Antibodies for Treating Cancer

Abstract

Antibodies to TREM2 and Gpnmb are provided, as are compositions comprising the antibodies and methods of using them.

Description

[Technical field]

[0001] Related Applications : This application claims priority to Israeli Patent Application No. 285416, filed August 5, 2021, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing A file entitled 93064.xml, created on Aug. 4, 2022, consisting of 649,124 bytes, submitted concurrently with the filing of this application, is incorporated herein by reference.

[0003] FIELD AND BACKGROUND OF THEINVENTION The present invention, in some embodiments thereof, relates to methods of treating cancer, and more particularly, but not exclusively, solid cancers, by reducing the immunosuppressive activity of myeloid cells. [Background technology]

[0004] Many of the determinants essential for immune function cannot be precisely characterized by traditional surface markers, and it is unclear how the internal processing and integration of these signals are translated into immune activation, suppression, and inflammation. Myeloid-derived suppressor cells (MDSCs) are known to promote a suppressive environment for effector T cells within the tumor microenvironment (TME), contributing to tumor growth and immune dysfunction. Despite their significant impact on treatment outcomes in a wide range of human diseases and cancer types, their exact functional role and molecular identity remain elusive and unclear. MDSCs do not fit traditional surface marker-based classification schemes and are classified using a wide range of myeloid surface markers, various cellular assays, and metabolic characteristics including expression of the immunosuppressive metabolic pathway, including expression of arginase 1 (Arg1). A thorough molecular understanding of this important and heterogeneous myeloid cell population based on their inhibitory metabolic capabilities may lead to the identification of their molecular markers, pathways, and activities, ultimately leading to more effective biomarkers and targeted immunotherapies.

[0005] Background art includes: Kim et al., Cancers (Basel).2019 Sep;11(9): 1315, International Publication No. 2017 / 058866, U.S. Patent Application Publication No. 20180043014, and Katzenelenbogen et al., Aμg 20;182(4):872-885.e19.doi: 10.1016 / j.cell.2020.06.032. Epub 2020 Aμg 11. Summary of the Invention

[0006] According to one aspect of some embodiments of the present invention, there is provided an antibody or fragment thereof comprising an antigen recognition domain capable of binding to triggering receptor expressed on myeloid cells 2 (TREM2), the antigen recognition domain comprising: 23A10A10 32F9E8 38C11H11 49A12D7 58B2A7 60A4F5 60H4A3 61B11C9 80E3H11 83E10B12 54H2C1 54H2C1B 23A10B10 23A10B11 38C11C10 60A4E10 60H4G2 80E3C7 The antibody comprises the complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, or the heavy and light chains, of the antibody selected from the group consisting of:

[0007] According to some embodiments of the invention, the TREM2 is human TREM2.

[0008] According to some embodiments of the invention, the antigen recognition domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the antibody 54H2C or the heavy and light chains.

[0009] According to some embodiments of the invention, the antigen recognition domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 or the heavy and light chains of antibody 80E3C7.

[0010] According to some embodiments of the invention, the antibody or fragment thereof can inhibit TREM2 in bone marrow-derived macrophages and generate activated macrophages in vitro.

[0011] According to an aspect of some embodiments of the present invention there is provided an antibody or fragment thereof comprising an antigen recognition domain capable of binding to the transmembrane glycoprotein NMB (Gpnmb), the antigen recognition domain comprising: g1-g2 g2-b6 g3-g2 g4-b4 g5-g2 g8-g2 g9-b4 b1-b2 b8-b8 b10-b9 b11-g2 b12-y8 b13-b7 b15-b7 b17-b17 b18-b19 b2-b2 b20-b21 b21-y8 b22-b23 b24-b26 b25-b26 y3-y22 y4-y3 y5-y5 y9-y6 y12-b4 y20-y19 y23-y20 y25-y21 y27-y22 The antibody comprises the complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, or the heavy and light chains, of the antibody selected from the group consisting of:

[0012] According to some embodiments of the invention, the Gpnmb is human Gpnmb.

[0013] According to some embodiments of the invention, the antibody or fragment thereof is capable of activating CD4 T cells.

[0014] According to some embodiments of the invention, the Gpnmb is human Gpnmb.

[0015] According to some embodiments of the invention, CD4 T cells can be activated.

[0016] According to an aspect of some embodiments of the present invention there is provided a bispecific antibody comprising in at least one arm an antigen recognition domain as described herein.

[0017] According to some embodiments of the invention, the antibody or fragment thereof comprises an antibody to TREM2 in one arm and an antibody Gpnmb in the other arm.

[0018] According to some embodiments of the invention, the effector function is ineffective or absent.

[0019] According to some embodiments of the invention, it is an IgG1.

[0020] According to some embodiments of the invention, the compound is formulated as an antibody drug conjugate (ADC).

[0021] According to some embodiments of the invention, the compound is formulated with a proinflammatory cytokine.

[0022] According to some embodiments of the invention, a proinflammatory cytokine is conjugated to form a conjugate.

[0023] According to some embodiments of the invention, the proinflammatory cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-21, and GM-CSF.

[0024] According to some embodiments of the invention, the conjugate comprises IL-2.

[0025] According to some embodiments of the invention, the conjugate is as set forth in SEQ ID NO:496 and SEQ ID NO:498, or SEQ ID NO:497 and SEQ ID NO:499.

[0026] According to some embodiments of the present invention, a chimeric antigen receptor (CAR) is formed.

[0027] According to some embodiments of the invention, the sequence is as set forth in SEQ ID NO:500.

[0028] According to an aspect of some embodiments of the present invention there is provided a cell expressing a fragment of an antibody described herein.

[0029] According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising the antibody or antibody fragment described herein.

[0030] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the antibody or antibody fragment or bispecific antibody or cell described herein and a pharma- ceutically acceptable carrier or diluent.

[0031] According to an aspect of some embodiments of the present invention there is provided a method of reducing immunosuppressive activity of a myeloid cell, the method comprising contacting a myeloid cell with an effective amount of an antibody or antibody fragment or bispecific antibody or cell described herein, thereby reducing the immunosuppressive activity of the myeloid cell.

[0032] According to an aspect of some embodiments of the invention there is provided a method of activating CD4 T cells, the method comprising contacting a CD4 T cell with an effective amount of the antibody or fragment thereof of claim 8, thereby activating the CD4 T cell.

[0033] According to an aspect of some embodiments of the present invention, there is provided a method of killing myeloid cells expressing TREM2, the method comprising contacting a cell population comprising contacting the TREM2-expressing myeloid cells with an effective amount of a cell described herein, thereby killing the myeloid cells expressing TREM2.

[0034] According to some embodiments of the invention, the contacting is effected in vivo.

[0035] According to some embodiments of the invention, the contacting is performed ex vivo.

[0036] According to an aspect of some embodiments of the invention there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody, antibody fragment, combination thereof, or bispecific antibody, or cell described herein, thereby treating the cancer.

[0037] According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof, the method comprising: (a) reducing the immunosuppressive activity of myeloid cells according to the methods described herein, the myeloid cells being derived from a subject, and thereafter (b) transplanting myeloid cells into a subject, thereby treating the cancer.

[0038] According to an aspect of some embodiments of the present invention there is provided an antibody, fragment thereof, bispecific antibody, or cell described herein for use in treating cancer.

[0039] According to some embodiments of the invention, the cancer is a solid tumor.

[0040] According to an aspect of some embodiments of the present invention there is provided a solid cancer selected from the group consisting of lung cancer, liver cancer, ovarian cancer, gastric cancer, and breast cancer.

[0041] According to some embodiments of the invention, the lung cancer is non-small cell lung cancer.

[0042] According to some embodiments of the invention, the lung cancer is small cell lung cancer.

[0043] According to some embodiments of the invention, the liver cancer is hepatocellular carcinoma.

[0044] According to some embodiments of the invention, the method or use further comprises a therapeutically effective amount of a checkpoint inhibitor.

[0045] According to some embodiments of the invention, the composition further comprises a therapeutically effective amount of a Bruton's tyrosine kinase (Btk) inhibitor.

[0046] According to some embodiments of the invention, the Bruton's tyrosine kinase (Btk) inhibitor is selected from the group consisting of ibrutinib, acalabrutinib, and spebrutinib.

[0047] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described in this application can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0048] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings. Reference will now be made specifically to the drawings in detail, with it being stressed that the particulars shown are by way of example and for the purpose of illustratively discussing embodiments of the present invention. In this regard, by reading the description in conjunction with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0049] The drawings are as follows: [Brief description of the drawings]

[0050] [Figure 1]Figure 1 shows SDS-PAGE analysis of hybridoma-derived monoclonal antibodies against human TREM2. Secondary antibody: Peroxidase-AffiniPure goat anti-mouse IgG, Fcg fragment specific (min X human, bovine, horse serum proteins). [Diagram 2] FIG. 2 shows the OD values ​​of HEK293 supernatant ELISA in a binding sensitivity test of seven anti-hTREM2 antibodies. [Diagram 3] FIG. 3 shows flow cytometer analysis of wild-type 293HEK cells (WT) and hTREM2-expressing HEK293 cells (hTREM2) stained with biotin-conjugated anti-hTREM2 antibody followed by incubation with APC-streptavidin. [Figure 4] Figure 4, A-B, shows the identification of lead antibodies. Mouse bone marrow cells from TREM2 knockout (KO) and hTREM2 transgenic (hTREM2) mice were cultured for 7 days in the presence of 30 ng / mL hM-CSF cytokine (Peprotech, 300-25) to generate bone marrow-derived macrophage cells (BMDMs). BMDMs were stained with biotin-conjugated anti-hTREM2 leader antibodies (83E10B12, 54H2C1, 80E3C7, or IgG control) followed by incubation with APC-streptavidin. A. Representative histograms show staining of 83E10B12, 54H2C1, 80E3C7 versus IgG control. B. Flow cytometry intensity of IgG control, 83E10B12, 54H2C1, and 80E3C7 versus TREM2 KO BMDMs or hTREM2 BMDMs. [Diagram 5] FIG. 5 shows SPR analysis of 83E10B12 anti-hTREM2 protein, 54H2C1 anti-hTREM2 protein, and 80E3C7 anti-hTREM2 protein. [Figure 6]6A-B show Western blot analysis of wild-type (WT) and hTREM2 overexpressing (OE)TREM2 293HEK cells (A), and hTREM2 BMDMs or KO BMDMs (B) with 83E10B12, 54H2C1, and 80E3C7 anti-hTREM2 antibodies. Secondary antibody: Peroxidase-AffiniPure goat anti-mouse IgG, Fcγ fragment specific (min X human, bovine, and horse serum proteins) (Jackson ImmunoResearch, 115-035-071). [Figure 7] Figure 7 shows immunohistochemistry of TREM2 KO BMDMs and hTREM2 BMDMs using 83E10B12, 54H2C1 and 80E3C7 anti-hTREM2 antibodies. Cells were fixed with cold methanol, washed with PBS and stained with anti-hTREM2 antibody. Secondary antibody: Alexa Fluor 647-AffiniPure F(ab')2 fragment donkey anti-mouse IgG(H+L) (Jackson ImmunoResearch, 715-606). [Figure 8] Figure 8, A-C, shows that mAb 54H2C1 and mAb 80E3C7 block hTREM2 activity in BMDM cultures. (A) Single cell maps of BMDM cultures of TREM2 KO and hTREM2 bone marrow cells. (B) Density plots highlighting cells from hTREM2 (cyan) or TREM2-KO (red) mice on single cell maps at days 2, 3, 4, 5, 6, and 7 during BMDM differentiation. (c) Quantification of TREM2+GPNMB+ and TREM2- macrophages at day 7 in BMDM cultures derived from wild-type or TREM2-KO cells, as well as wild-type cells treated with mAb 54H2C1, mAb 80E3C7, or IgG control at day 2. [Figure 9] FIG. 9 shows ELISA analysis of the penetration of biotin-conjugated hTREM2 antibodies in humanized TREM2 mice bearing MCA-205-induced tumors. [Figure 10]Figure 10A-B shows that hGPNMB protein suppresses CD4 T cell activation. CFSE-stained human CD4 T cells were incubated for 3 days in pre-coated anti-CD3, anti-CD2, and anti-CD28 antibodies for activation and proliferation. Copy numbers were calculated from CFSE intensity measurements by flow cytometry and IFNg secretion measurements by ELISA (Biolegend, BLG-430104). [Figure 11] Figure 11 shows the mean fluorescence intensity (MFI) curves of E3C7 antibody (yellow) and IgG control (grey) staining human M2 macrophages at the indicated antibody concentrations. Biotinylated antibody was used followed by PE-streptavidin conjugation. [Figure 12] Figure 12A-B show the effect of E3C7 on macrophage polarization. Human CD14+ monocytes were purified from peripheral blood of three healthy donors, differentiated into macrophages by administration of hM-CSF for 5 days, and subsequently polarized into "M2" macrophages by administration of IL-4. Anti-TREM2 antibody, E3C7 Ab, was added to the cultures on days 3 and 5 of the assay. (A) qPCR and (B) ELISA were performed 24 hours after treatment with IL4. [Figure 13] Figure 13, A-C, shows that mAb E3C7 blocks hTREM2 activity in differentiating human macrophages in culture. (A) Single-cell maps of human in vitro differentiated macrophages in the presence or absence of IL-4 cytokine and with or without anti-TREM2 / IgG control antibodies. (B) Density plots highlighting cells in the M-CSF (pink) or M-CSF+IL-4 (blue) samples on single-cell maps. (C) Density plots highlighting cells in the anti-TREM2 (pink), IgG control (green), or no antibody (blue) samples on single-cell maps. [Figure 14]Figure 14 shows that mAb E3C7 blocks hTREM2 activity in differentiating human macrophages in culture. Dot plot visualization shows the expression of differentially expressed genes in two differentiation conditions: M-SCF, M-CSF+IL4 differentiated macrophages treated with either anti-TREM2 mAb, IgG control, or no antibody treatment. [Figure 15] Figures 15A-B show that antibody E3C7 remodels the mouse tumor microenvironment (TME). (A) Percentage of TAMs with high type I interferon signaling in E3C7-treated mice compared to IgG controls. (B) Percentage of dysfunctional CD8 T cells (PD1+LAG3+) in E3C7-treated mice compared to IgG controls. [Figure 16] 16 is a volcano plot showing differential gene expression in tumor-associated macrophages from E3C7-treated mice. The plot shows genes that were differentially expressed in TME macrophages treated with E3C7 and IgG control. [Figure 17A] Figures 17A and 17B show that anti-TREM2 antibody E3C7 and inflammatory cytokines (IL2, IL-15) synergize to increase the pro-inflammatory phenotype of macrophages. (A) Gene expression analysis after treatment with anti-TREM2, GM-CSF, IL12, IL15, IL2, or a combination of anti-TREM2 and one of the cytokines. (B) M2 macrophage cytokine secretion analysis after treatment with anti-TREM2 and IL-2 / IL-15 cytokines. [Figure 17B] Same as above [Figure 18]Figure 18A-B shows that anti-TREM2 antibody E3C7 and inflammatory cytokines (IL2, IL-15) synergistically increase the proinflammatory phenotype of M2 macrophages, thereby relieving their inhibition of CD8 T cell activation. (A) Human CD8 T cells stained with the cell tracking dye CFSE were co-cultured with M2 macrophages treated with anti-TREM2 antibody and IL2 / IL-15 cytokines for 3 days in pre-coated anti-CD3 and CD28 antibodies for activation and proliferation. The percentage of proliferating cells was calculated by measuring CFSE intensity by flow cytometry. (B) Human CD8 T cells stained with CFSE were co-cultured with M2 macrophages in pre-coated anti-CD3 and CD28 antibodies for activation and proliferation in the presence of IL-2 / IL-15 cytokines for 3 days in pre-coated anti-CD3 and CD28 antibodies for activation and proliferation. The percentage of proliferating cells was calculated by measuring CFSE intensity by flow cytometry. [Figure 19] Figure 19, A-D, are graphs showing binding of anti-hTREM2-cytokine fusions. Plates were coated with recombinant hTREM2 (2 μg / ml) and used in a direct ELISA assay with various concentrations of recombinant antibody. Alternatively, hM2 cell supernatants were used in a soluble TREM2 protein binding assay (sandwich Elisa). Different secondary antibodies were used: A, C: anti-human IgG, B, D: anti-hIL2 (BLG-500302). [Figure 20] Figures 20A-B show that anti-hTrem-IL2 Ab and its fusion with IL-2 according to an embodiment of the invention activate hCD8+ / CD4+ cells in culture. hCD8 / CD4+ cells were cultured with antibody (10 μg / ml) or recombinant protein (100 ng / ml) with or without activation. A. Percentage of proliferating CD8+ cells on day 4. B. IFNg secretion (pg / ml) at 24 hours and on day 4. [Figure 21] Figure 21 shows IFNg release under different co-culture conditions as determined by IFNg concentration in the medium. Statistical significance was tested using Holm-Sidak's multiple comparison test (16 replicates per condition). [Figure 22-1]Figure 22 A-F are graphs showing the activity of TREM2 CAR-T cells. (A) IFN-γ ELISA assay of TREM2-CAR T cells co-cultured with TREM2+HEK293. (B) Flow cytometry analysis of activation and killing of TREM2+HEK293 by TREM2-CAR T cells co-cultured with TREM2+HEK293. (C) FSC-SSC gating of flow cytometry of human TAM-like cells after 24 hours of co-culture with TREM2-CAR T cells and mock CAR T cells. (D) IFN-γ ELISA assay of TREM2-CAR T cells co-cultured with human TAM-like cells. (E) Flow cytometry analysis of activation and killing of TREM2-CAR T cells co-cultured with human TAM-like cells. (F) IFN-γ ELISA assay of TREM2-CAR T cells co-cultured with BMDMs and BMDCs from humanized TREM2, TREM2ko, and wt mice. [Figure 22-2] Same as above [Figure 23] FIG. 23 is a schematic diagram of a TREM2-chimeric antigen receptor according to some embodiments of the invention. [Figure 24] FIG. 24 shows schematics and sequences of anti-TREM2-IL2 fusions according to some embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Some embodiments of the present invention relate to methods of treating cancer, and more particularly, but not exclusively, solid cancers, by reducing the immunosuppressive activity of myeloid cells.

[0052] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0053] The present inventors have +Using direct targeting of myeloid cells, we analyzed the inhibitory metabolic circuitry within the tumor microenvironment. + TREM2 + We identified two distinct populations of cells: a tumor-associated macrophage population and a unique Mreg population characterized by defined surface markers (e.g., Gpnmb) and signaling (including hypoxia). + We demonstrated the suppressive activity of TAM and Mreg populations. Our findings identify TREM2 as a marker and potential regulator of suppressive myeloid cells. Genetic ablation of TREM2 in mice results in dysfunctional CD8 + A dramatic decrease in the Mreg population was accompanied by a decrease in T cells and an increase in immune reactivity against the tumor, including an increase in NK cells and cytotoxic T cells. The results suggest that specifically targeting the Mreg population may be more beneficial than targeting the tumor-associated macrophage population for treating cancer.

[0054] Thus, we have previously proposed modulating the regulatory myeloid cell population (Mreg) by co-targeting triggering receptor expressed on myeloid cells 2 (TREM2) and the transmembrane glycoprotein NMB (Gpnmb) to treat cancer. We have now identified antibodies against TREM2 and Gpnmb that can be used for such co-targeting.

[0055] Anti-TREM2 antibodies were screened by using a unique screening assay in which bone marrow-derived macrophages are activated in the presence of the screened antibody to acquire an M1 profile. This activation is a direct consequence of TREM2 blockade (loss of function) and mimics the TREM2 knockout phenotype, as disclosed in the Examples section below. On the other hand, anti-Gpnmb binders are selected based on their ability to inhibit Mregs, which suppress the activation of CD4 T cells, and thereby activate CD4 T cells.

[0056] The ability to activate CD4 T cells and macrophages makes the antibodies of the invention advantageous for clinical use, particularly in the treatment of cancer.

[0057] Functional characterization of anti-TREM2 antibodies of some embodiments of the present invention demonstrated high affinity recognition (low nanomolar to picomolar range) and inhibition of TREM2 function in myeloid cells as evidenced by their ability to reprogram immunosuppressive macrophages into proinflammatory monocyte-like cells. Comparative single-cell RNAseq analysis demonstrated transcriptome patterns of antibody-treated reprogrammed BMDMs and TREM2 - / - It was found that the transcriptome patterns from the mice were nearly identical. The anti-TREM2 antibodies of some embodiments of the present invention attenuate tumor growth in tumor-bearing humanized mice and reprogram tumor macrophages as evidenced by the differential increase in type I IFN genes. The combination of anti-TREM2 antibodies with inflammatory cytokines or the conjugation of anti-TREM2 antibodies with inflammatory cytokines was shown to synergistically enhance myeloid reprogramming and T cell activation. In addition, TREM2-CAR T cells generated using the antibodies of some embodiments of the present invention can mount an effective cytotoxic response and deplete human TREM2-expressing myeloid cells and immunosuppressive macrophages. The observed efficacy of TREM2-CAR T cells is an important milestone towards the development of myeloid-focused cancer immunotherapy.

[0058] Thus, according to one aspect of the present invention, there is provided an antibody or fragment thereof comprising an antigen recognition domain capable of binding to triggering receptor expressed on myeloid cells 2 (TREM2), said antigen recognition domain comprising: 23A10A10 32F9E8 38C11H11 49A12D7 58B2A7 60A4F5 60H4A3 61B11C9 80E3H11 83E10B12 54H2C1 54H2C1B 23A10B10 23A10B11 38C11C10 60A4E10 60H4G2 80E3C7 The antibody comprises the complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, or the heavy and light chains, of the antibody selected from the group consisting of:

[0059] TREM2 is an immunoglobulin-like receptor that is expressed primarily on myeloid lineage cells, including, but not limited to, macrophages, dendritic cells, osteoclasts, microglia, monocytes, dermal Langerhans cells, and Kupffer cells. In some embodiments, TREM2 forms a receptor-signaling complex with DAP12. In some embodiments, TREM2 phosphorylates and signals through DAP12, an ITAM domain adaptor protein. In some embodiments, TREM2 signaling results in downstream activation of PI3K. In some embodiments, TREM2 signaling results in downstream phosphorylation of spleen tyrosine kinase (stk).

[0060] TREM2 proteins of the present disclosure include, without limitation, mammalian TREM2 proteins, including, but not limited to, human TREM2 protein (Uniprot Accession No. Q9NZC2), mouse TREM2 protein (Uniprot Accession No. Q99NH8), rat TREM2 protein (Uniprot Accession No. D3ZZ89), rhesus monkey TREM2 protein (Uniprot Accession No. F6QVF2), bovine TREM2 protein (Uniprot Accession No. Q05B59), equine TREM2 protein (Uniprot Accession No. F7D6L0), porcine TREM2 protein (Uniprot Accession No. H2EZZ3), and canine TREM2 protein (Uniprot Accession No. E2RP46).

[0061] An exemplary human TREM2 amino acid sequence is shown below as SEQ ID NO:1.

[0062] In some embodiments, human TREM2 is a preprotein that includes a signal peptide. In some embodiments, human TREM2 is a mature protein. In some embodiments, the mature TREM2 protein does not include a signal peptide. In some embodiments, the mature TREM2 protein is expressed on a cell. In some embodiments, TREM2 contains a signal peptide located at amino acid residues 1-18 of human TREM2 (SEQ ID NO:1), an extracellular immunoglobulin-like variable (IgV) domain located at amino acid residues 29-112 of human TREM2 (SEQ ID NO:1), an additional extracellular sequence located at amino acid residues 113-174 of human TREM2 (SEQ ID NO:1), a transmembrane domain located at amino acid residues 175-195 of human TREM2 (SEQ ID NO:1), and an intracellular domain located at amino acid residues 196-230 of human TREM2 (SEQ ID NO:1). According to a specific embodiment, TREM2 is human TREM2.

[0063] According to a specific embodiment, the antigen recognition domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the antibody 54H2C or the heavy and light chains.

[0064] According to a specific embodiment, the antigen recognition domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, or the heavy and light chains, of antibody 80E3C7 (also referred to as "E3C7").

[0065] According to specific embodiments, the antibody or antibody fragment is capable of inhibiting TREM2 in bone marrow-derived macrophages in vitro, resulting in activated macrophages, which is typical of a TREM2 knockout.

[0066] In other words, the antibody or antibody fragment or bispecific antibody is an inhibitory antibody against TREM2, which can be explained by the similar phenotype after incubation to TREM2 knockout cells (see the Examples section).

[0067] Specifically, the inventors have found that bone marrow-derived macrophages (BMDMs) express high amounts of TREM2. Antibodies of some embodiments of the invention also bind to TREM2-BMDMs.

[0068] BMDMs are known to produce high levels of inhibitory cytokines such as IL-10 and TGF-β. The effect of antibodies on the chronological maturation process of BMDMs can be determined using single cell RNA sequencing. The effect can be seen 2-7 days after activation. The effect is typically the acquisition of an M1 phenotype. Thus, as can be seen in the Examples section below, wild type hTREM2 BM cells exhibit an M2 phenotype with high expression of Gpnmb, Lpl, Anxa1, Mmp12, Adam8, Lgals1, Lgals3, Spp1, and Lilrb4a at day 7, while cells of the TREM2-KO BM genotype displayed an activated M1 phenotype including Selenop, Ms4a4a, Fcgr2b, Ms4a7, and Lyz2 (Figure 8A-B). To screen for antibodies with antagonistic activity against TREM2, hTREM2 mouse bone marrow cells are cultured in culture with M-CSF and anti-hTREM2 antibodies or IgG isotypes on days 2 and 5 of culture. Single-cell RNA-seq is used to characterize and quantify cells at day 7 for cellular distribution between M2 (TREM2+Gpnmb+) and M1 (TREM2-) phenotypes in each condition.

[0069] As can be seen in Figure 8C, over 70% of TREM2-KO cells reached the M1 phenotype and less than 10% displayed the M2 phenotype, in contrast to 40% of hTREM2 cells, which displayed the M2 phenotype and only 24% displayed the M1 phenotype. Addition of IgG isotype mAbs to the cultures did not significantly alter the M1 / M2 ratio, showing results similar to untreated cultures, whereas addition of anti-hTREM2 mAbs 54H2C1 or 80E3C7 dramatically reduced the percentage of M2 phenotype to 12% and 16%, respectively, and increased the M1 phenotype to 69% and 63%, respectively (Figure 8C), showing a maturation course very similar to that of TREM2-KO cells.

[0070] As used herein, an "M1 macrophage" is a macrophage that expresses Selenop, Ms4a4a, Fcgr2b, Ms4a7, and Lyz2.

[0071] As used herein, "M2 macrophages" are macrophages that express Gpnmb, Lpl, Anxa1, Mmp12, Adam8, Lgals1, Lgals3, Spp1, and Lilrb4a.

[0072] According to further or alternative embodiments, there is provided an antibody or fragment thereof comprising an antigen recognition domain capable of binding to the transmembrane glycoprotein NMB (Gpnmb), said antigen recognition domain comprising: g1-g2 g2-b6 g3-g2 g4-b4 g5-g2 g8-g2 g9-b4 b1-b2 b8-b8 b10-b9 b11-g2 b12-y8 b13-b7 b15-b7 b17-b17 b18-b19 b2-b2 b20-b21 b21-y8 b22-b23 b24-b26 b25-b26 y3-y22 y4-y3 y5-y5 y9-y6 y12-b4 y20-y19 y23-y20 y25-y21 y27-y22 The antibody comprises the complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, or the heavy and light chains, of the antibody selected from the group consisting of:

[0073] Tables A and B below list the SEQ ID NO for each antibody sequence. Each antibody should be considered an individual embodiment.

[0074]

Table 1-1

[0075]

Table 1-2

[0076]

Table 1-3

[0077]

Table 1-4

[0078]

Table 1-5

[0079]

Table 1-6

[0080]

Table 1-7

[0081]

Table 1-8

[0082]

Table 1-9

[0083]

Table 1-10

[0084] [Table 1-11]

[0085] [Table 1-12]

[0086] Transmembrane glycoprotein NMB (GPNMB) is a type IA cell surface glycoprotein that is encoded by the GPNMB gene in humans. In humans, two transcript variants have been characterized for this gene, encoding 560 and 572 amino acid isoforms. The 470 aa long fragment is the extracellular domain used for mouse immunization. The mouse and rat orthologues of GPNMB are known as DC-HIL and osteoactivin, respectively. An exemplary GPNMB has the amino acid sequence set forth in SEQ ID NO:2.

[0087] According to a specific embodiment, the Gpnmb is human Gpnmb (SEQ ID NO:2).

[0088] According to a specific embodiment, the antibody, fragment thereof, or bispecific antibody is capable of activating CD4 T cells.

[0089] CD4 + T cell activation occurs through the simultaneous engagement of the T cell receptor and costimulatory molecules (such as CD28 or ICOS) on the T cell by major histocompatibility complex (MHCII) peptides and costimulatory molecules on the APC. Both are required for the production of an effective immune response, and in the absence of costimulation, T cell receptor signaling alone leads to anergy. The signaling pathway downstream of the costimulatory molecules usually involves the PI3K pathway, which generates PIP3 at the plasma membrane and recruits PH domain containing signaling molecules such as PDK1, which are essential for the activation of PKC-θ and ultimately IL-2 production. Optimal CD8 + T cell responses are CD4 + Depends on signal transduction. CD4+ The cells are involved in the initial antigen activation of naive CD8 T cells and in the activation of memory CD8 T cells following acute infection. + It is useful for maintaining T cells. Therefore, CD4 + T cell activation is mediated by CD8 + May be beneficial for T cell action.

[0090] According to specific embodiments, the antibody is a homologue of any of the antibodies in Table A above comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of the VH and / or VL chain, so long as it is capable of binding to TREM2 and preferably inhibiting the activity of TREM2 as evidenced by macrophage activation.

[0091] According to specific embodiments, the antibody is a homologue of any of the antibodies in Table B above comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of the VH and / or VL chain, so long as it is capable of binding to Gpnmb and preferably inhibiting the activity of Gpnmb as evidenced by CD4 T cell activation.

[0092] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to residues in the two sequences that are the same when aligned. When percentages of sequence identity are used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity) and thus the functional properties of the molecule are not altered. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thus increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0. Scoring of conservative substitutions is calculated, for example, according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 992, 89(22): 095-9].

[0093] Identity (eg, percent homology) can be determined using any homology comparison software, including, for example, the BlastN or BlastP software of the National Center for Biotechnology Information (NCBI), such as using default parameters.

[0094] When reference is made to "at least 90% identity," the claimed invention also refers to at least 9%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or 00% identity, each of which represents a different embodiment.

[0095] According to a specific embodiment, the level of identity is at least 90% across the entire sequence (either the VH chain and / or the VL chain as described herein) as determined as described herein.

[0096] According to specific embodiments, the level of identity is at least 90%, 9%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% across at least one (or at least two, three, four or five) of the CDR sequences of an antibody of Table A or B described herein.

[0097] Exemplary CDR sequences and complete light and heavy chains of human antibodies are provided in Tables A or B above.

[0098] The term "antibody" as used in the present invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv, or single domain molecules, such as VH and VL, directed against an epitope of an antigen. These functional antibody fragments are defined as follows: (1) Fab, which is a fragment containing a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digesting a whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', which is the fragment of an antibody molecule that can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of a heavy chain; two Fab' fragments are obtained per antibody molecule; and (3) Fab', which is the fragment of an antibody that can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction, without subsequent reduction. 2, F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains; (5) Single-chain antibody ("SCA"), a genetically engineered molecule containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable polypeptide linker as a genetically fused single-chain molecule; and (6) Single-domain antibody, which is composed of a single VH or VL domain that exhibits sufficient affinity for an antigen.

[0099] In certain embodiments, the antibody is a monoclonal antibody.

[0100] Methods for producing polyclonal and monoclonal antibodies, and fragments thereof, are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, which is incorporated herein by reference, and the Examples section below).

[0101] Antibody fragments according to the invention can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems). Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fc fragment. These methods are described, for example, in Goldenberg U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein, which are incorporated herein by reference in their entirety. See also Porter, RR [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separating the heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques can be used, so long as the fragment binds to the antigen recognized by the intact antibody.

[0102] An Fv fragment consists of an association of a VH chain and a VL chain. The association can be noncovalent, as described by Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720)]. Alternatively, the variable chains can be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises a VH chain and a VL chain connected by a peptide linker. These single-chain antigen-binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, and the expression vector is then introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, in Whitlow and Filpula, Methods 2: 97-105 (1991), Bird et al., Science 242:423-426. (1988), Pack et al., Bio / Technology 11:1271-77 (1993), and U.S. Pat. No. 4,946,778, the entireties of which are incorporated herein by reference.

[0103] Another form of antibody fragment is a peptide encoding a single complementarity determining region (CDR). A CDR peptide ("minimal recognition unit") can be obtained by constructing a gene encoding the CDR of an antibody of interest. Such a gene is prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using the polymerase chain reaction. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0104] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequences derived from the non-human immunoglobulin. Humanized antibodies comprise a human immunoglobulin (recipient antibody) in which residues forming the complementarity determining regions (CDRs) of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0105] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as import residues and are typically taken from an import variable domain. Humanization can be essentially performed according to the method of Winter and coworkers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)] by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Such humanized antibodies are thus chimeric antibodies (US Pat. No. 4,816,567) in which significantly less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from similar sites in rodent antibodies.

[0106] Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95). (1991)]. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which resembles in every respect that seen in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 856-859 (1995); 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0107] According to one embodiment, the antibody is a monospecific antibody.

[0108] According to one embodiment, the antibody is a bispecific, multivariant or chimeric antibody recognizing two different antigens, TREM2 and Gpnmb.

[0109] A "bispecific antibody" of the invention has two different antigen-binding sites such that the antibody specifically binds to two different antigens. Such antibodies can be generated by combining portions of two separate antibodies or antibody fragments that recognize two different antigenic groups, or by modifying a single antibody molecule to contain two specificities (as discussed in detail above).

[0110] According to one embodiment, a bispecific antibody is a hybrid antibody having two different heavy / light chain pairs and two different binding sites.

[0111] According to one embodiment, a bispecific antibody comprises an antigen recognition domain in a structural loop region of the antibody (e.g., the CH3 region of the heavy chain). Thus, a bispecific antibody may comprise an antibody fragment comprising the Fc region of the antibody, referred to as "Fcab". Such an antibody fragment typically comprises the CH2-CH3 domain of the antibody. Fcabs are engineered to comprise at least one modification in the structural loop region of the antibody, i.e., the CH3 region of the heavy chain. Such an antibody fragment may be produced, for example, as follows: provide a nucleic acid encoding an antibody comprising at least one structural loop region (e.g., the Fc region), modify at least one nucleotide residue of at least one structural loop region, transfer the modified nucleic acid into an expression system, express the modified antibody, contact the expressed modified antibody with an epitope, and determine whether the modified antibody binds to the epitope. See, for example, U.S. Pat. Nos. 9,045,528 and 9,133,274, which are incorporated herein by reference in their entirety.

[0112] Antibodies with higher valencies (ie, the ability to bind to three or more antigens) can also be prepared and are referred to as multispecific antibodies.

[0113] According to specific embodiments, the bispecific antibody comprises in at least one arm thereof the antigen recognition domain of any one of the above antibodies, or in particular among the CDRs of an antibody of Table A or B.

[0114] According to a specific embodiment, the antibody comprises an anti-TREM2 antibody in one arm and an anti-Gpmnb antibody in the other arm.

[0115] According to a specific embodiment, the antibody comprises in one arm an anti-TREM2 antibody described herein and in the other arm an anti-Gpmnb antibody described herein.

[0116] To generate the multispecific antibodies of some embodiments of the invention, moieties of the invention can be modified in the Fc region, e.g., in the CH3 domain (according to Kabat), as is well known in the art, to ensure correct assembly of the multispecific antibody through its heavy chains.

[0117] Thus, the CH3 domain of one heavy chain is modified such that, in the original interface of the CH3 domain of one heavy chain that borders the original interface of the CH3 domain of the other heavy chain in the multispecific antibody, amino acid residues are replaced by amino acid residues with a larger side chain volume, generating a protrusion in the interface of the CH3 domain of one heavy chain, which can be positioned in a cavity in the interface of the CH3 domain of the other heavy chain, and the CH3 domain of the other heavy chain is modified such that, in the original interface of the second CH3 domain that borders the original interface of the first CH3 domain in the trivalent bispecific antibody, amino acid residues are replaced by amino acid residues with a smaller side chain volume, generating a cavity in the interface of the second CH3 domain, into which the protrusion in the interface of the first CH3 domain can be positioned (also known as the "knobs-into-holes" approach by Genentech).

[0118] According to a specific embodiment, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0119] According to a specific embodiment, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0120] According to a specific embodiment, both CH3 domains are further modified by introducing a cysteine ​​(C) as an amino acid at the corresponding position of each CH3 domain, such that a disulfide bridge can be formed between both CH3 domains.

[0121] In a specific embodiment, the bispecific comprises a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain". Additional interchain disulfide bridges between the CH3 domains can also be used, for example by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C or S354C mutation in the CH3 domain of the "hole chain" (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681). Thus, in another preferred embodiment, the bispecific antibody comprises a Y349C mutation, a T366W mutation in one of the two CH3 domains and an E356C mutation, a T366S mutation, a L368A mutation, a Y407V mutation in the other of the two CH3 domains, or a Y349C mutation, a T366W mutation in one of the two CH3 domains and an S354C mutation, a T366S mutation, a L368A mutation, a Y407V mutation in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form an interchain disulfide bridge) (numbering always according to the EU index of Kabat). However, other knobs-in-holes techniques described in EP1870459A1 may alternatively or additionally be used. Specific examples of bispecific antibodies are the R409D and K370E mutations in the CH3 domain of the "knob chain" and the D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering always according to the EU index of Kabat).

[0122] In another embodiment the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain", a T366S mutation, an L368A mutation and a Y407V mutation in the CH3 domain of the "hole chain", and further comprises an R409D mutation and a K370E mutation in the CH3 domain of the "knob chain", a D399K mutation and an E357K mutation in the CH3 domain of the "hole chain".

[0123] In another embodiment the bispecific antibody comprises a Y349C mutation, a T366W mutation in one of the two CH3 domains and a S354C mutation, a T366S mutation, a L368A mutation, a Y407V mutation in the other of the two CH3 domains, or the bispecific antibody comprises a Y349C mutation, a T366W mutation in one of the two CH3 domains and a S354C mutation, a T366S mutation, a L368A mutation, a Y407V mutation in the other of the two CH3 domains and further comprises an R409D mutation, a K370E mutation in the CH3 domain of the "knob strand" and a D399K mutation, an E357K mutation in the CH3 domain of the "hole strand".

[0124] According to a specific embodiment, Y349C / T366S / L368A / Y407V mutations are introduced into a first mAb (e.g., anti-TREM2) and S354C / T366W into a second mAb (e.g., anti-Gpnmb) (Merchant et al., 1998; Ridgway et al., 1996).

[0125] Alternatively or additionally, at least one of the moieties can be expressed in a CrossMab format (CH1-CL swapping) for correct heavy-light chain pairing.

[0126] The basis of the CrossMab technology is the crossover of antibody domains within one arm of a bispecific IgG antibody that allows correct chain association, while the correct heterodimerization of the heavy chains can be achieved by the knob-into-hole technique or charge interactions described above. This can be achieved by exchanging different domains within a Fab fragment. Fab domains within a Fab fragment (CrossMab Fab format), or the variable VH-VL domains alone (CrossMab VH-VL format), or the constant CH1-CL domain (CrossMab CH1-CL format) may be exchanged for this purpose. CH1-CLIn this format, the respective original light chains and the new VL-CH1 light chains do not undergo undesired interactions with the respective original heavy chains and the VH-CL-containing heavy chains, and theoretically no by-products can be formed. Fab In this format, non-functional monovalent antibodies (MoAbs) and non-functional Fab fragments may be formed. These by-products can be removed by chromatographic techniques. CrossMab VH-VL In this format, undesirable by-products due to the association of the VL-CH1 / VL-CL domains known from Bence-Jones proteins may occur between the VL-CH1-containing heavy chain and the original unmodified VL-CL light chain. Based on the existing conserved charge pairs in the wild-type antibody framework, the introduction of repulsive charge pairs into the constant CH1 and CL domains of the wild-type uncrosslinked Fab fragments allows the CrossMab VH-VL+ / - This Bence-Jones-like by-product formation can be overcome in the form. Further details of the CrossMab technology can be found in Klein et al. Methods 154, 1 February 2019, Pages 21-31c.

[0127] Alternatively, multispecific antibodies, e.g., bispecific antibodies, described herein can be prepared by conjugating the moieties using methods known in the art. For example, each moiety of the multispecific antibody can be produced separately and then conjugated to one another. A variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132, Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0128] Alternatively or additionally, the conjugation of the portions of the multispecific antibody can be via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a specific embodiment, the hinge region is modified prior to conjugation to contain an odd number of sulfhydryl residues, preferably one.

[0129] According to one aspect of the invention, there is provided a method for producing an antibody, the method comprising: (a) expressing in a host cell a heterologous polynucleotide encoding an antibody described herein; and, optionally, (b) recovering the antibody from the host cell.

[0130] Thus, polynucleotides encoding the antibodies of some embodiments of the present invention are cloned into an expression construct selected according to the expression system to be used. Exemplary polynucleotide sequences are shown in SEQ ID NOs: 6-23, 42-59, 186-216, 248-278.

[0131] A variety of prokaryotic or eukaryotic cells can be used as host expression systems for expressing the antibodies of some embodiments of the present invention. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence, yeast transformed with recombinant yeast expression vectors containing the coding sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors such as Ti plasmids containing the coding sequence. Mammalian expression systems can also be used to express the antibodies of some embodiments of the present invention.

[0132] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3., pSinRep5, DH26S, DHBB, pNMT, pNMT4, pNMT8 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof. According to a specific embodiment, the vectors used are pFUSE2-CLIg-mk, pFUSE2-CHIg-mG1 for the light and heavy chains, respectively. According to a specific embodiment, the antibody is transiently expressed in Expi293F cells.

[0133] Expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, can also be used. SV40 vectors include pSVT7 and pMT2. Bovine papilloma virus-derived vectors include pBV-MTHA, and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A, pAV009 / B, pAV009 / C, pAV009 / D, pAV009 / E, pAV009 / F, pAV009 / G, pAV009 / H, pAV009 / I, pAV009 / L, pAV009 / N ... + , pMTO0 / A + , pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0134] Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (990) Methods in Enzymol. 85:60-89].

[0135] In yeast, a number of vectors containing constitutive or inducible promoters may be used, as disclosed in U.S. Patent Application Publication No. 5,932,447, or vectors that facilitate integration of foreign DNA sequences into the yeast chromosome may be used.

[0136] When a plant expression vector is used, the expression of the coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA promoter and the 9S RNA promoter of CaMV [Brisson et al. (984) Nature 30:5-54] or the coat protein promoter for TMV [Takamatsu et al. (987) EMBO J. 6:307-3] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (984) EMBO J. 3:-680 and Brogli et al., (984) Science 224:838-843] or heat shock promoters such as soybean hsp7.5-E or hsp7.3-B [Gurley et al. (986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to those of skill in the art. See, for example, Weissbach & Weissbach, 988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 42-463.

[0137] Other expression systems, such as insect and mammalian host cell systems, which are well known in the art and described further below, may also be used in accordance with some embodiments of the present invention.

[0138] It will be appreciated that antibodies can also be produced in in vivo systems, for example in mammals, such as goats, rabbits, and the like.

[0139] Harvesting of the recombinant antibody is performed after an appropriate (cultivation) time. The phrase "recovering the antibody" refers to recovering the entire fermentation medium containing the antibody and does not necessarily imply an additional step of separation or purification. Notwithstanding the above, the antibodies of some embodiments of the invention may be purified using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization.

[0140] Once antibodies are obtained, they can be tested for activity as described above.

[0141] In some embodiments, the antibodies described herein contain modifications to improve their ability to mediate effector functions. Such modifications are known in the art and include afucosylation or engineering the affinity of Fc for activating receptors (mainly FCGR3a for ADCC and C1q for CDC). Table B of US 10,428,143 summarizes the various designs reported in the literature for effector function engineering.

[0142] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn297 in EU numbering) without modifying the amino acid sequence are well known in the art. GlymaX® technology (ProBioGen AG) is based on introducing into the cells used for antibody production a gene for an enzyme that diverts the cellular pathway of fucose biosynthesis. This prevents the antibody-producing cells from adding the sugar "fucose" to the carbohydrate moiety of N-linked antibodies (von Horsten et al. (2010) Glycobiology. 2010 December;20 (12): 1607-18). Another approach to obtain antibodies with reduced levels of fucosylation can be found in U.S. Pat. No. 8,409,572, which teaches the selection of cell lines for antibody production based on their ability to reduce the fucosylation level of antibodies. An antibody can be fully afucosylated, meaning that the antibody contains no detectable fucose, or partially afucosylated, meaning that the isolated antibody contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose typically found in a similar antibody produced in a mammalian expression system.

[0143] According to another specific embodiment, the antibody has an Fc domain with ineffective or no effector function. It is known in the art that the IgG1 isoform of human antibodies has little or no ADCC or CDC activity.

[0144] However, other isotypes are contemplated, e.g., IgG2, IgG3, or IgG4.

[0145] The antibody may be soluble or insoluble.

[0146] The insoluble antibody can be part of a particle (synthetic or non-synthetic, e.g., a liposome) or a cell (e.g., a CAR-T cell, in which the antibody is part of a chimeric antigen receptor (CAR), typically as an scFv fragment).

[0147] Thus, in some embodiments, the antibody sequences of the present invention can be used to develop chimeric antigen receptors (CARs). CARs are transmembrane receptors expressed on immune cells that drive the recognition and killing of target cells (e.g., myeloid cells expressing TREM2). CARs typically contain three basic parts. These include an ectodomain (also known as a recognition domain), a transmembrane domain, and an intracellular (signaling) domain. The ectodomain drives binding to cellular antigens on the target cell, while the intracellular domain typically contains cell signaling functions to promote the killing of the bound target cell. In addition, they may have an extracellular domain with one or more of the antibody variable domains or fragments thereof described herein. The CARs of the present invention also contain a transmembrane domain and a cytoplasmic tail. CARs can be designed to contain one or more segments of an antibody, an antibody variable domain, and / or an antibody CDR, such that when such a CAR is expressed on an immune effector cell, the immune effector cell will bind to and eliminate any cell that is recognized by the antibody portion of the CAR.

[0148] The features of CARs include their ability to exploit the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and reactivity to selected targets in an MHC-unrestricted manner. MHC-unrestricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thus circumventing a major mechanism of tumor evasion. Furthermore, CARs, when expressed in T cells, advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0149] According to some embodiments of the present invention, the CARs engineered to target tumors have specificity for TREM2. In some embodiments, the ectodomain of these CARs may comprise one or more antibody variable domains or fragments thereof. In some embodiments, the CARs are expressed in T cells, which may be referred to as "CAR-engineered T cells" or "CAR-Ts." CAR-Ts may be engineered with a CAR ectodomain that has one or more antibody variable domains.

[0150] Thus, in some embodiments of the present disclosure, the antibody sequences of the present invention may be used to develop chimeric antigen receptors (CARs). In some embodiments, CARs are transmembrane receptors expressed on immune cells that drive the recognition and killing of target cells, such as myeloid cells expressing TREM2 (e.g., as exemplified for TREM2-expressing HEK293 cells, humanized TREM2 bone marrow-derived macrophages (BMDMs), and human monocyte-derived macrophages (hMac) lines).

[0151] Immune cells expressing the CAR of the present invention (see, for example, FIG. 23) can be generated by well-known techniques, such as those described in the Examples. Immune cells expressing the CAR of the present invention can be used to kill TREM2-expressing myeloid cells. Thus, the present invention encompasses a method of killing TREM2-expressing myeloid cells, comprising contacting a cell population comprising TREM2-expressing myeloid cells with immune cells, preferably T cells, comprising the CAR of the present invention, whereby the TREM2-expressing myeloid cells are killed.

[0152] In one embodiment, the contacting is performed ex vivo or in vitro.

[0153] Thus, for example, immune effector cells can be harvested from a subject in need thereof and then the cells can be engineered to express a disclosed CAR. The engineered cells can then be infused back into the subject.

[0154] The disclosed CAR-modified immune effector cells can be used or administered either alone or as a pharmaceutical composition in combination with other components, such as diluents and / or IL-2, IL-15, IL-21, or other cytokines or cell populations. These components can be added as protein components or can be expressed by the CAR-modified immune effector cells, for example, by genetically engineering the CAR-modified immune effector cells to express the disclosed cytokines.

[0155] Pharmaceutical compositions can include the CAR-modified immune effector cells described herein in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients.

[0156] In some embodiments, the compositions for use in the disclosed methods are formulated for intravenous administration.The pharmaceutical compositions can be administered in any manner suitable for killing TREM2-expressing myeloid cells.The amount and frequency of administration are determined by factors such as the condition of the patient and the severity of the patient's disease, but the appropriate dosage may be determined by clinical trials.

[0157] In various embodiments, a therapeutic amount of CAR-modified immune effector cells is administered to a patient. The "therapeutic amount" can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). The pharmaceutical composition comprising the T cells described in the present application can be administered in an amount of 10 to 20 mg / kg / day. 4 ~10 9 Cells / kg body weight, e.g. 10 5 ~10 6The T cell compositions may be administered in doses of cells / kg body weight, including all integer values ​​within these ranges. The T cell compositions may also be administered multiple times at these doses. The cells may be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can be easily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly. Increasing the cytotoxic or therapeutic activity of the antibody, when necessary, can also be achieved, such as by using the concept of antibody-drug conjugates (ADCs). In such configurations, the antibody is attached to a heterologous effector moiety that can be used to increase its toxicity or make the antibody detectable.

[0158] In some embodiments, the antibody of the present invention can be developed for antibody drug conjugate (ADC) therapeutic use. ADC is an antibody to which one or more cargoes (e.g., therapeutic agents) are attached [e.g., directly or via a linker (e.g., cleavable or non-cleavable linker)]. ADC is useful for delivery of therapeutic agents (e.g., drugs or cytotoxic agents) to one or more target cells or target tissues (Panowski, S. et al., 204. mAbs 6:, 34-45). In some cases, ADC can be designed to bind to a surface antigen on a target cell. Upon binding, the entire antibody-antigen complex can be internalized and directed to cell lysosomes. The ADC can then be degraded, releasing the attached cargo.

[0159] Therapeutic agents can be small molecule drugs, proteinaceous agents (e.g., cytokines or chemokines, such as tumor necrosis factor (TNF) or IL12), nucleic acid agents, radioisotopes, carbohydrates, etc. These can function as cytotoxic agents, e.g., chemotherapeutic agents.

[0160] According to a specific embodiment, the therapeutic agent is a nucleic acid sequence (e.g., DNA or RNA, e.g., mRNA) encoding a viral antigen for inducing an antiviral immune response against tumors. Examples of viral antigens include, but are not limited to, CMV antigens, EBV antigens, coronavirus antigens, etc.

[0161] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells.

[0162] If the cargo is a cytotoxic agent, the target cell is killed or otherwise incapacitated. The cytotoxic agent may include, but is not limited to, cytoskeleton inhibitors (e.g., tubulin polymerization inhibitors and kinesin spindle protein (KSP) inhibitors), DNA damaging agents (e.g., calicheamicin, duocarmycin, and pyrrolobenzodiazepine dimers such as taliline and tesirin), topoisomerase inhibitors (e.g., camptothecin compounds or derivatives such as 7-ethyl-0-hydroxycamptothecin (SN-38) and exatecan derivative DXd), transcription inhibitors (e.g., RNA polymerase inhibitors such as amanitin), and kinase inhibitors (e.g., phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase kinase (MEK) inhibitors).

[0163] Tubulysin inhibitors may include, but are not limited to, maytansines (e.g., emtansine [DM] and ravtansine [DM4]), auristatins, tubulysins, and vinca alkaloids or derivatives thereof. Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-0), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F, and dolastatin. Exemplary tubulysin compounds include naturally occurring tubulysins A, B, C, D, E, F, G, H, I, U, and V, as well as tubulysin analogs such as pretubulysin D (PTb-D43) and N4-desacetoxytubulysin H (Tbl). Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). In some embodiments, cytotoxic agents may include auristatin derivatives (e.g., -aminopropan-2-yl-auristatin F, auristatin F-hydroxypropylamide, auristatin F-propylamide, auristatin F phenylenediamine (AFP)); tubulysin derivatives; vinca alkaloid derivatives (e.g., N-(3-hydroxypropyl)vindesine (HPV)); and any of those described in U.S. Pat. Nos. 8,524,24, 8,685,383, 8,808,9, and 9,254,339, U.S. Patent Publication Nos. 205034008, 2060220696, and 2060022829, the contents of each of which are incorporated herein by reference in their entirety.

[0164] The term refers to radioisotopes (e.g. 211 At, 131 I, 125 I, 32 P, 35 S, and 177 Radioactive isotopes of Lu, including Lu 86 Y, 90 Y, 111 In, 177 Lu,225 Ac, 212 Bi, 213 Bi, 66 Ga, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 71 As, 72 As, 76 As, 77 As, 65 Zn, 48 V, 203 Pb, 209 Pb, 212 Pb, 166 Ho, 149 Pm, 153 Sm, 201 Tl, 188 Re, 186 Re, and 99 mTc), enzymes such as nucleases and fragments thereof, antibiotics, therapeutic RNA molecules (e.g., siRNAs, antisense oligonucleotides, microRNAs, ribozymes, RNA decoys, aptamers), DNAzymes, and toxins such as small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin, e.g., pokeweed antiviral protein (PAP), ricin toxin A, abrin, gelonin, saporin, cholera toxin A, diphtheria toxin, Pseudomonas exotoxin, and alpha-sarcin (including fragments and / or variants thereof).

[0165] In some embodiments, the antibody-drug conjugates (ADCs) of the invention may further comprise one or more polymeric carriers connecting the antibody and the therapeutic agent (e.g., antibody-polymer-drug conjugates). As used herein, the term "polymeric carrier" refers to a polymer or modified polymer that may be covalently attached to one or more therapeutic agents and / or antibodies. The polymeric carrier may provide additional attachment sites for the therapeutic agent, increase the drug-to-antibody ratio, and enhance the therapeutic effect of the ADC. In some embodiments, the polymeric carriers used in the invention may be water-soluble and / or biodegradable. Such polymeric carriers include poly(ethylene glycol) (PEG), poly(N-(2-hydroxypropyl)methacrylamide) (polyHPMA), poly(α-amino acids) [e.g., poly(L-lysine), poly(L-glutamic acid), and poly((N-hydroxyalkyl)glutamine)], carbohydrate polymers [e.g., dextrin, hydroxyethyl starch (HES), and polysialic acid], glycopolysaccharides (e.g., homopolysaccharides such as cellulose, amylose, dextran, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen, and lixenan; or agarose. Polysaccharides such as homopolysaccharides, such as sucrose, hyaluronan, chondroitin sulfate, dermatan sulfate, keratan sulfate, alginic acid, and heparin, glycolipids, complex carbohydrates, polyglycerols, polyvinyl alcohols, poly(acrylic acid), polyketals, and polyacetals (e.g., poly(1-hydroxymethylethylene hydroxymethyl formal, also known as PHF or FLEXIMER®, described in U.S. Pat. Nos. 5,850, 5,863,990, and 5,958,398, the contents of each of which are incorporated herein by reference in their entirety) and derivatives, dendrimers, copolymers, and mixtures thereof.For example, polymeric carriers can include polyacetal / polyketal copolymers (eg, PHF), and hydrophilic polymers such as polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, polypeptides, and derivatives thereof.

[0166] In some embodiments, a therapeutic agent is attached (e.g., covalently bonded) to an antibody of the invention directly or via a linker. In some embodiments, a therapeutic agent is attached directly or via a linker to a polymeric carrier, which is attached directly or via a linker to an antibody. In some embodiments, a linker may comprise an oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, phthalic acid, isophthalic acid, terephthalic acid, diglycolic acid, tartaric acid, glutamic acid, fumaric acid, or aspartic acid moiety (including amide, imide, or cyclic imide derivatives of each, and each optionally substituted). Exemplary linkers may include any of those disclosed in U.S. Pat. Nos. 8,524,24, 8,685,383, 8,808,9, 9,254,339, and / or 9,555,2, the contents of each of which are incorporated herein by reference in their entirety.

[0167] In some embodiments, the linker can be a cleavable linker that can be degraded under certain conditions (such as a change in pH, temperature, or reduction) or cleaved by enzymes (e.g., proteases and glucuronidases) to release the therapeutic agent from the ADC. Such linkers can include labile bonds such as ester, amide, or disulfide bonds. Non-limiting examples of cleavable linkers include pH-sensitive linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, thioethers, orthoesters, acetals, or ketals); reduction-sensitive linkers [e.g., N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-S-acetylthioacetate (SATA), and N-succinimidyl-oxycarbonyl-alpha-methyl-alanine (ALA)-. photolabile linkers; and enzymatically cleavable linkers (e.g., peptide linkers such as valine-citrulline, valine-citrulline-p-aminobenzoyloxycarbonyl (vc-PAB), maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB), glucuronidase-cleavable linkers such as glucuronide-MABC, or esterase-cleavable linkers).

[0168] In other embodiments, the linker may be a non-cleavable linker. A non-cleavable linker may increase the plasma stability of the ADC compared to a cleavable linker. Exemplary non-cleavable linkers include maleimidoalkanes and maleimidocyclohexane (MCC).

[0169] The antibody-drug conjugates (ADCs) of the present invention can be prepared using any method known in the art. For example, the therapeutic agent can be modified to contain a functional group capable of reacting with a functional group on the antibody. The antibody-drug conjugates (ADCs) can be prepared by reacting two functional groups to form a conjugate. In some cases, the polymeric carrier can be modified to contain a functional group capable of reacting with a functional group on the therapeutic agent and a functional group on the antibody under different chemical conditions. The antibody, the polymeric carrier, and the therapeutic agent can be linked to form an antibody-polymer-drug conjugate through sequential chemical reactions. For conjugation to the antibody, lysine or cysteine ​​residues can be used as conjugation sites. In some embodiments, the antibody can be engineered to have additional lysine or cysteine ​​residues. Such an approach can avoid disruption of the antibody structure (e.g., interchain disulfide bonds) and maintain the stability and / or activity of the antibody.

[0170] Alternatively or additionally, various agents may be used to increase the therapeutic effectiveness of the antibody, such as combining the antibody with a proinflammatory cytokine, such as an inflammatory cytokine of the TNF family or IL12, e.g., IFNα, IFNβ, IFNγ, IL-2, IL-11, IL-21, G-CSF, GM-CSF, and / or TNFα.

[0171] According to a specific embodiment, the cytokine is conjugated to an antibody.

[0172] According to a specific embodiment, the bond is covalent.

[0173] According to a specific embodiment, the conjugate is a chimeric protein in which the antibody is translationally fused to the cytokine (upstream or downstream thereof, with or without a linker, as described herein).

[0174] According to a specific embodiment, the cytokine is IL-2.

[0175] According to specific embodiments, the conjugate is as set forth in SEQ ID NOs: 496 and 498 or 497 and 499.

[0176] According to a specific embodiment, the cytokine is IL-15.

[0177] The present invention encompasses uses and methods of enhancing myeloid reprogramming using the antibodies of the present invention.

[0178] In one embodiment, the invention encompasses a method of enhancing myeloid reprogramming comprising contacting a TREM2-expressing cell population with an antibody of the invention and a cytokine, e.g., a cytokine selected from the TNF family, IL-21, IL-12, IL-15, IFNα, IFNβ, IFNγ, IL-2, IL-11, G-CSF, GM-CSF, and / or TNFα. The method may enhance myeloid reprogramming of a TREM2-expressing cell population. As detailed in the Examples, the method may result in a synergistic effect between the antibody and the cytokine.

[0179] Chimeric proteins in which the antibodies of the invention are translationally fused to a cytokine, such as a cytokine selected from the TNF family, IL-12, IL-15, IFNα, IFNβ, IFNγ, IL-2, IL-11, IL-21, G-CSF, GM-CSF, and / or TNF, can be produced by well-known techniques, such as those described in the Examples. The chimeric proteins can be used to enhance myeloid reprogramming. In one embodiment, the invention encompasses a method of enhancing myeloid reprogramming, comprising contacting a population of TREM2-expressing cells with a chimeric protein of the invention. As detailed in the Examples, the method can result in a synergistic effect of the chimeric protein.

[0180] The antibodies of some embodiments of the invention have immunomodulatory activity.

[0181] Thus, according to one aspect of the invention there is provided a method of reducing the immunosuppressive activity of a myeloid cell, the method comprising contacting a myeloid cell with an effective amount of an antibody or antibody fragment or bispecific antibody described herein, thereby reducing the immunosuppressive activity of the myeloid cell.

[0182] According to another aspect, there is provided a method of activating a CD4 T cell, the method comprising contacting a CD4 T cell with an effective amount of an antibody or fragment thereof, thereby activating the CD4 T cell.

[0183] According to one embodiment, the contacting is performed in vivo.

[0184] According to another embodiment, the contacting is performed ex vivo.

[0185] The term "myeloid cells" as used herein refers to cells arising from the common myeloid progenitor cell (CMP). In one embodiment, myeloid cells are cells arising from the lineage of myeloblasts and their daughter types (e.g., basophils, neutrophils, eosinophils, monocytes, and macrophages). One subgroup of myeloid cells is the immunosuppressant myeloid cells.

[0186] According to a specific embodiment, the myeloid cells are M2 macrophages that acquire an M1 phenotype upon incubation with the antibodies (against TREM2) of some embodiments of the invention.

[0187] As mentioned, the antibody is contacted with myeloid cells of the subject in order to reduce the amount and / or activity of a specific subpopulation of said myeloid cells (those that express both TREM2 and Gpnmb).

[0188] In one embodiment, the contacting is carried out in vivo.

[0189] In another embodiment, the contacting is performed ex vivo, i.e., the myeloid cells are removed from a subject and then contacted with the agent.

[0190] Myeloid cells are typically removed from a subject by bone marrow biopsy. Mobilizing agents such as Plerixafor® and G-CSF can be used to mobilize the cells to the periphery.

[0191] The antibodies of this aspect of the invention specifically increase the activity of macrophages that express both triggering receptor expressed on myeloid cells 2 (TREM2) and the transmembrane glycoprotein NMB (Gpnmb).

[0192] In one embodiment, the antibody enhances the activity of cells expressing both markers by at least 2-fold compared to cells expressing only one of the markers (i.e., cells expressing only TREM2 and not Gpnmb, or vice versa) (activated macrophages). In another embodiment, the antibody enhances the activity of cells expressing both markers by at least 5-fold compared to cells expressing only one of the markers (i.e., cells expressing only TREM2 and not Gpnmb, or vice versa). In another embodiment, the antibody enhances the activity of cells expressing both markers by at least 10-fold compared to cells expressing only one of the markers (i.e., cells expressing only TREM2 and not Gpnmb, or vice versa).

[0193] Because the methods described herein are used to reduce the immunosuppressive activity of myeloid cells, the inventors believe that the methods can be used in the treatment of cancer.

[0194] Thus, according to another aspect of the present invention there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody, antibody fragment, combination thereof (anti-TREM2 and anti-Gpnmb), or bispecific antibody described herein, thereby treating the cancer.

[0195] According to another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising: (a) reducing the immunosuppressive activity of myeloid cells according to the above method, wherein the myeloid cells are derived from a subject, and thereafter (b) transplanting myeloid cells into a subject, thereby treating the cancer.

[0196] As used herein, "reducing" refers to at least a 10%, 20%, 30%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 5-fold, 10-fold reduction in immunosuppressive activity in the presence of the antibody compared to a control (negative, e.g., untreated cells) sample.

[0197] As used herein, "subject" refers to a mammal, such as a human, who has been diagnosed with cancer.

[0198] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated malignant cell growth.

[0199] Examples of cancers that may be analyzed and treated according to some embodiments of the present invention include tumors of the digestive tract (colon cancer, rectal cancer, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis 1, hereditary nonpolyposis 2, hereditary nonpolyposis 3, hereditary nonpolyposis 6; colorectal cancer, hereditary nonpolyposis 7, small intestine and / or large intestine cancer, esophageal cancer, tylosis with esophageal cancer, gastric cancer, pancreatic cancer, pancreatic endocrine tumors), endometrial cancer, dermatofibrosarcoma protuberans, gallbladder cancer, biliary tract tumors, prostate cancer, prostate adenocarcinoma, kidney cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcin ... cancer), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cell tumor, ovarian immature teratoma, uterine tumor, epithelial ovarian tumor, sacrococcygeal tumor, choriocarcinoma, placental trophoblastic tumor, epithelial adult tumor, ovarian cancer, serous ovarian cancer, ovarian sex cord tumor, cervical carcinoma, uterine cervix carcinoma, small cell lung cancer and non-small cell lung cancer, nasopharyngeal carcinoma, breast carcinoma (e.g., ductal carcinoma, invasive intraductal carcinoma, sporadic; breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3;breast cancer-ovarian cancer), squamous cell carcinoma (e.g., in the head and neck), neurogenic tumors, astrocytoma, ganglioblastoma, neuroblastoma, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B-cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma, thymic lymphoma), glioma, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, brain malignancies (tumors), other various carcinomas (e.g., Bronchogenic large cell carcinoma, ductal carcinoma, Ehrlich-Lettre ascites carcinoma, epidermoid carcinoma, large cell carcinoma, Lewis lung carcinoma, medullary carcinoma, mucoepidermoid carcinoma, oat cell carcinoma, small cell carcinoma, spindle cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, undifferentiated carcinoma, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblastic), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), hepatocellular carcinoma (glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B-cell), adrenal tumor, insulinoma, pancreatic islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, lymphosarcoma, melanoma, breast tumor, mast cell tumor, medulloblastoma, mesothelioma, metastatic tumor, monocytic tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, neural tissue glial tumor, neural tissue neuronal tumor, schwannoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's osteosarcoma), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., These include, but are not limited to, Ewing's sarcoma, histiocytic cell sarcoma, Jensen's sarcoma, osteogenic sarcoma, reticulum cell sarcoma), Schwannoma, subcutaneous tumors, teratocarcinomas (e.g., multipotent teratocarcinoma), teratomas, testicular tumors, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, Lynch family of cancer syndrome II, male germ cell tumors, mast cell leukemia, medullary thyroid, multiple meningiomas, endocrine tumor myxosarcoma, familial nonchromaffin paraganglioma, pilomatrixoma, papillary, familial and sporadic, familial rhabdoid predisposition syndrome, rhabdoid tumor, soft tissue sarcoma, and Turcot's syndrome with glioblastoma;

[0200] According to a specific embodiment, the cancer is melanoma.

[0201] According to a particular embodiment, the cancer is a solid tumor (lung cancer, liver cancer, ovarian cancer, gastric cancer, and breast cancer).

[0202] According to a specific embodiment, the cancer is a primary tumor.

[0203] According to a specific embodiment, the cancer is metastatic.

[0204] According to a specific embodiment, the cancer is a secondary tumor.

[0205] According to a specific embodiment, the lung cancer is non-small cell lung cancer.

[0206] According to a specific embodiment, the lung cancer is small cell lung cancer.

[0207] According to a specific embodiment, the liver cancer is hepatocellular carcinoma.

[0208] According to another aspect of the present invention, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of (i) a first antigen recognition domain that downregulates the activity of TREM2; and (ii) administering to the subject a second antigen recognition domain that specifically downregulates the activity of Gpnmb, thereby treating the cancer.

[0209] According to one embodiment, the first antigen recognition domain specifically binds to TREM2 expressed on myeloid cells. According to another embodiment, the second antigen recognition domain specifically binds to Gpnmb. The phrases "specifically bind(s)" or "bind(s) specifically" when referring to a binding molecule refer to a binding molecule that has a moderate or high binding affinity, exclusively or predominantly, to a target molecule, such as TREM2 or Gpnmb. The phrase "specifically binds" refers to a binding reaction that determines the presence of a target protein (such as TREM2 or Gpnmb) in the presence of a heterogeneous population of proteins and other biologics. Thus, under defined assay conditions, a designated binding molecule preferentially binds to a particular target protein (e.g., TREM2 or Gpnmb) and does not bind in any meaningful amount to other components present in the test sample. Specific binding to a target protein under such conditions may require a binding molecule selected for its specificity for a particular target protein. A variety of assay formats can be used to select binding molecules that specifically react with a particular target protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore, and Western blots can be used to identify binding molecules that specifically bind to TREM2 or Gpnmb. Typically, a specific or selective reaction is at least twice the background signal or noise, and more typically more than 10 times the background. Assuming that the binding molecule is an antibody, the phrase "specifically binds" refers to a binding reaction that determines the presence of an antigen (such as TREM2 or Gpnmb) in a heterogeneous population of proteins and other biologics. Typically, an agent that specifically binds to an antigen has a specific binding activity of at least about 1×10 -6 ~1×10 -7 , or about 1×10 -8 ~1×10 -9 M, or approximately 1 x 10 -10 ~1×10 -11 Dissociation constant (K D) and / or binds to a given antigen (e.g., an antigen of TREM2 or Gpnmb) with an affinity that is at least 2-fold, 5-fold, 10-fold, or 20-fold greater than the binding affinity of the given antigen or a non-specific antigen other than a closely related antigen (e.g., BSA, casein).

[0210] According to certain embodiments, the antigen recognition domain that reduces the amount and / or activity of TREM2 is an inhibitor antibody, also referred to herein as an antagonist antibody.

[0211] According to a specific embodiment, the affinity of the selected antibody is within 10 as determined by surface plasmon resonance (SPR) assay. -8 M~10 -14 M (see conditions in the Examples section).

[0212] According to some embodiments, the affinity range is 10 -8 M~10 -14 It's M.

[0213] According to some embodiments, the affinity range is 10 -8 M~10 -13 It's M.

[0214] According to some embodiments, the affinity range is 10 -8 M~10 -12 It's M.

[0215] According to some embodiments, the affinity range is 10 -8 M~10 -11 It's M.

[0216] According to some embodiments, the affinity range is 10 -8 M~10 -10 It's M.

[0217] According to some embodiments, the affinity range is 10 -8 M~10 -19 It's M.

[0218] According to some embodiments, the affinity range is 10 -9 M~10 -14 It's M.

[0219] According to some embodiments, the affinity range is 10 -9 M~10 -13 It's M.

[0220] According to some embodiments, the affinity range is 10 -9 M~10 -12 It's M.

[0221] According to some embodiments, the affinity range is 10 -9 M~10 -11 It's M.

[0222] According to some embodiments, the affinity range is 10 -9 M~10 -10 It's M.

[0223] According to some embodiments, the affinity range is 10 -10 M~10 -13 It's M.

[0224] According to some embodiments, the affinity range is 10 -10 M~10 -12 It's M.

[0225] According to some embodiments, the affinity range is 10 -10 M~10 -11 It's M.

[0226] Affinity is determined using a variety of techniques, one example of which is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the association rate constant (ka) and the dissociation rate constant (kd) can be measured. The equilibrium dissociation constant (KD(M)) can then be calculated from the ratio of the kinetic rate constants (kd / ka). In some embodiments, affinity is determined by a kinetic method, such as the equilibrium exclusion binding assay (KinExA) as described in Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. Using the KinExA assay, the equilibrium dissociation constant (KD(M)) and the association rate constant (ka(M'V 1 ) can be measured. The dissociation rate constant (kd) can be calculated from these values ​​(KD x ka). In other embodiments, the affinity is determined by biolayer interferometry, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015 and employed in the Octet® system (Pall ForteBio). The kinetic constants (ka and kd) and affinity constants (KD) can be calculated in real time using biolayer interferometry. In some embodiments, the antigen binding proteins described herein have an affinity of about 10 as measured by kd (dissociation rate constant) for human TREM2 and human Gpnmb. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 or lower (lower values ​​indicate higher binding affinity), and / or a binding affinity of about 10 as measured by a KD (equilibrium dissociation constant) for human TREM2 and human Gpnmb. -8 M, about 10 -9 M, about 10 -10 M, about 10 -11M or lower binding affinity (lower values ​​indicate higher binding affinity). In certain embodiments, the antigen binding proteins of the invention specifically bind to human TREM2 and human Gpnmb with a KD of about 1 pM to about 100 nM when measured by biolayer interferometry at 25° C. For example, in some embodiments, the antigen binding proteins of the invention specifically bind to human TREM2 and human Gpnmb with a KD of less than 100 nM when measured by biolayer interferometry at 25° C. In other embodiments, the antigen binding proteins of the invention specifically bind to human TREM2 and human Gpnmb with a KD of less than 50 nM when measured by biolayer interferometry at 25° C. In yet other embodiments, the antigen binding proteins of the invention specifically bind to human TREM2 and human Gpnmb with a KD of less than 25 nM when measured by biolayer interferometry at 25° C. In one particular embodiment, the antigen binding protein of the invention specifically binds to human TREM2 and human Gpnmb with a KD of less than 10 nM as measured by biolayer interferometry at 25° C. In another particular embodiment, the antigen binding protein of the invention specifically binds to human TREM2 and human Gpnmb with a KD of less than 5 nM as measured by biolayer interferometry at 25° C. In another particular embodiment, the antigen binding protein of the invention specifically binds to human TREM2 and human Gpnmb with a KD of less than 1 nM as measured by biolayer interferometry at 25° C.

[0227] It will be appreciated that the antibodies of the present invention may be administered to a subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0228] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0229] As used herein, the term "active ingredient" refers to an antibody of the invention (eg, the antibody) that is responsible for the biological effect.

[0230] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound. These terms include adjuvants.

[0231] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0232] Techniques for formulating and administering drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0233] Suitable routes of administration can include, for example, oral, rectal, neurosurgical strategies (e.g., intracerebral injection, intrastriatal or intraventricular injection, intraspinal, epidural), transmucosal, intestinal, or parenteral delivery (including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intracardiac, intravenous, intraperitoneal, intranasal, or intraocular injections).

[0234] Alternatively, the pharmaceutical composition can be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue area (eg, adipose tissue) of the patient.

[0235] According to a preferred embodiment, the antibody is not administered to the brain of the subject.

[0236] The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0237] Thus, pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate the processing of the active ingredient into a pharma- ceutically usable preparation. The appropriate formulation depends on the chosen route of administration.

[0238] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0239] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and the like, for oral ingestion by the patient. Pharmacological preparations for oral use can be made by using solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries as necessary, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose, and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0240] Dragee cores are provided with suitable coatings.For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.Dyes or pigments can be added to tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0241] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally mixed with a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All formulations for oral administration should be in a dosage suitable for the selected route of administration.

[0242] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0243] For administration by nasal inhalation, the active ingredient for use according to the invention is conveniently delivered in the form of an aerosol spray provided by a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., made of gelatin) for use in a dispenser can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0244] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Preparations for injection may be provided in unit dosage form, for example, in ampoules or in multi-dose containers with optional addition of preservatives. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0245] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form.Furthermore, suspensions of active ingredients can be prepared as suitable oil-based or water-based injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0246] Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0247] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water-based solution, before use.

[0248] Pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0249] Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve the intended purpose (e.g., reducing the number or size of adipocytes, or reducing visceral fat mass).

[0250] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0251] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be initially estimated from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0252] Toxicity and therapeutic efficacy of the active ingredients described herein may be determined by standard pharmaceutical procedures in vitro in cell cultures or experimental animals. Data obtained from these in vitro and cell culture assays and animal studies may be used in formulating a range of dosages for use in humans. Dosages may vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage may be chosen by the individual physician in view of the patient's condition. (See, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1.)

[0253] Dosage and interval can be adjusted individually to provide tissue levels of active ingredient (minimal effective concentration, MEC) sufficient to reduce the number or size of adipocytes or reduce visceral fat. MEC varies from preparation to preparation, but can be estimated from in vitro data. The dosage required to achieve MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentration.

[0254] Depending on the severity and responsiveness of the condition to be treated, dosage may be single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the pathology is achieved.

[0255] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0256] The compositions of the present invention may be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also conform to a notice associated with the container, in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice indicates that the composition form for human or veterinary administration is approved by that agency. Such notice may, for example, be that of a label approved by the U.S. Food and Drug Administration for prescription drugs, or that of an approved product insert. Compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of an indicated condition, as further detailed above.

[0257] The inventors contemplate administering to the subject an additional chemotherapeutic agent (in combination with the above-described antibodies targeting TREM2 / Gpnmb expressing cells), which may act synergistically with the above-described antibodies for the treatment of cancer.

[0258] The treatment may be combined with any anti-cancer treatment known in the art, including, but not limited to, chemotherapeutic agents, radiotherapeutic agents, hormonal therapy, immunomodulatory agents, engineered immune cell therapy (e.g., CAR-T), and other therapeutic regimens known in the art (e.g., surgery, cell transplantation, e.g., hematopoietic stem cell transplantation).

[0259] Chemotherapeutic agents of the present invention include cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), asprin, sulindac, curcumin, alkylating agents (nitrogen mustards (e.g., mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU) ; thylenimines / methylmelamines (e.g., triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine)); alkyl sulfonates (e.g., busulfan); triazines (e.g., dacarbazine (DTIC)); folic acid analogs (e.g., methotrexate and trimetrexate), pyrimidine analogs (e.g., 5-fluorouracil, fluorodeoxyuridine, gefitinib, etc.); antimetabolites, including mucitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2 difluorodeoxycytidine), purine analogs (e.g., 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA)); antimitotics (e.g., paclitaxel), vincas Alkaloids (including vinblastine (VLB), vincristine, and vinorelbine), natural products, including taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins (e.g., etoposide and teniposide); antibiotics (e.g., actinomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin);Enzymes (e.g., L-asparaginase), cytokines (e.g., interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta, and GM-CSF), anti-angiogenic factors (e.g., angiostatin and endostatin), inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors (including soluble VGF / VEGF receptors), platinum coordination complexes (e.g., cisplatin and carboplatin), anthracenediones (e.g., mitoxantrone), ), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives, including N-methylhydrazine (MIH) and procarbazine, adrenal cortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); hormones and antagonists, including corticosteroid antagonists such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); esophageal corticosteroids (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); androgens, including testosterone propionate and fluoxymesterone / equivalents; antiandrogens (e.g., flutamide, gonadotropin releasing hormone analogs, and leuprolide); nonsteroidal antiandrogens (e.g., flutamide); kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors, and receptor tyrosine kinase inhibitors (e.g., imatinib mesylate (commercially available as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor), currently marketed as Tarveca); and antiviral agents (e.g., oseltamivir phosphate, amphotericin B, and palivizumab);

[0260] In some embodiments, the chemotherapeutic agent of the present invention is selected from the group consisting of cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), quizartinib (AC220), sorafenib (BAY 43-9006), lestaurtinib (CEP-701), midostaurin (PKC412), carboplatin, carmustine, chlorambucil, dacarbazine, ifosfamide, lomustine, mechlorethamine, procarbazine, pentostatin, (2' deoxycoformycin), etoposide, teniposide, topotecan, vinblastine, vincristine, paclitaxel, dexamethasone, methylprednisolone, prednisone, all-trans retinoic acid, arsenic trioxide, interferon-alpha, rituximab (Ri tuxan®), gemtuzumab ozogamicin, imatinib mesylate, Cytosar-U), melphalan, busulfan (Myleran®), thiotepa, bleomycin, platinum (cisplatin), cyclophosphamide (Cytoxan®), daunorubicin, doxorubicin, idarubicin, mitoxantrone, 5-azacytidine, cladribine, fludarabine, hydroxyurea, 6-mercaptopurine, methotrexate, 6-thioguanine, or any combination thereof.

[0261] According to a specific embodiment, the treatment is combined with an immune checkpoint inhibitor as described below.

[0262] As used herein, "immune checkpoint blockade" refers to cancer immunotherapy. This therapy targets immune checkpoints, which are key regulators of the immune system that stimulate or inhibit the actions of the immune system, which tumors can use to protect themselves from attacks by the immune system. Checkpoint therapy can block inhibitory checkpoints and activate stimulatory functions, thereby restoring immune system function. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is a transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1 or CD274).

[0263] Examples of immune checkpoint inhibitors include cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed death 1 (PD-1) or its ligand, lymphocyte activation gene-3 (LAG3), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), indoleamine (2,3)-dioxygenase (IDO), adenosine A2a receptor, neuritin, B and T lymphocyte attenuator (BTLA), killer immunoglobulin-like receptors (KIR), T cell immunoglobulin-mucin domain-containing protein 3 (TIM-3), inducible T cell costimulator (ICOS), CD27, CD2 8, CD40, CD244 (2B4), CD160, GARP, OX40, CD137 (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, CCR2, CCRS, CCR6, CD39, CD73, CD4, CD18, CD49b, CD1d, CDS, CD21, TIMI, CD19, CD20, CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, CD11b, Ly6G, ICAM-1, FAP, PDGFR, podoplanin, as well as immune checkpoint inhibitors of TIGIT.

[0264] Examples of clinically approved immune checkpoint inhibitors include, but are not limited to, Ipilimumab (anti-CTLA-4), Nivolimumab (anti-PD-1), and Pembrolizumab (anti-PD1).

[0265] According to another embodiment, the treatment is combined with a Bruton's tyrosine kinase (Btk) inhibitor (e.g., ibrutinib, acalabrutinib, or spebrutinib).

[0266] The inventors also contemplate selecting a type of treatment based on the presence of myeloid cells that express both TREM2 and Gpnmb.

[0267] Thus, according to yet another aspect of the present invention there is provided a method of treating cancer in a subject, comprising: (a) analyzing the presence of myeloid cells expressing both TREM2 and Gpnmb in a sample from a subject; and (b) treating the subject with a therapeutically effective amount of an antibody targeting TREM2 and / or Gpnmb described herein if the amount of said myeloid cells is above a predetermined amount, or treating the subject with a therapeutically effective amount of a chemotherapeutic agent other than said antibody targeting TREM2 and / or Gpnmb if the amount of said cells is below a predetermined amount; A method is provided that includes:

[0268] Methods for determining the gene expression profile can be performed at the RNA level or at the protein level.

[0269] Below is a more detailed description of the methods that can be used to analyze the expression of multiple genes at the single cell level.

[0270] Methods for Analyzing and / or Quantifying RNA Northern Blot Analysis: This method involves the detection of specific RNAs in a mixture of RNAs. The RNA sample is denatured by treatment with an agent that prevents hydrogen bonding between base pairs (e.g., formaldehyde), ensuring that all RNA molecules have an unfolded, linear conformation. Individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or nylon-based membrane to which the denatured RNA adheres. The membrane is then exposed to a labeled DNA probe. The probe can be labeled using radioisotopes or enzyme-linked nucleotides. Detection can use autoradiography, colorimetric reactions, or chemiluminescence. This method allows both quantification of the amount of a specific RNA molecule and the determination of its identity by its relative position on the membrane, which indicates the distance it traveled in the gel during electrophoresis.

[0271] RT-PCR analysis: This method uses PCR amplification of relatively rare RNA molecules. First, RNA molecules are purified from cells and converted into complementary DNA (cDNA) using reverse transcriptase (such as MMLV-RT) and primers, such as oligo-dT, random hexamers, or gene-specific primers. Then, PCR amplification reaction is carried out in a PCR machine by applying gene-specific primers and Taq DNA polymerase. Those skilled in the art can select the length and sequence of gene-specific primers and PCR conditions (i.e., annealing temperature, number of cycles, etc.) that are suitable for detecting specific RNA molecules. It will be understood that semi-quantitative RT-PCR reaction can be used by adjusting the number of PCR cycles and comparing the amplified products with known controls.

[0272] RNA in situ hybridization staining: In this method, DNA or RNA probes are attached to RNA molecules present in cells. Generally, to preserve the cell structure and prevent the degradation of RNA molecules, cells are first fixed on a microscope slide and then subjected to a hybridization buffer containing a labeled probe. The hybridization buffer contains reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) that allow the DNA or RNA probe to specifically hybridize with its target mRNA molecule in situ while avoiding non-specific binding of the probe. Those skilled in the art can adjust the hybridization conditions (i.e., temperature, salt and formamide concentration, etc.) for the specific probe and cell type. After hybridization, unbound probes are washed away and bound probes are detected using known methods. For example, if a radiolabeled probe is used, the slide is subjected to a photographic emulsion which reveals the signal generated using the radiolabeled probe; if the probe is labeled with an enzyme, an enzyme-specific substrate is added for the formation of a colorimetric reaction; if the probe is labeled with a fluorescent label, a fluorescent microscope is used to reveal the bound probe; if the probe is labeled with a tag (e.g., digoxigenin, biotin, etc.), the bound probe can be detected following interaction with a tag-specific antibody which can be detected using known methods.

[0273] In situ RT-PCR staining: This method is described in Nuovo GJ, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, RT-PCR reactions are performed on fixed cells by incorporating labeled nucleotides into the PCR reaction. The reaction is carried out using a specific in situ RT-PCR device such as the laser capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountain View, Calif.).

[0274] Single-cell transcriptome analysis This method relies on sequencing the transcriptome of a single cell. In one embodiment, a high-throughput method is used that allows RNA from different cells to be individually tagged, thereby creating a single library while preserving the cell identity of each read. This method can be carried out in a number of ways. For example, see US Patent Application Publication No. 20100203597 and US Patent Application Publication No. 20180100201, the contents of which are incorporated herein by reference.

[0275] One particular method for performing single-cell transcriptome analysis is summarized below.

[0276] Typically, the cells are aliquoted into wells so that there is only one cell in each well. The cells are treated with an agent that disrupts the cell and nuclear membranes, making the cellular RNA available for the sequencing reaction.

[0277] According to one embodiment, RNA is amplified using the following in vitro transcription amplification protocol.

[0278] (Step 1) Under conditions that allow synthesis of a single-stranded DNA molecule from the RNA, the RNA of a single cell is contacted with an oligonucleotide that includes a poly dT sequence at its terminal 3' end, a T7 RNA polymerase promoter sequence at its terminal 5' end, and a barcode sequence located between the poly dT sequence and the RNA polymerase promoter sequence, where the barcode sequence includes a cell barcode and a molecular identifier.

[0279] The poly dT oligonucleotides of this embodiment may optionally include adapter sequences required for sequencing (see, for example, FIG. 5).

[0280] RNA polymerase promoter sequences are known in the art and include, for example, the T7 RNA polymerase promoter sequence, e.g., SCGATTGAGGCCGGTAATACGACTCACTATAGGGGC (SEQ ID NO:3).

[0281] Preferably, the poly dT sequence comprises at least 5 nucleotides. According to another embodiment, the poly dT sequence is about 5 to 50 nucleotides, more preferably about 5 to 25 nucleotides, even more preferably about 12 to 14 nucleotides.

[0282] Barcode sequences are useful during multiplex reactions when multiple samples are pooled into a single reaction. Barcode sequences can be used to identify a particular molecule, sample, or library. The barcode sequence is attached to the 5' end of the poly dT sequence and the 3' end of the T7 RNA polymerase sequence. The barcode sequence can be 3-400 nucleotides, more preferably 3-200 nucleotides, and even more preferably 3-100 nucleotides. Thus, the barcode sequence can be 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides.

[0283] In one embodiment, the barcode sequence is used to identify a cell type or cell source (e.g., a patient).

[0284] Molecular identifiers are useful for correcting amplification biases that reduce the quantitative accuracy of the method. Molecular identifiers consist of 4 to 20 bases. The molecular identifiers are of such length that each RNA molecule in a sample is tagged (labeled) with a molecular identifier that has a unique sequence.

[0285] After annealing a primer (e.g., poly dT primer) to the RNA sample, an RNA-DNA hybrid can be synthesized by reverse transcription using an RNA-dependent DNA polymerase. Suitable RNA-dependent DNA polymerases for use in the methods and compositions of the present invention include reverse transcriptases (RTs). RTs are well known in the art. Examples of RTs include, but are not limited to, Moloney murine leukemia virus (M-MLV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase, Rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, Rous-associated virus (RAV) reverse transcriptase, and myeloblastosis-associated virus (MAV) reverse transcriptase, or other avian sarcoma leukemia virus (ASLV) reverse transcriptase, and modified RTs derived therefrom. See, for example, U.S. Pat. No. 7,056,716. Many reverse transcriptases, such as those from avian myeloblastosis virus (AMV-RT) and Moloney murine leukemia virus (MMLV-RT), contain multiple activities (e.g., polymerase activity and ribonuclease activity) and can function in forming double-stranded cDNA molecules. However, in some instances, it is preferable to use an RT that lacks RNase H activity or has substantially reduced RNase H activity.

[0286] RTs lacking RNase H activity are known in the art, including those that contain a mutation of wild-type reverse transcriptase that eliminates RNase H activity. Examples of RTs with reduced RNase H activity are described in US Patent Publication No. 20100203597. In these cases, the addition of RNase H from other sources, such as that isolated from E. coli, can be used to form single-stranded cDNA. Combinations of RTs are also contemplated, including combinations of different non-mutated RTs, combinations of different mutant RTs, and combinations of one or more non-mutated RTs with one or more mutant RTs.

[0287] Examples of suitable enzymes include, but are not limited to, AffinityScript from Agilent or Superscript III from Invitrogen. Preferably, the reverse transcriptase lacks terminal deoxynucleotidyl transferase (TdT) activity.

[0288] Additional components required for the reverse transcription reaction include dNTPs (dATP, dCTP, dGTP, and dTTP), and optionally reducing agents such as dithiothreitol (DTT) and MnCl2.

[0289] Poly dT oligonucleotides can be attached to a solid support (eg, beads) so that the synthesized cDNA can be purified.

[0290] Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated.

[0291] The annealing temperature is typically selected to provide optimal efficiency and specificity, and typically ranges from about 50° C. to about 80° C., usually from about 55° C. to about 70° C., and more usually from about 60° C. to about 68° C. Annealing conditions are typically maintained for a period ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 5 minutes.

[0292] (Step 2): Once the cDNA is generated, it can be pooled from cDNA generated from other single cells (using the same methods described herein above).

[0293] To remove excess primers, the sample can be optionally treated with an enzyme such as exonuclease I. Other options for purifying single-stranded DNA are also contemplated, including, for example, the use of paramagnetic microparticles. This can be performed after or before pooling the samples.

[0294] (Step 3): Second strand synthesis. Synthesis of the second strand of cDNA can be performed by incubating the sample in the presence of nucleotide triphosphates and DNA polymerase. Commercial kits are available for this step, including additional enzymes such as RNase H (to remove the RNA strand) and buffers. This reaction can optionally be performed in the presence of DNA ligase. Following second strand synthesis, the product can be purified using methods known in the art, including, for example, the use of paramagnetic microparticles.

[0295] (Step 4): Following the synthesis of the second strand of cDNA, RNA can be synthesized by incubating with the corresponding RNA polymerase. Commercially available kits such as the T7 High Yield RNA Polymerase IVT Kit (New England Biolabs) can be used.

[0296] (Step 5): Before fragmenting the amplified RNA, DNA can be removed using DNase enzyme. The RNA can be purified before fragmentation. RNA fragmentation can be carried out as known in the art. Fragmentation kits, such as the Ambion fragmentation kit, are commercially available.

[0297] (Step 6): The 3' end of the amplified and fragmented RNA is now labeled. For this, a ligase reaction is performed that essentially ligates single-stranded DNA (ssDNA) to the RNA. Other methods of labeling amplified and fragmented RNA are described in US Patent Publication No. 20170137806, the contents of which are incorporated herein by reference. The single-stranded DNA has a free phosphate at the 5' end and, optionally, a blocking moiety at the 3' end to prevent head-to-tail ligation. Examples of blocking moieties include a C3 spacer or a biotin moiety. Typically, the ssDNA is 10-50 nucleotides long, more preferably 15-25 nucleotides long.

[0298] (Step 7): Reverse transcription is then performed using primers complementary to those used in the previous step. The library can then be completed and amplified through nested PCR reactions, as illustrated in FIG.

[0299] (Step 8): Amplification Once the adapter polynucleotides of the invention have been ligated to the single-stranded DNA (i.e., following extension of the single-stranded DNA), an amplification reaction can be performed.

[0300] (Step 9): Sequencing Methods for sequencing are generally known to those skilled in the art. A preferred sequencing method is next-generation sequencing or parallel high-throughput sequencing, such as massively parallel signature sequencing (MPSS). An example of a sequencing method envisioned is pyrosequencing, in particular 454 pyrosequencing, for example based on the Roche 454 Genome Sequencer. This method amplifies DNA in water droplets in an oil solution, with each droplet containing a single DNA template attached to a bead coated with a single primer, which subsequently forms a clonal colony. In pyrosequencing, luciferase is used to generate light to detect individual nucleotides added to the nascent DNA, and the aggregated data is used to generate sequence readouts. Yet another envisioned example is Illumina or Solexa sequencing, for example by using the Illumina Genome Analyzer technology based on reversible dye terminators. Typically, DNA molecules are attached to primers on a slide and amplified so that local clonal colonies are formed. Then, one nucleotide at a time can be added, and unincorporated nucleotides are washed away. Images of the fluorescently labeled nucleotides can then be taken, and the dye is chemically removed from the DNA to allow for the next cycle. Yet another example is the use of Applied Biosystems' SOLiD technology, which uses sequencing by ligation. This method is based on the use of a pool of all possible oligonucleotides of fixed length, labeled according to the sequenced position. Such oligonucleotides are annealed and ligated. Preferential ligation by DNA ligase to matching sequences then typically results in a signal that conveys information about the nucleotide at that position. DNA is typically amplified by emulsion PCR, so that the resulting beads, each containing only copies of the same DNA molecule, can be placed on a glass slide to obtain sequences of comparable quantity and length to Illumina sequencing.A further method is based on Helicos' Heliscope technology, which captures fragments by poly-T oligomers tethered to an array. In each sequencing cycle, polymerase and a single fluorescently labeled nucleotide are added, and the array is imaged. The fluorescent tag is then removed, and the cycle is repeated. Further examples of sequencing techniques that are included in the method of the present invention are sequencing by hybridization, sequencing by using nanopores, microscope-based sequencing techniques, microfluidic Sanger sequencing, or microchip-based sequencing methods. The present invention also envisions further development of these techniques, such as further improvements in the accuracy of sequencing or the time required for sequencing the genome of an organism.

[0301] According to one embodiment, the sequencing method comprises deep sequencing.

[0302] As used herein, the term "deep sequencing" refers to a sequencing method in which a target sequence is read multiple times in one test. A single deep sequencing run is composed of multiple sequencing reactions that are performed on the same target sequence and each generate an independent sequence readout.

[0303] It will be appreciated that methods that rely on microfluidics can also be used to perform single cell transcriptome analysis.

[0304] Thus, a combination of molecular barcoding and emulsion-based microfluidics can be used to isolate, lyse, barcode and prepare nucleic acids from individual cells in a high-throughput manner. A microfluidic device (e.g., fabricated with polydimethylsiloxane) produces sub-nanoliter inverse emulsion droplets. These droplets are used to co-encapsulate barcoded capture beads and nucleic acids. For example, each bead is uniquely barcoded, allowing each droplet and its contents to be distinguished. Nucleic acids can come from any source known in the art, e.g., from a single cell, a pair of cells, a cell lysate, or from a solution. The cells are lysed as they are encapsulated in the droplets. To load single cells and barcoded beads into these droplets using Poisson statistics, 100,000 to 10 million such beads are needed to barcode approximately 10,000 to 100,000 cells. In this regard, a single cell sequencing library may exist, which may include combining one uniquely barcoded mRNA capture microbead with a single cell in an emulsion droplet of 75-125 μm in diameter, lysing the cell and allowing its RNA to be captured by hybridization onto the RNA capture microbead, performing reverse transcription either inside or outside the emulsion droplet to convert the cell's mRNA into first strand cDNA covalently linked to the mRNA capture microbead, pooling the cDNA-attached microbeads from all the cells, and preparing and sequencing a single composite RNA-Seq library as described herein above.In this regard, see Macosko et al., 2015, "Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets" Cell 161, 1202-1214, the specification of International Patent Application No. PCT / US2015 / 049178, published on March 17, 2016 as WO 2016 / 040476, Klein et al., 2015, "Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells" Cell 161, 1187-1201, Zheng, et al., 2016, "Haplotyping germline and cancer genomes with high-throμghput linked-read sequencing" Nature Biotechnology 34, 303-311, and WO 2014210353. The entire contents and disclosures of each of these are incorporated herein by reference in their entirety.

[0305] Methods for detecting protein expression and / or activity The expression and / or activity levels of proteins expressed in the cells of the cultures of some embodiments of the invention can be determined using methods known in the art.

[0306] Enzyme-Linked Immunosorbent Assay (ELISA): This method involves immobilizing a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate onto a surface, such as the well of a microtiter plate. A substrate-specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. The presence of the antibody is then detected and quantified by a colorimetric reaction using the antibody-coupled enzyme. Enzymes commonly used in this method include horseradish peroxidase and alkaline phosphatase. When well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. Substrate standards are generally used to improve the accuracy of quantification.

[0307] Western Blot: This method involves separating a substrate from other proteins by an acrylamide gel, followed by transferring the substrate to a membrane (e.g., nylon or PVDF). The presence of the substrate is then detected by an antibody specific for the substrate, which is then detected by an antibody-binding reagent. The antibody-binding reagent can be, for example, Protein A or other antibodies. The antibody-binding reagent can be radiolabeled or linked to an enzyme, as described above. Detection can be by autoradiography, colorimetric reaction, or chemiluminescence. This method allows both quantification of the amount of substrate and determination of its identity by its relative position on the membrane, which indicates the distance traveled in the acrylamide gel during electrophoresis.

[0308] Radioimmunoassay (RIA): In one version, this method uses a specific antibody immobilized on a precipitable support, such as agarose beads, and a radiolabeled antibody-binding protein (e.g., I 125 The technique involves precipitating the desired protein (i.e., substrate) with 5'-labeled protein A. The number of counts in the precipitated pellet is proportional to the amount of substrate.

[0309] An alternative version of the RIA uses a labeled substrate and an unlabeled antibody-binding protein. Samples containing unknown amounts of substrate are added in various amounts. The decrease in precipitate counts from the labeled substrate is proportional to the amount of substrate in the added sample.

[0310] Fluorescence Activated Cell Sorting (FACS): This method involves the in situ detection of a substrate within a cell by a substrate-specific antibody. The substrate-specific antibody is linked to a fluorophore. It is detected by a cell sorter that reads the wavelength of light emitted by each cell as it passes through a beam of light. Two or more antibodies can be used simultaneously in this method.

[0311] Immunohistochemical analysis: this method involves the in situ detection of substrates in fixed cells by substrate-specific antibodies, which may be linked to an enzyme or to a fluorophore. Detection is performed by microscopy and subjective or automated evaluation. When enzyme-linked antibodies are used, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei, for example using hematoxylin or Giemsa stains.

[0312] In situ activity assay: According to this method, a chromogenic substrate is applied onto cells containing active enzyme and the enzyme catalyzes a reaction in which the substrate is cleaved to produce a chromogenic product that can be visualized by light or fluorescence microscopy.

[0313] In vitro activity assays: These methods measure the activity of a particular enzyme in a protein mixture extracted from cells. Activity can be measured in spectrophotometer wells using colorimetric methods or in non-denaturing acrylamide gels (i.e. activity gels). After electrophoresis, the gel is immersed in a solution containing a substrate and a colorimetric reagent. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of color produced. Enzyme standards are generally used to improve the accuracy of quantification.

[0314] According to a specific embodiment, gene expression is determined by transcriptome analysis.

[0315] According to a specific embodiment, gene expression is determined by single cell transcriptome analysis as described above.

[0316] Thus, if a particular level of cells is observed, e.g., greater than 5%, greater than 10% of myeloid cells in the derived sample, the subject may be considered a candidate for a therapy that targets these cells. If an insufficient number of myeloid cells of this characteristic is observed, the subject is not considered a candidate for this therapy.

[0317] As used herein, the term "about" refers to ±10%.

[0318] The terms "comprises," "comprising," "includes," "including," "having," and their conjugations mean "including but not limited to."

[0319] The term "consisting of" means "including and limited to."

[0320] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0321] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately, or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features that are described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment would not work without those elements.

[0322] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0323] Working Example Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0324] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998), U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassays are widely described in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879, 262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, "Oligonucleotide "Synthesis" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986), "Immobilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout the specification. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0325] Materials and Methods Production of anti-human TREM2 monoclonal antibodies Five BalB / C and five SJL mice were immunized with a recombinant protein consisting of the extracellular domain of human TREM2 fused to a His tag (SEQ ID NO: 4, MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSHHHHHH). Spleens of mice were harvested and fused with Sp2 / 0 myeloma cells. Ab-producing clones were selected by ELISA and screened against 293HEK cells stably transfected with hTREM2.

[0326] direct ELISA 96-well ELISA microplates were coated with His-tagged hTREM2 at 0.5 μg / mL, 100 μl / well, diluted in PBS, pH 7.4, and incubated overnight at 4°C. Plates were rinsed three times with 0.05% Tween 20 in PBS, blocked with 1% BSA in PBS for 1 h at room temperature (RT), and rinsed again. Plates were incubated with anti-hTREM2 antibody (100 μl / well) at the indicated concentrations for 2 h at RT. Plates were rinsed and incubated with Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fcg Fragment Specific (min X Human, Bovine, Horse Serum Proteins, Jackson ImmunoResearch) for 20 min at RT. Plates were rinsed and incubated with TMB reagent (TM4500, Scytek) for 20 min at RT, followed by the addition of 2N sulfuric acid (DY994, R&D) as a stop solution. OD was measured at two wavelengths (450 nm and 570 nm) using an ELISA plate reader.

[0327] Supernatant Elisa Wild-type 293HEK cells or 293HEK cells overexpressing hTREM2 were cultured for 24 h (3 million cells in a 10 cm plate). The supernatant was collected, centrifuged at 900 g for 5 min and filtered (0.45 μm). 96-well ELISA microplates were coated with 0.2 μg / well of anti-human TREM2 antibody (AF1828, R&D) diluted in PBS and incubated overnight at 4°C. The plates were rinsed three times with 0.05% Tween 20 in PBS, blocked with 1% BSA in PBS for 1 h at room temperature (RT) and rinsed again. The plates were incubated with cell culture supernatant (100 μl / well) for 2 h at RT and rinsed again. The plates were incubated with 0.1 μg of biotinylated anti-hTREM2 antibody for 2 h at RT. After incubation, the plates were rinsed and incubated with streptavidin-HRP (DY998, R&D) for 20 min at RT. The plates were rinsed and incubated with TMB reagent (TM4500, Scytek) for 20 min at RT, followed by the addition of 2N sulfuric acid (DY994, R&D) as a stop solution. The OD was measured at two wavelengths (450 nm and 570 nm) using an ELISA plate reader.

[0328] Cell-based ELISA Wild-type 293HEK cells or 293HEK cells overexpressing hTREM2 were seeded on poly-L-lysine-coated 96-well tissue culture plates and cultured for 48 h in the presence of MMP inhibitor GM-6001 (25 μM, Enzo). Cells were washed three times with PBS and blocked for 2 h at RT (5% FBS, 1% BSA in PBS). Cells were washed with 1% BSA in PBS and incubated with 0.1 μg (or the indicated amount) of biotinylated anti-hTREM2 antibody for 4 h at RT. After incubation, cells were incubated with streptavidin-HRP (DY998, R&D) for 20 min at RT. Cells were washed and incubated with TMB reagent (TM4500, Scytek) for 30 min at RT, followed by the addition of 2N sulfuric acid (DY994, R&D) as a stop solution. OD was measured at two wavelengths (450 nm and 570 nm) using an ELISA plate reader. Antibody biotinylation was performed using the EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0329] Western blot (SDS PAGE) for TREM2 detection Cells were washed with ice-cold PBS, resuspended in cold hypotonic lysis buffer (0.01 M Tris, pH 7, 1 mM EDTA, 1 mM EGTA) supplemented with protease inhibitor cocktail (cOmplete™, Roche) and incubated on ice for 30 min. Cells were flash frozen in liquid nitrogen, thawed and centrifuged at 16,000 g for 45 min at 4° C. The pellet was resuspended in STE lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.6, 2 mM EDTA, 1% Triton X-100), incubated on ice for 20 min and centrifuged at 16,000 g for 30 min at 4° C. Supernatants were collected and protein concentrations were measured using the BCA protein assay. Proteins (50 μg) were separated on a 12% Bis-Tris gel and transferred to a nitrocellulose membrane (Thermo Fisher Scientific). The membrane was blocked with 3% BSA diluted in TBS-Tween for 1 h at room temperature. The membrane was incubated with anti-hTREM2 antibody (1 μg / ml) overnight at 4°C. Peroxidase-AffiniPure goat anti-mouse IgG was used as the secondary antibody (Jackson ImmunoResearch, 115-035-071). Bound antibodies were visualized using SignalFire Elite ECL reagent (Cell Signaling Technology).

[0330] Differentiation of bone marrow-derived macrophages (BMDMs) Mouse bone marrow cells from TREM2 knockout (KO) and hTREM2 transgenic (hTREM2) mice were cultured for 7 days in the presence of 30 ng / mL hM-CSF cytokine (Peprotech, 300-25) to generate bone marrow-derived macrophage cells (BMDMs).

[0331] Flow cytometry analysis TREM2 KO BMDMs and hTREM2 BMDMs were washed with MACS buffer (PBS pH 7.2, 0.5% BSA, and 2 mM EDTA), stained with biotin-conjugated anti-hTREM2 antibody (10 μg / mL), followed by washing with MACS buffer and incubation with APC-streptavidin (Biolegend, 405207), and then analyzed by flow cytometer (LSRII, BD).

[0332] Surface plasmon resonance (SPR) analysis Affinity measurements were obtained by SPR performed on a BIAcore T200 instrument equipped with a series S sensor chip CM5 (Cytiva). hTREM2-His protein was captured on the chip and anti-hTREM2 antibody was used as the analyte. Steady-state affinity binding was used to fit sensograms to obtain equilibrium dissociation constant (KD) values.

[0333] Immunofluorescence analysis TREM2 KO BMDMs and hTREM2 BMDMs were seeded on poly-L-lysine-coated coverslips and cultured for 24 h. Cells were washed twice with PBS, fixed with cold methanol, washed, and blocked with 5% FBS in PBS for 1 h. Cells were incubated with anti-hTREM2 antibody (2 μg / ml) overnight at 4°C. Cells were washed with PBS, followed by secondary antibody staining (Alexa Fluor 647-AffiniPure F(ab')2 fragment donkey anti-mouse IgG(H+L), Jackson ImmunoResearch, 715-606) and DAPI.

[0334] Bone marrow derived macrophage (BMDM) differentiation perturbation assay Mouse bone marrow cells were differentiated into macrophages as indicated, while anti-hTREM2 antibody was added to the culture medium (10 μg / mL) on days 2 and 5 of culture.

[0335] ELISA analysis of penetration of biotin-conjugated hTREM2 antibodies MCA-205 cells were washed, resuspended in PBS, and injected subcutaneously (0.5 million cells / mouse in 100 μl PBS). The flanks of mice were pre-shaved and injected subcutaneously (sc). On day 9, mice were treated intraperitoneally (ip) with biotin-conjugated anti-hTREM2 antibody (70 μg / mouse) and sacrificed 24 hours later. For ELISA analysis, the indicated organs were harvested and total protein was extracted by homogenization in RIPA lysis buffer supplemented with cOmplete™ protease inhibitor cocktail. Samples were left on ice for 20 min and centrifuged at 13,000×g for 20 min at 4° C. The supernatant was collected and protein concentration was determined using the BCA method. 100 μg protein was loaded onto pre-coated and blocked (1% BSA in PBS) hTREM2 protein followed by 5 washes to bind HRP-streptavidin. Plates were rinsed and incubated with TMB reagent (TM4500, Scytek) for 20 min at RT, followed by the addition of 2N sulfuric acid (DY994, R&D) as a stop solution. OD was measured at two wavelengths (450 nm and 570 nm) using an ELISA plate reader.

[0336] Production of anti-human GPNMB monoclonal antibodies Five C57BL / 6J mice were transfected with a recombinant protein consisting of the extracellular domain of human GPNMB fused to a His tag (SEQ ID NO: 5, MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWSSDENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNLIFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQSHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTANVTLGPQLMEVTVYRRHGRAYVPIAQ Mice were immunized with IgG1-specific BCR1 (VKDVYVVTDQIPVFVTMFQKNDRNSSDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVNHTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPSKPTPSLATTLKSYDSNTPGPAGDNPLELSRIPDENCQINRYGHFQATITIVEGILEVNIIQMTDVLMPVPWPESSLIDFVVTCQGSIPTEVCTIISDPTCEITQNTVCSPVDVDEMCLLTVRRTFNGSGTYCVNLTLGDDTSLALTSTLISVPDRDPASPLRMANHHHHHH). Mice spleens were harvested and single B cells were analyzed for GPNMB binding by flow cytometry and selected for BCR sequencing. Productive BCRs were cloned into OG527 (IgG) and OG528 (Igk) for antibody production.

[0337] Binding screening of anti-human GPNMB monoclonal antibodies Anti-human GPNMB antibodies will be screened for binding to hGPNMB-expressing 293HEK and human peripheral blood derived macrophages by direct ELISA and flow cytometry.

[0338] Functional screening of anti-human GPNMB monoclonal antibodies Human leukocytes were extracted from peripheral blood by Ficoll separation. CD4 T cells were isolated using CD4 microbeads (Miltenyi Biotec, 130-045-101). CD4 cells were incubated with recombinant hGPNMB protein for 1-3 days in pre-coated anti-CD3, anti-CD28, and anti-CD2 antibodies (Miltenyi Biotec, 130-091-441). Copy numbers were measured by CFSE staining using flow cytometry. IFNg secretion was measured by ELISA (Biolegend, BLG-430104). Anti-hGPNMB antibodies were tested for their ability to suppress CD4 T cell activation by adding 10 μg / mL of antibody during T cell incubation.

[0339] Differentiation of human monocyte-derived macrophages (hMDM) and human monocyte-derived dendritic cells (hMDC) CD14+ cells were isolated from human blood using a CD14+ isolation kit (Miltenyi Biotech #130-050201). Cells were cultured for 5 days in the presence of 30ng / mL hM-CSF cytokine (Peprotech, 300-25) followed by administration of 20ng / mL IL4 to generate M2 macrophages. To generate hMDCs, cells were cultured for 7 days in the presence of 30ng / mL hGM-CSF cytokine (Peprotech, 300-03).

[0340] Stimulation of hMDMs Primary hMDMs were generated as described above. 10 μg / mL anti-TREM2 and / or 20 ng / mL IL2 or IL15 cytokines were added to the cultures on days 3, 5, and 6.

[0341] Gene expression analysis For gene expression analysis, cells were washed twice with PBS, followed by cell lysis and RNA purification with Dynabeads mRNA DIRECT purification kit (Thermo Fisher, 61012) according to the manufacturer's instructions. cDNA was generated using the SuperScript III kit (Thermo Fisher, 18080044) according to the manufacturer's instructions.

[0342] qPCR analysis was performed using SYBR Green (LightCycler 480 SYBR Green, Roche, 04-887-352) reagents and the following primers (Table C).

[0343] [Table 2]

[0344] Cytokine secretion analysis Cytokine secretion analysis was performed on macrophage supernatants 24 hours after the M2 polarization process. Supernatants were diluted 1:20 and cytokine levels were measured using the CBA Human Inflammatory Cytokine Kit (BD551811) according to the manufacturer's instructions.

[0345] Isolation, activation, and co-culture of human T cells and hMDMs Primary hMDMs were generated as described previously. After selection of CD14+ cells, CD14- were frozen during macrophage differentiation. After 6 days, cells were thawed and CD8 T cells were selected by human CD8 microbeads (Miltenyi Bitec, 130-045-201). CD8 T cells were stained with a proliferation tracking dye (65-0842-85, Thermo Fisher) and seeded on plates precoated with anti-CD3 (10 μg / mL, OKT, BLG-317326) and anti-CD28 (2 μg / mL, BLG-302934). Differentiated and treated M2 macrophages were added to the co-culture at a ratio of 1:3 (macrophages:T cells). After 4 days, the proliferation rate of the cells was analyzed by flow cytometry.

[0346] Recombinant antibody binding assay Direct Elisa was performed as previously described. Briefly, recombinant human TREM2 (2 μg / ml) was coated onto Elisa plates. After blocking, plates were incubated with various concentrations of recombinant antibodies. The following secondary antibodies were used for antibody detection: anti-human IgG-HRP (709-035-149, Jackson ImmunoResearch), human IL2 (BLG-500302, BioLegend), followed by anti-rat IgG-HRP (ab97057, Abcam). Supernatant Elisa was performed as previously described. Anti-TREM2 antibody AF1828 (R&D) was used as capture antibody, followed by blocking and incubation with hMDM supernatants. Anti-TREM2 recombinant antibody was used as the detection antibody, followed by detection with secondary antibodies (anti-human IgG-HRP (709-035-149, Jackson ImmunoResearch), human IL2 (BLG-500302, BioLegend), followed by anti-rat IgG-HRP (ab97057, Abcam)).

[0347] Differentiation and processing of bone marrow derived macrophages (BMDM) Mouse bone marrow cells were extracted from femurs and tibias of hTREM2 transgenic (hTREM2) mice (female, 12 weeks old) and seeded (20K cells in 100 μl C10 medium) in 96-well non-tissue culture plates in the presence of 30 ng / mL hM-CSF cytokine (Peprotech, 300-25) to generate bone marrow derived macrophage cells (BMDMs). On day 2, the medium was replaced with 100 μl C10 medium supplemented with 30 ng / mL hM-CSF (control) or 30 ng / mL hM-CSF + 10 μg / ml of various antibodies (recE3C7 (recombinant anti-human TREM2), E3C7 (anti-human TREM2), recE3C7-long-IL2, recE3C7-short-IL2, or IgG (isotype control)) (see Table 1). On day 5, 20ng / ml mouse IL-4 (Peprotech, 214-14) was added to all conditions and the procedure on day 2 was repeated. On day 7, the medium was aspirated, cells were washed once with 200μl PBS (- / -), and 44K activated pan T cells were added to BMDMs of different conditions in 100μl unsupplemented C10 medium.

[0348] Isolation and activation of mouse T cells T cells were isolated from the spleens of WT mice (female, 12 weeks old) using the Miltenyi (cat. no. 130-095-130) mouse pan T cell isolation kit. Isolated T cells were cultured in U-shaped 96-well tissue culture plates (pre-coated with 0.5 μg / well anti-mouse CD3 (BLG-100340)) at 3 × 10 in 200 μl C10 medium supplemented with 2 μg / ml anti-mouse CD28 (BLG-102116). 5 The cells were cultured for 24 hours. As a control, 3 × 10 5 Naïve T cells were cultured in a different well in the same plate without CD3 coating and without adding CD28 to the medium. The cells were kept in culture for 24 hours.

[0349] Coculture of T cells and BMDM One day after T cell activation, activated and naïve T cells were removed from the wells with a pipette and transferred separately to 15 ml tubes. Cells were centrifuged at 400 g for 5 min and then resuspended in C10 medium at a concentration of 44K cells per 100 μl. Activated T cells were added to wells of BMDMs (44K cells in 100 μl volume) washed with PBS (- / -). As an activated / naïve T cell only control, activated and naïve T cells were seeded in U-shaped 96-well tissue culture plates (44K cells per 100 μl C10). After 42 hours, supernatants were collected from all wells to measure IFNg release.

[0350] Mouse interferon gamma secretion assay Interferon gamma (IFNg) concentrations were measured using ELISA MAX Deluxe Set for mouse IFN-gamma (BGL-430804, Biolegend) and human IFN-gamma (BLG-430115, Biolegend). 96-well ELISA microplates were coated with 0.2 μg / well of anti-mouse IFN-gamma antibody diluted in PBS and incubated overnight at 4°C. Plates were rinsed three times with 0.05% Tween-20 in PBS, blocked with 1% BSA in PBS for 1 h at room temperature (RT) and rinsed again. Coated plates were incubated with cell culture supernatant (100 μl / well) for 2 h at RT and rinsed again. Plates were incubated with 0.1 μg of biotinylated anti-mouse IFN-gamma antibody for 2 h at RT. After incubation, plates were rinsed and incubated with streptavidin-HRP for 20 min at RT. Plates were rinsed and incubated with solution F substrate for 10 min at RT, followed by addition of 2N sulfuric acid (DY994, R&D) as a stop solution. OD was measured at two wavelengths (450 nm and 570 nm) using an ELISA plate reader.

[0351] Design and cloning of anti-human TREM2 CAR T cells For the design of anti-human TREM2 CAR T cells, the sequence of the recognition domain of anti-human TREM2 antibody #80E3C7 was used to replace the corresponding domain in MSGV-1D3-28Z All ITAMs intact (Addgene #107226). A gblock containing the entire variable region sequence of the TREM2 CAR construct and T2A-BFP was ordered and cloned into MSGV-1D3-28Z using the restriction enzymes NcoI (NEB #R3193S) and SalI (NEB #R3138S).

[0352] Generation of anti-human TREM2 CAR T cells Retroviral particles were produced by transfecting retroviral packaging PLAT-E cells grown to 70-80% confluence in 6-well or 10 cm plates. Five hours prior to transfection, PLAT-E cells were provided with supplemented DMEM without penicillin and streptomycin. Transfection was performed using Lipofectamine 2000 (ThermoFisher #11668027) under the manufacturer's instructions and included the retroviral packaging vector Pcl-Eco (Addgene #12371) and retroviral CAR T expression plasmid. The medium was changed 5-13 hours after transfection. After 48 hours, transfection efficiency was investigated using a fluorescent microscope. The medium containing retroviral particles was harvested 48-72 hours after transfection. T cells were isolated from the spleens of 8-12 week old female WT mice using the Mouse Pan T Cell Isolation Kit II (Miltenyi Biotech #130-095-130). Cells were incubated in RPMI medium supplemented with 100U / ml rIL2 and 2μg / ml anti-CD28 (Biolegend #102102) on 24 well tissue culture plates coated with 250ng / well anti-CD3e (ThermoFisher #16-0031-82). Retroviral transduction of T cells was performed by adding retroviral particles to 24 well non-tissue culture plates coated with RetroNectin (Takara Bio #T100A) and centrifuging at 2000g for 2 hours at 32°C. T cells were added on top of the viral soup followed by centrifugation at 400g for 10 minutes. The cells were further grown for 2-5 days to reach sufficient cell numbers.

[0353] Differentiation of bone marrow derived macrophages (BMDM) and bone marrow derived dendritic cells (BMDC) Mouse bone marrow cells from WT, TREM2 knockout (KO), and hTREM2 transgenic (hTREM2) mice were cultured for 7 days in the presence of 30 ng / mL hM-CSF cytokine (Peprotech, 300-25) to generate bone marrow-derived macrophage cells (BMDMs). To generate bone marrow-derived dendritic cells (BMDCs), cells were cultured for 7 days in the presence of 30 ng / mL hGM-CSF cytokine (Peprotech, 300-03).

[0354] Co-culture of TREM2 CAR T cells TREM2 CAR T cells were co-cultured at a 1:1 ratio with hM2 and hDCs from wild-type 293HEK or hTREM2-overexpressing 293HEK, CD14+, and BMDM or BMDC from WT, TREM2 knockout (KO), or hTREM2 transgenic (hTREM2) mice. BFP-expressing T cells were used as an ac control for T cell responses. All cells were plated on poly-L-lysine-coated 96-well tissue culture plates and cultured for 24 hours. Supernatants were harvested for IFN-γ ELISA and cells were harvested for further analysis using flow cytometry.

[0355] Human interferon gamma secretion assay ELISA was performed using ELISA MAX Deluxe Set Mouse IFN-γ (Biolegend #430804). 96-well ELISA microplates were coated with 0.2 μg / well of anti-mouse IFN-γ antibody diluted in PBS and incubated overnight at 4°C. Plates were rinsed three times with 0.05% tween20 in PBS, blocked with 1% BSA in PBS for 1 h at room temperature (RT) and rinsed again. Plates were incubated with cell culture supernatant (100 μl / well) for 2 h at RT and rinsed again. Plates were incubated with 0.1 μg of biotinylated anti-mouse IFN-γ antibody for 2 h at RT. After incubation, plates were rinsed and incubated with streptavidin-HRP for 20 min at RT. Plates were rinsed and incubated with TMB reagent for 20 min at RT followed by the addition of Stop Solution 2N Sulfuric Acid (DY994, R&D). OD was measured at two wavelengths (450 nm and 570 nm) using an ELISA plate reader.

[0356] Flow cytometry analysis - CART analysis Mouse T cells were harvested, washed with MACS buffer (PBS pH 7.2, 0.5% BSA, and 2 mM EDTA), stained with PE / Cy7-conjugated anti-CD8a antibody, PE-conjugated anti-CD25 antibody, APC / Cy7-conjugated anti-CD107 antibody, and APC-conjugated anti-CD279 (PD1) antibody, followed by washing with MACS buffer and then analyzed by flow cytometer (Symphony S6 BD). EXAMPLES

[0357] Screening for monoclonal anti-TREM2 binding We have produced and purified mouse anti-human TREM2 (hTREM2) monoclonal antibodies (see above) and screened these mAbs for sensitivity and specificity binding to hTREM2. Screening was performed by first measuring binding of TREM2 recombinant protein (Table 2, Figure 5) and additionally comparing mAb binding ability to hTREM2-expressing 293HEK cells with WT (Table 3, Figures 2-3, Figure 6A-B). Bone marrow derived macrophages (BMDMs) were found to express high amounts of TREM2. We extracted bone marrow cells from human TREM2 (hTREM2) transgenic mice and used them for differentiation of bone marrow derived macrophages (BMDMs). hTREM2 BMDMs were used for additional binding screening of antibodies (Figures 4A-B, Figures 6A-B to Figure 7).

[0358] Table 1 Summary of requirements for 18 hybridoma-derived monoclonal antibodies against human TREM2 protein. Purity is measured by SDS-PAGE. Endotoxin content (EU / mg) is measured by LAL assay. Titer is measured by direct ELISA, title value is the highest dilution with S / B (signal / blank) ≧2.1.

[0359] [Table 3]

[0360] Table 2 Direct ELISA analysis of 18 hybridoma-derived monoclonal antibodies against human TREM2 protein Coating antigen: His-TREM2, 0.5 μg / mL in PBS, pH 7.4, 100 μl / well. Antibody stock concentration: 1mg / mL Secondary antibody: Peroxidase-AffiniPure Goat anti Mouse IgG, Fcg fragment specific (min x human, bovine, horse serum proteins) The titer is the highest dilution at which S / B (signal / blank) is ≧2.1.

[0361] [Table 4]

[0362] Table 3 - 293 HEK cells were stably transfected with the hTREM2 gene followed by puromycin selection. A. Cell culture supernatants (Sup) were used to test the soluble TREM2 protein binding ability of 18 hybridoma-derived monoclonal antibodies by direct ELISA. B. Cells were captured in 96-well plates and used in a cell-based ELISA of 18 hybridoma-derived monoclonal antibodies. The OD values ​​of positive cells (hTREM2-expressing HEK293) and negative cells (wild-type HEK293) are shown in the table.

[0363] [Table 5] EXAMPLES

[0364] Screening of monoclonal anti-TREM2 antagonists Since loss of function of TREM2 showed reduced tumor growth in several mouse syngeneic tumor models, we designed an assay to effectively screen the blocking activity of anti-TREM2 monoclonal antibodies. Bone marrow-derived macrophages (BMDMs) are known to produce high levels of inhibitory cytokines such as IL-10 and TGF-β (Wang, BMC Immunology 2013 14:6). To determine whether TREM2 plays a role in the maturation and suppressive function of BMDM cells, we cultured bone marrow cells from femurs and tibias of TREM2 knockout (KO) and hTREM2 transgenic (hTREM2) mice in the presence of M-SCF for 7 days (Methods) and characterized the maturation course over time using single-cell RNA-seq. Clustering analysis and 2d projections of cells from each time point / genotype showed a distinct maturation course that differed between the TREM2-KO genotype and that of hTREM2, starting at day 5 and showing maximum phenotype at day 7. Wild-type hTREM2 BM cells exhibited an M2 phenotype with high expression of Gpnmb, Lpl, Anxa1, Mmp12, Adam8, Lgals1, Lgals3, Spp1, and Lilrb4a at day 7, while TREM2-KO BM genotypes displayed an activated M1 phenotype including Selenop, Ms4a4a, Fcgr2b, Ms4a7, and Lyz2 (Figure 8A-B). To screen for antibodies with antagonistic activity against TREM2, we cultured hTREM2 mouse bone marrow cells with M-CSF and added anti-hTREM2 antibodies or IgG isotypes to the medium (10 μg / mL) on days 2 and 5 of culture. Single-cell RNA-seq was used to characterize the cells at day 7 and the cellular distribution between M2 (TREM2+Gpnmb+) and M1 (TREM2-) phenotypes in each condition. More than 70% of TREM2-KO cells achieved the M1 phenotype and less than 10% displayed the M2 phenotype, in contrast to 40% of hTREM2 cells and only 24% displayed the M2 phenotype.Addition of IgG isotype mAbs to the cultures did not significantly alter the M1 / M2 ratio, showing results similar to those of untreated cultures, whereas addition of anti-hTREM2 mAbs 54H2C1 or 80E3C7 dramatically reduced the percentage of M2 phenotype to 12% and 16%, respectively, and increased the M1 phenotype to 69% and 63% (Figure 8C), showing a maturation course very similar to that of TREM2-KO cells.

[0365] To quantify the binding specificity of anti-hTREM2 mAbs to tumor-associated macrophages and Mregs in vivo, we performed ELISA analysis of biotin-conjugated anti-hTREM2 mAbs 54H2C1, 80E3C7, and 83E10B12 in humanized TREM2 mice bearing MCA-205-induced tumors. For mAbs 54H2C1, 80E3C7, we detected a more than two-fold enrichment of mAb concentrations in the tumor TME compared to LN, liver, kidney, lung, heart, brain, and spleen. mAb 80E3C7 was mostly concentrated in the liver and kidney (see Figures 9-10A-B). EXAMPLES

[0366] Anti-TREM2 antibodies of some embodiments of the invention reprogram human monocyte-derived macrophages To investigate the effect of the antagonist TREM2 Ab E3C7 in human myeloid cells, purified CD14+ monocytes from the blood of three healthy donors were differentiated into macrophages in the presence of human M-CSF for 5 days. The resulting monocyte-derived macrophages (MDMs) were stimulated with the "M2" activating cytokine IL4 for 24 hours, resulting in MDM polarization into TAM-like cells. TREM2 expression was investigated by flow cytometric analysis of antibody binding of TREM2 Ab to the cell surface of these TAM-like cells (Figure 11). Cell cultures were treated with TREM2 Ab or comparable IgG control from day 3 onwards, followed by analysis of monocyte-to-TAM progression markers (qPCR) and protein secretion (ELISA).

[0367] Representative genes were selected from three major categories: (a) type I interferon activity-IFI6 (interferon alpha-inducible protein 6), (b) proinflammatory chemokines IL-8 and CCL23, and (c) S100 calcium-binding proteins A8 and A9 (S100A8 and S100A9), which are actively released during inflammation and play a crucial role by stimulating leukocyte recruitment and inducing cytokine secretion. As shown in Figure 12A-B, treatment of cell cultures with E3C7 induced the expression of CCL23, IFI6, S100A8, and S100A9 (A), as well as increased secretion of IL8 (B) in three out of three donors tested.

[0368] The results demonstrate that antagonist TREM2 Abs interfere with MDM differentiation and reprogram immunosuppressive TAMs into proinflammatory monocyte-like cells (also referred to herein as "M1-like macrophages").

[0369] To further characterize the effect of anti-TREM2 mAb on monocyte-derived macrophages treated with anti-TREM2 mAb, we profiled MDM cultures using single-cell RNA sequencing with two differentiation conditions (M-CSF only or M-CSF+IL4) and three treatments (anti-TREM2 E3C7, IgG control, or no mAb). 2d projections of cells from six conditions showed a specific IL-4 effect and a specific anti-TREM2 effect (Figure 13A-C). Increased expression of S100A8, CCL8, CCL23, and other proinflammatory genes was observed in cells treated with anti-TREM2 mAb, indicating a strong reprogramming to more immune-active M1-like macrophages (Figure 14). EXAMPLES

[0370] Anti-TREM2 antibodies attenuate tumor growth and reprogram tumor macrophages To test whether TREM2 blockade reprograms macrophages in vivo, tumor-bearing mice (MCA205 syngeneic model) were treated with anti-TREM2 antibody (E3C7) or IgG control. 500K MCA-205 cells were injected subcutaneously into humanized TREM2 mice, mice were treated with anti-TREM2 antibody or IgG control on days 6 and 9, tumors were harvested on day 10, and CD45+ positive cells were sequenced using scRNA-seq. Mice treated with anti-TREM2 (E3C7) showed an increased percentage of type I interferon TAMs compared to controls, while CD8 dysfunctional T cells (high PDCD1 and LAG3) were reduced in E3C7-treated animals compared to controls (Figure 15A-B). Differential gene expression analysis of E3C7-treated tumor macrophages compared to IgG controls revealed an increase in type I IFN genes, including Ifit1, Ifit2, and Irf7, in addition to the Ccl7, Ccl2, Ccl6, and Ccl12 chemokines (Figure 16). EXAMPLES

[0371] Cytokine conjugation of anti-TREM2 antibodies enhances myeloid reprogramming and T cell activation To investigate the synergistic effect of anti-hTREM2 antibodies and inflammatory cytokines, human monocyte-derived macrophages (hMDMs) were treated during differentiation with either anti-TREM2 antibodies, human IL2 (200-02-50, PeproTech), human IL15 (200-15-50, PeproTech), human GM-CSF (PeproTech, AF-315-03-1000), human IL12 (R&D, 10018-IL), or a combination of anti-TREM2 with one of the cytokines (Figure 17A-B). Gene expression analysis (Figure 17A) shows that monocyte genes (S100A9, S100A8) and inflammation-related genes (CCL23, IFI6, CCL18) were elevated after anti-TREM2 treatment and increased after treatment with a combination of anti-TREM2 and IL2 or IL15. In addition, secretion of inflammatory cytokines (FIG. 17B) was elevated following administration of anti-TREM2 or cytokines, and was increased following treatment with a combination of anti-TREM2 and IL2 or IL15.

[0372] Anti-TREM2 treated hMDMs exhibit a reduction in T cell suppressive capacity, as can be seen by the increased percentage of proliferating T cells in Figure 18A-B. Furthermore, this reduction is observed following the addition of IL2 or IL15 cytokines to either M2 macrophages or T cell-macrophage co-cultures.

[0373] Recombinant anti-human TREM2 antibodies were produced either unconjugated or conjugated to the human IL2 cytokine (the table in Figure 24 shows two versions, one with a short linker and one with a long linker, SEQ ID NOs: 496 and 498 or SEQ ID NOs: 497 and 499, respectively).

[0374] The binding properties of the generated antibodies against TREM2 were tested (Figure 19A-D). The conjugated antibodies were shown to bind to both recombinant TREM2 (Figure 19A) and soluble TREM2 (Figure 19D) similar to the recombinant anti-hTREM2 antibody. To confirm the presence of cytokines on the generated molecules, hIL2 was detected on the antibodies bound to hTREM2 (BLG-500302, BioLegend, Figure 19B and E).

[0375] The conjugated antibodies were then tested for their ability to activate primary human CD8+ and CD4+ cells (Figure 20A-B). Cells were cultured in the presence of antibody (10 μg / ml) or with recombinant protein (100 ng / ml), with or without activation.

[0376] The percentage of proliferating CD8+ cells was found to be increased by IL2-conjugated antibodies, similar to stimulation with recombinant IL2 (Figure 20A). This was true for both activated and non-activated CD8+ cells. Stimulation with recombinant non-conjugated anti-hTREM2 did not affect proliferation. IL2-conjugated antibodies stimulated IFN-g secretion by CD8+ cells compared to anti-TREM2 antibodies after 24 hours and 4 days of simulation, similar to stimulation with recombinant IL2 (Figure 20B). Similarly, IL2-conjugated antibodies stimulated IFN-g secretion by CD4+ cells compared to anti-TREM2 antibodies at 24 hours (Figure 20B).

[0377] To validate the ability of the antibodies to deliver IL2 cytokine to TREM2-expressing macrophages to exert the desired activity (enhancing T cell responses), in vitro experiments were performed as follows: Bone marrow-derived macrophages (BMDMs) were isolated from humanized TREM2 mice. At two time points, these cells were treated with anti-human TREM2 antibodies (both conjugated and not conjugated to human IL2), and then the ability of the variously treated BMDMs to suppress / activate T cell responses was tested.

[0378] Bone marrow-derived macrophages stimulated with IL-4 have an immunosuppressive phenotype (M2 state) and therefore inhibit T cell responses. IFNg release by T cells is a direct indicator of T cell activity and response, as T cells are more activated to release IFNg and vice versa. Thus, the results show that activated T cells co-cultured with untreated BMDMs were indeed more inhibited compared to the activated T cell only control. Furthermore, BMDMs treated with anti-human TREM2 (both recE3C7 and E3C7) did not inhibit T cells as much as the untreated control, and then the isotype IgG control, and the IgG isotype control did not produce the same effect (Figure 21). Furthermore, the results showed that conjugated anti-TREM2-IL2 treatment (both the short and long forms of human IL2) enhanced T cell responses 4.2-5 fold higher when compared to activated T cells co-cultured with recE3C7 BMDMs (Figure 21). EXAMPLES

[0379] Targeting tumor-associated macrophages (TAMs) with TREM2 CAR-T cells We generated CART cells aimed at recognizing and depleting TREM2-expressing myeloid cells. In this setting, we used the high affinity (low nanomolar to picomolar range) recognition domain of the 80E3C7 monoclonal Ab. Results demonstrated that TREM2-CAR T cells can mount an effective cytotoxic response and deplete human TREM2-expressing myeloid cells and immunosuppressive macrophages.

[0380] First, a TREM2-expressing cell line was used to verify that TREM2-CAR T cells recognize TREM2. TREM2-CAR T cells were co-cultured with HEK293 overexpressing TREM2 at a 1:1 ratio (Figure 22A-B). As positive controls, activated TREM2-CAR T was used with CD3 / CD28 beads and CD19-CAR cultures were used with A20 cell line. Additionally, mock CAR transduced T cells (BFP T cells) and HEK293 with no TREM2 expression were used. IFN-γ ELISA was performed after 24 hours, showing dramatic IFN-γ secretion of CAR-TREM2 T cells when co-cultured with HEK293 overexpressing TREM2. Results were verified using flow cytometry with markers for activation (CD25 and PD1) and death (CD107) showing a significant increase consistent only with CD19-CAR T cell and bead activation. No non-specific response with HEK293 was observed. These results demonstrated that the CAR-TREM2 T cell response to TREM2 was highly specific and potent.

[0381] To validate this finding in a human environment, CD14+ monocytes were purified from human blood and the cells were differentiated into TAM-like and dendritic cells for one week. TREM2-CAR T cells were cultured at a 1:1 ratio with both products and undifferentiated human CD14+ (Figure 22C-E). As a positive control, activated TREM2-CAR T cells were used with CD3 / CD28 beads and also cultured with HEK293 TREM2+. As before, mock CAR-transduced T cells (BFP T cells) were used as a negative control. As shown in Figure 22C, after 24 hours, it was observed that all macrophages exhibiting human TAM-like cells cultured with TREM2-CAR T cells were completely wiped out, while mock T cells did not show any response. IFNg ELISA and flow cytometry analysis were performed again as described above. The results indicate the high specificity and cytotoxicity of TREM2-CAR T cells in depleting human TAM-like cells, even in a human environment.

[0382] To further validate the specificity and efficacy of TREM2-CAR T cells, humanized TREM2 mice, TREM2 KO We compared BMDMs and BMDCs (Figure 12F) differentiated from bone marrow-derived cells of humanized TREM2 mice and wt mice. The same experimental setup as above was used. The results showed a significantly stronger killing response only when TREM2-CAR T cells were cultured with differentiated cells from humanized TREM2 mice.

[0383] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0384] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

[0385] Additionally, any priority documents to this application are incorporated herein by reference in their entirety.

Claims

1. An antibody or a fragment thereof comprising an antigen recognition domain capable of binding to triggering receptor expressed on myeloid cells 2 (TREM2), wherein the antigen recognition domain is 80E3C7 23A10A10 32F9E8 38C11H11 49A12D7 58B2A7 60A4F5 60H4A3 61B11C9 80E3H11 83E10B12 54H2C1 54H2C1B 23A10B10 23A10B11 38C11C10 60A4E10 60H4G2 CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 which are complementarity determining regions (CDRs) of an antibody selected from the group consisting of, or an antibody or a fragment thereof comprising a heavy chain and a light chain.

2. An antibody or a fragment thereof comprising an antigen recognition domain capable of binding to transmembrane glycoprotein NMB (GpnmB), wherein the antigen recognition domain is g1 - g2 g2 - b6 g3 - g2 g4 - b4 g5 - g2 g8 - g2 g9 - b4 b1 - b2 b8 - b8 b10 - b9 b11 - g2 b12 - y8 b13 - b7 b15 - b7 b17 - b17 b18 - b19 b2 - b2 b20 - b21 b21 - y8 b22 - b23 b24 - b26 b25 - b26 y3 - y22 y4 - y3 y5 - y5 y9 - y6 y12 - b4 y20 - y19 y23 - y20 y25 - y21 y27 - y22 CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 which are complementarity determining regions (CDRs) of an antibody selected from the group consisting of, or an antibody or a fragment thereof comprising a heavy chain and a light chain.

3. The antibody or a fragment thereof according to claim 1 or 2, wherein the antibody has an inactivated effector function or no effector function.

4. The antibody or a fragment thereof according to claim 1 or 2, wherein the antibody is IgG1.

5. The antibody or a fragment thereof according to claim 1 or 2, formulated as an antibody-drug conjugate (ADC).

6. A bispecific antibody comprising the antigen recognition domain of the antibody according to claim 1 or 2 in at least one arm.

7. A bispecific antibody comprising the antibody according to claim 1 in one arm and the antibody according to claim 2 in the other arm.

8. The bispecific antibody according to claim 6, wherein the effector function is inactivated or has no effector function.

9. The bispecific antibody according to claim 6, wherein the antibody is IgG1.

10. The bispecific antibody according to claim 6, formulated as an antibody-drug conjugate (ADC). **Claim 11**: The antibody or fragment thereof according to claim 1 or 2, wherein the fragment forms a chimeric antigen receptor (CAR). **Claim 12**: A cell expressing the fragment of the antibody according to claim 11. **Claim 13**: A product comprising the antibody or fragment thereof according to claim 1 or 2. **Claim 14**: A pharmaceutical composition comprising the antibody or fragment thereof according to claim 1 or 2 and a pharmaceutically acceptable carrier or diluent. **Claim 15**: A pharmaceutical composition comprising the bispecific antibody according to claim 6 and a pharmaceutically acceptable carrier or diluent. **Claim 16**: A pharmaceutical composition comprising the cell according to claim 12 and a pharmaceutically acceptable carrier or diluent. **Claim 17**: A method for reducing the immunosuppressive activity of myeloid cells ex vivo, comprising contacting myeloid cells with an effective amount of the antibody or fragment thereof according to claim 1 or 2, thereby reducing the immunosuppressive activity of the myeloid cells. **Claim 18**: A method for reducing the immunosuppressive activity of myeloid cells ex vivo, comprising contacting myeloid cells with an effective amount of the bispecific antibody according to claim 6, thereby reducing the immunosuppressive activity of the myeloid cells. **Claim 19**: A method for reducing the immunosuppressive activity of myeloid cells ex vivo, comprising contacting myeloid cells with an effective amount of the cell according to claim 12, thereby reducing the immunosuppressive activity of the myeloid cells. **Claim 20**: A method for activating CD4 T cells ex vivo, comprising contacting CD4 T cells with an effective amount of the antibody or fragment thereof according to claim 2, thereby activating the CD4 T cells. **Claim 21**: A method for killing myeloid cells expressing TREM2 ex vivo, comprising contacting a cell population comprising myeloid cells expressing TREM2 with an effective amount of the cell according to claim 12, thereby killing the myeloid cells expressing TREM2. **Claim 22**: The antibody or fragment thereof according to claim 1 or 2, for use in the treatment of cancer in a subject in need thereof. **Claim 23**: The bispecific antibody according to claim 6, for use in the treatment of cancer in a subject in need thereof. The cell according to claim 12, which is for treating cancer in a subject in need of cancer treatment.

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