claims

1. An isolated polynucleotide or a set of polynucleotides encoding an antibody that binds to human TREM1 (SEQ ID NO: 1), wherein the antibody comprises three heavy chain complementarity determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3), wherein the CDR-H1, CDR-H2, and CDR-H3 are from a heavy chain variable domain (VH) comprising the amino acid sequences set forth in SEQ ID NO: 4, 5, or 6, and wherein X is glutamine (Q), leucine (L), isoleucine (I), or glutamic acid (E), and the CDR-L1, CDR-L2, and CDR-L3 are from a light chain variable domain (VL) comprising the amino acid sequences set forth in SEQ ID NO: 20, 21, or 22.

2. a. the CDR-H1 comprises the sequence set forth in SEQ ID NO: 23, b. the CDR-H2 comprises the sequence set forth in SEQ ID NO:24; c. the CDR-H3 comprises the sequence set forth in SEQ ID NO:29, and X is glutamine (Q), leucine (L), isoleucine (I), or glutamic acid (E); d. the CDR-L1 comprises the sequence set forth in SEQ ID NO:26; e. the CDR-L2 comprises the sequence set forth in SEQ ID NO:27; and f. the CDR-L3 comprises the sequence set forth in SEQ ID NO: 28; 2. The isolated polynucleotide or set of polynucleotides of claim 1.

3. The isolated polynucleotide or set of polynucleotides described in claim 2, wherein the CDR-H3 comprises the sequence set forth in SEQ ID NO:

33.

4. The isolated polynucleotide or set of polynucleotides described in claim 2, wherein the CDR-H3 comprises the sequence set forth in SEQ ID NO:

32.

5. An isolated polynucleotide or set of polynucleotides described in claim 1, wherein the VH comprises the sequence set forth in SEQ ID NO: 17 and the VL comprises the sequence set forth in SEQ ID NO:

20.

6. An isolated polynucleotide or set of polynucleotides as described in claim 1, wherein the VH comprises a sequence selected from the sequences set forth in SEQ ID NO: 16, 17, or 18, and the VL comprises a sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22.

7. The isolated polynucleotide or set of polynucleotides of claim 1, wherein the antibody comprises a heavy chain sequence set forth in SEQ ID NO: 34 and a light chain sequence set forth in SEQ ID NO:

35.

8. An isolated polynucleotide or set of polynucleotides described in claim 1, wherein the VH comprises a sequence selected from the sequences set forth in SEQ ID NO: 12, 13, or 14, and the VL comprises a sequence selected from the sequences set forth in SEQ ID NO: 20, 21, or 22.

9. An isolated polynucleotide or set of polynucleotides described in claim 1, wherein the VH comprises the sequence set forth in SEQ ID NO: 13 and the VL comprises the sequence set forth in SEQ ID NO:

20.

10. The isolated polynucleotide or set of polynucleotides of claim 1, wherein the antibody comprises a heavy chain sequence set forth in SEQ ID NO: 36 and a light chain sequence set forth in SEQ ID NO:

37.

11. The isolated polynucleotide or set of polynucleotides of claim 1, wherein the antibody comprises a human Fc.

12. The isolated polynucleotide or set of polynucleotides described in claim 11, wherein the human Fc is wild-type human IgG1 Fc.

13. The isolated polynucleotide or set of polynucleotides of claim 12, wherein the VH comprises the sequence set forth in SEQ ID NO: 17, the VL comprises the sequence set forth in SEQ ID NO: 20, and the human Fc comprises wild-type human IgG1 Fc.

14. The isolated polynucleotide or set of polynucleotides of claim 12, wherein the VH comprises the sequence set forth in SEQ ID NO: 13, the VL comprises the sequence set forth in SEQ ID NO: 20, and the human Fc comprises wild-type human IgG1 Fc.

15. The isolated polynucleotide or set of polynucleotides of claim 1, wherein the antibody is humanized.

16. A vector or set of vectors comprising the isolated polynucleotide or set of polynucleotides described in claim 1.

17. A host cell comprising the isolated polynucleotide or set of polynucleotides described in claim 1.

18. A host cell comprising the vector or set of vectors described in claim 16.

Images