Lilrb2 antibody products and methods

Abstract

The present disclosure relates to LILRB2 antibody products and methods of use thereof. The antibody product specifically binds to LILRB2 on cells, such as myeloid cells or cancer cells. The antibody products can be used in methods of treating disease, such as methods of cancer immunotherapy.

Description

[0001] Cross-reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 380,034, filed Oct. 18, 2022, U.S. Provisional Application No. 63 / 490,705, filed Mar. 16, 2023, and U.S. Provisional Application No. 63 / 514,057, filed Jul. 17, 2023, the entireties of which are incorporated herein by reference.

[0003] Incorporation of Sequence Listing by Reference

[0004] This application contains a Sequence Listing (filename: 58365_Seqlisting.XML; 100,193 bytes; date: Oct. 13, 2023) in computer-readable form as a separate part of this disclosure, which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0005] The present disclosure relates to LILRB2 antibody products and related methods. The products disclosed herein bind to LILRB2 on cells such as macrophages or cancer cells. The products can be used in methods of treating diseases, such as methods of treating cancer. BACKGROUND ART

[0006] Cancer is characterized by the accumulation of growth-modifying genetic alterations and the ability to evade detection or elimination by the immune system. Cancer immunotherapy allows for the overcoming of this immune tolerance, resulting in immune recognition and anti-tumor responses. However, tumors typically use multiple mechanisms to mediate immune escape, such as the reduction or loss of tumor antigenicity, immune escape, or the presence of inhibitory cells or cytokines in the tumor microenvironment (TME) that inhibit the killing of cancer cells by CD8+ T cells.

[0007] Recently developed antibodies targeting immune checkpoints have revolutionized the way malignant solid tumors are treated and have raised the hope that even subjects with advanced disease may achieve cure or long-term remission. Checkpoint inhibitor (CPI) antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1), and programmed death ligand-1 (PD-L1) have now established clear efficacy for first-line treatment of metastatic disease in multiple tumor types, including lung, melanoma, triple-negative breast cancer, head and neck, and others.

[0008] Despite this success, most subjects treated with these CPIs either do not respond or eventually progress or relapse. In addition, CPIs have failed to improve the outcomes of those subjects with tumors that are not infiltrated by immune cells (so-called "cold" tumors). This lack of responsiveness and treatment failure in cold tumors is thought to be due in part to the immunosuppressive TME. Immunosuppressive myeloid cells such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) are important components of the TME and contribute to immune escape by many solid tumors. MDSC and TAM suppress the anti-tumor immune response and promote the tumorigenic environment. High levels of tumor infiltration by MDSC and TAM generally portend a poor prognosis for subjects with solid tumors. Mitigating the immunosuppression of myeloid cells in the TME to improve T cell-mediated responses is a rational adjunct to CPI therapy.

[0009] Leukocyte immunoglobulin-like receptor B2 (LILRB2, also known as ILT4, LIR2, MIR-10, CD85d) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing immunosuppressive member of the leukocyte immunoglobulin-like receptor family that is expressed on myeloid cells (monocytes, macrophages, dendritic cells, and granulocytes) but not on lymphocytes. In the TME, LILRB2 is present on myeloid-derived suppressor cells (MDSC) and tumor-supporting tumor-associated macrophages (TAM). LILRB2 appears to have a dual role in cancer biology, serving as an immune checkpoint on myeloid cells and as a tumor-supporting factor when expressed on tumor cells. Trans or cis interactions of LILRB2 with its ligands mediate immunosuppression by myeloid cells and promote tumor immune escape in the TME. Such ligands include human leukocyte antigen G (HLA-G), which belongs to the non-classical MHC class I molecules, and classical HLA class I molecules HLA-A and HLA-B. Targeting this pathway in the TME may enhance the efficacy of T cell checkpoint inhibitors. In addition, ANGPTL2 and ANGPTL5 promote lung cancer development and survival via tumor-expressed LILRB2 by signaling LILRB2 expression through SHP1 on myeloid cells in the TME or via HLA-G expression by tumors that is associated with poor survival in multiple cancers.

[0010] Recent data indicate that the combination of LILRB2 blockade with cytokines (e.g., IL-4, IL-10) or innate differentiation / polarization signals promotes the development of anti-tumor myeloid cells in vitro. In addition, the combination of anti-LILRB2 antibody with anti-PD-1 therapy enhanced tumor growth inhibition (TGI) in a humanized tumor model.

[0011] Antibodies that target and antagonize LILRB2 are currently being evaluated in clinical trials for the treatment of cancer, such as MK-4830 (IgG4) (Agenus and Merck) and JTX-8064 (IgG4) (Jounce). Preliminary clinical data for the first-in-class anti-LILRB2 MK-4830 indicate that LILRB2 blockade abrogates the PD-1 resistance mechanism in patients with advanced solid tumors. MK-4830 is well tolerated when administered as a single agent or in combination with pembrolizumab (anti-PD-1) and has shown dose-dependent evidence of target engagement and anti-tumor activity in patients lacking predictive biomarkers associated with response to anti-PD-1 monotherapy. The overall response rate for the MK-4830 / pembrolizumab combination treatment was 24%. The MK-4830 clinical trial data support the development of anti-LILRB2 antibodies in combination with CPI therapies.

[0012] PCT Publication No. WO 2021 / 138079 A1 discloses the use of a combination of a PD-1 antagonist, an ILT4 (LILRB2) antagonist, and lenvatinib (a kinase inhibitor) for the treatment of cancer.

[0013] There is a need in the art for LILRB2 antibody products and methods for their therapeutic use. SUMMARY OF THE INVENTION

[0014] The present disclosure provides LILRB2 antibody products (sometimes also referred to herein as "anti-LILRB2 antibody products" herein), compositions comprising such antibody products, and methods for their preparation and use in the treatment of diseases including cancer.

[0015] The LILRB2 antibody products provided herein can be full antibodies or can comprise antibody fragments having immunological function, and thus the LILRB2 antibody products include antibodies having a naturally occurring or recombinant structure, as well as other polypeptides having an antigen-binding domain, such as antibody fragments. Nucleic acid molecules, vectors, and host cells useful for producing the antibody products are also provided. The antibody products and their compositions can be used to prevent or treat a variety of different disease conditions, including but not limited to preventing or treating disease conditions such as cancer. The LILRB2 antibodies provided herein have a useful half-life and exhibit anti-tumor activity in a humanized mouse model. The humanized LILRB2 antibodies provided herein restore the effector function of activated and exhausted T cells by M2c-mediated immunosuppression and enhance the secretion of pro-inflammatory cytokines by M0 macrophages or LPS-stimulated PBMCs.

[0016] It is believed that the LILRB2 antibody products provided herein bind to LILRB2 at epitopes different from those bound by antibodies currently in clinical use. In preclinical studies, the antibody products enhanced LPS-induced IFN-γ production, reduced IL-10 release by peripheral blood mononuclear cells (PBMCs), and alleviated the immunosuppression of tumor-promoting macrophages to induce T cell proliferation and IFN-γ and perforin secretion by said T cells. In addition, the antibody products inhibited the development of immunosuppressive macrophages. Compared with a control LILRB2 antibody, a representative antibody product has demonstrated the ability to restore the ability of exhausted T cells to secrete IFN-γ in the presence of tumor-promoting macrophages, and also significantly enhanced the activity of pembrolizumab in combination studies. Importantly, in a humanized mouse tumor model, the chimeric antibody product demonstrated superior anti-tumor activity, with significant tumor growth inhibition and tumor regression.

[0017] Without wishing to be bound by any particular theory, it appears that the anti-immunosuppressive activity of the antibodies provided herein stems from two distinct but coordinated mechanisms: the involvement of the variable region of LILRB2 and the involvement of the heavy chain of the Fc receptor.

[0018] On the one hand, it is believed that LILRB2 expressed on myeloid cells negatively regulates anti-tumor immunity by binding to HLA-G on tumor cells and MHC class I on myeloid cells. Binding of LILRB2 to these ligands promotes immunosuppressive myeloid cells in the tumor microenvironment by inhibiting Ca 2+ signaling across the cell membrane of myeloid cells, recruiting Src homology 2 domains containing protein tyrosine phosphatase-1 (SHP1) and SHP2 phosphatases within the myeloid cell membrane, and producing cytokines by myeloid cells, which can prevent checkpoint inhibitor (CPI) therapy from enhancing anti-tumor T cell responses. Individually or in combination, these phenomena can result in reduced killing of cancer cells. It is also believed that LILRB2 further hinders tumor cell killing by competing with cytotoxic T lymphocytes for binding to MHC class I.

[0019] On the other hand, it is known that activation of Fc receptors (FcγRIA, FcγRIIA, and FcγRIIIA) on myeloid cells plays a crucial role in promoting cell activation, differentiation, and induction of adaptive immune responses by regulating antigen presentation. These receptors signal via SRC family kinases and spleen tyrosine kinase through immunoreceptor tyrosine-based activation motifs, resulting in transcriptional activation of several pro-inflammatory cytokines and chemokines, which drive cell movement, migration, differentiation, and survival.

[0020] The antibodies disclosed herein appear to be able to couple LILRB2 antagonism to Fc receptor-mediated activation. Binding of the antibody to a unique epitope on LILRB2 not only blocks the interaction of myeloid cells with tumor cell HLA-G but also blocks cis-interaction with MHC class I, thereby inactivating both inhibitory signals in myeloid cells. This effect can promote the polarization of tumor-infiltrating myeloid cells towards an inflammatory phenotype while releasing MHC class I on these cells to engage receptors required for optimal activation of cytotoxic T lymphocytes. In addition, the antibody can enhance anti-tumor immunity by participating in the activation of FcγRIIIA to provide an immune-stimulatory signal. This dual or co-participation mechanism provides these antibodies with a new property of targeting myeloid cells to reverse CPI resistance, enhance tumor cell killing and improve patient prognosis.

[0021] Non-clinical data of representative antibody products demonstrated typical human IgG PK characteristics in humanized FcRn mice. The antibody products did not cross-react with non-human primate LILRB2 and did not show cross-reactivity to other inhibitory or activating LILR family members. Treatment with representative antibody products did not trigger the release of inflammatory cytokines or activation of neutrophils in human whole blood, and representative antibody products did not deplete human monocytes or neutrophils, thus indicating a tolerable safety profile.

[0022] The present disclosure provides an antibody product that binds to human LILRB2, the antibody product comprising CDR-H1 as shown in SEQ ID NO:16, CDR-H2 as shown in SEQ ID NO:17, CDR-H3 as shown in SEQ ID NO:24, CDR-L1 as shown in SEQ ID NO:19, CDR-L2 as shown in SEQ ID NO:20, and CDR-L3 as shown in SEQ ID NO:21. The antibody product may comprise CDR-H1 as shown in SEQ ID NO:16, CDR-H2 as shown in SEQ ID NO:17, CDR-H3 as shown in SEQ ID NO:18, CDR-L1 as shown in SEQ ID NO:19, CDR-L2 as shown in SEQ ID NO:20, and CDR-L3 as shown in SEQ ID NO:21. The antibody product may comprise CDR-H1 as shown in SEQ ID NO:22, CDR-H2 as shown in SEQ ID NO:23, CDR-H3 as shown in SEQ ID NO:24, CDR-L1 as shown in SEQ ID NO:25, CDR-L2 as shown in SEQ ID NO:26, and CDR-L3 as shown in SEQ ID NO:21. The antibody product may comprise a heavy chain variable region comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:1, 6, 7, 8, 9, or 10; or (b) the amino acid sequence shown in SEQ ID NO:1, 6, 7, 8, 9, or 10. The antibody product may comprise a light chain variable region comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 11, 12, 13, 14, or 15; or (b) the amino acid sequence shown in SEQ ID NO:2, 11, 12, 13, 14, or 15. The antibody product may comprise: (a) a heavy chain variable region comprising SEQ ID NO:1; and a light chain variable region SEQ ID NO:2; (b) a heavy chain variable region SEQ ID NO:6; and a light chain variable region comprising SEQ ID NO:11; (c) a heavy chain variable region comprising SEQ ID NO:6; and a light chain variable region comprising SEQ ID NO:12; (d) a heavy chain variable region comprising SEQ ID NO:6; and a light chain variable region comprising SEQ ID NO:13; (e) a heavy chain variable region comprising SEQ ID NO:6; and a light chain variable region comprising SEQ ID NO:14; (f) a heavy chain variable region comprising SEQ ID NO:6;and a light chain variable region, the light chain variable region comprising SEQ ID NO:15; (g) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:7; and a light chain variable, the light chain variable comprising SEQ ID NO:11; (h) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region, the light chain variable region comprising SEQ ID NO:12; (i) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region, the light chain variable region comprising SEQ ID NO:13; (j) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:7; a light chain variable region, the light chain variable region comprising SEQ ID NO:14; (k) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region, the light chain variable region comprising SEQ ID NO:15; (l) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region, the light chain variable region comprising SEQ ID NO:11; (m) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region, the light chain variable region comprising SEQ ID NO:12; (n) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region, the light chain variable region comprising SEQ ID NO:13; (o) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region, the light chain variable region comprising SEQ ID NO:14; (p) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region, the light chain variable region comprising SEQ ID NO:15; (q) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:9; a light chain variable region, the light chain variable region comprising SEQ ID NO:11; (r) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region, the light chain variable region comprising SEQ ID NO:12; (s) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region, the light chain variable region comprising SEQ ID NO:13; (t) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region, the light chain variable region comprising SEQ ID NO:14; (u) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region, the light chain variable region comprising SEQ ID NO:15; (v) a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region, the light chain variable region comprising SEQ ID NO:11;(w) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:12; (x) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:13; (y) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:14; or (z) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:15.;

[0023] The antibody product may comprise a heavy chain comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH). The antibody product may comprise a light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL). The antibody product may comprise a heavy chain and a light chain, the heavy chain comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH), and the light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL). The antibody product may comprise a heavy chain region comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:32; or (b) the amino acid sequence set forth in SEQ ID NO:32. The antibody product may comprise a light chain region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:42; or (b) the amino acid sequence set forth in SEQ ID NO:42. An illustrative antibody product comprises a heavy chain and a light chain region, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO:32 and the light chain region comprising the amino acid sequence set forth in SEQ ID NO:42.

[0024] The antibody product may comprise an IgA, IgD, IgE, IgG or IgM heavy chain constant domain. The antibody product may comprise an IgG1 constant domain, an IgG2 constant domain or an IgG4 constant domain. The antibody product may comprise an IgG1 constant domain. The antibody product may comprise an IgG1 heavy chain amino acid sequence, the IgG1 heavy chain amino acid sequence comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:3, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32; or (b) the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32. The antibody product may comprise an IgG4 constant domain. The antibody product may comprise an IgG4 heavy chain amino acid sequence, the IgG4 heavy chain amino acid sequence comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37; or (b) the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37.

[0025] The antibody product may comprise: (a) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:1; and a κ light chain having an amino acid sequence comprising SEQ ID NO:2; (b) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (c) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (d) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (e) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (f) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (g) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (h) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (i) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (j) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (k) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (l) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (m) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12;(n)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (o)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (p)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (q)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (r)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (s)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (t)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (u)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (v)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (w)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (x)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (y)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; or (z)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15.;

[0026] The antibody product can specifically bind to human LILRB2 with a KD of, for example, 0.5 nM to 500 nM.

[0027] The antibody product can specifically bind to human LILRB2 expressed by myeloid cells or cancer cells, such as cells expressing LILRB2. The antibody product can specifically bind to human immunosuppressive myeloid cells. The immunosuppressive myeloid cells can be in the tumor microenvironment. The immunosuppressive myeloid cells can be macrophages, myeloid dendritic cells, or myeloid-derived suppressor cells. The immunosuppressive myeloid cells can be M2a, M2b, M2c, or M2d macrophages. The antibody product can specifically bind to human M2c macrophages with a KD of, for example, 0.5 nM to 500 nM.

[0028] The antibody product can be a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single-chain antibody. The antibody product can be a monospecific, bispecific, trispecific, or multispecific antibody.

[0029] The antibody product can be bound by Fc receptors expressed on immunosuppressive macrophages or other myeloid cells. The antibody product can be bound by CD16 (FcγRIII) (e.g., FcγRIIIa, FcγRIIIb), CD32 (FcγRII), or CD64 (FcγRI) expressed on immunosuppressive macrophages or other myeloid cells.

[0030] The present disclosure provides a method of providing cancer immunotherapy to a subject in need thereof, wherein the cancer is associated with the presence of immunosuppressive macrophages, the method comprising administering to the subject a therapeutically effective amount of an antibody product provided herein. In the method, the antibody product binds to macrophages, and the binding of the antibody product to macrophages can produce at least one of the following effects: (a) promoting the activation of CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof; (b) promoting the proliferation of CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof; (c) preventing macrophages from polarizing into an immunosuppressive phenotype; and (d) enhancing the innate anti-tumor response. The activation of the CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof can be measured as an increase in the level of IFN-γ, TNF-α, or perforin, or any combination thereof. In the method, the binding of the antibody product to macrophages may or may not be cytotoxic to the macrophages. In the method, the binding of the antibody product to macrophages can produce at least one of the following effects: (a) internalization of the antibody product by the macrophages; (b) secretion of TNFα, IL-6, perforin, or any combination thereof; (c) reducing the release of IL-10; (d) activating CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof; (e) proliferating CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof; and (f) promoting tumor cell killing in the tumor microenvironment. The method can produce: two or more of (a) to (f); three or more of (a) to (f); four or more of (a) to (f); five or more of (a) to (f); or all of (a) to (f). In the method, the binding of the antibody product to macrophages can increase the immunostimulatory activity in the tumor microenvironment. The binding of the antibody product to macrophages can reduce the immunosuppressive activity of the macrophages. The binding of the antibody product to macrophages can reduce the pro-tumor activity of the macrophages. The binding of the antibody product can promote CD4+ T cell activation, CD4+ T cell proliferation, or both CD4+ T cell activation and proliferation. The binding of the antibody product can promote CD8+ T cell activation, CD8+ T cell proliferation, or both CD8+ T cell activation and proliferation. The binding of the antibody product can promote cytotoxic lymphocyte-mediated cancer cell killing. The binding of the antibody product can promote NK cell-mediated tumor cell killing. The binding of the antibody product to macrophages can reduce the inhibition of cytotoxic T cell-mediated tumor cell killing in the tumor microenvironment. For example, the cancer can be sarcoma, carcinoma, or hematological cancer.For example, the cancer can be glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, testicular germ cell carcinoma, gastric cancer, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid carcinoma, cutaneous squamous cell carcinoma, or ovarian cancer. The cells of the cancer can express or overexpress LILRB2.

[0031] The methods provided herein can further comprise administering to the subject an effective amount of an anti-cancer therapeutic agent. The anti-cancer therapeutic agent can be an immune checkpoint inhibitor including but not limited to a PD-1 antagonist. The effective amount of the PD-1 antagonist can be an amount effective to relieve the immunosuppression of T cells. The immunosuppression of the T cells can be mediated by the interaction of the T cells with myeloid cells expressing PD-L1.

[0032] The present disclosure provides a composition comprising (a) an antibody product provided herein, and (b) an excipient. The present disclosure provides an article of manufacture comprising the composition provided herein and a container.

[0033] The present disclosure contemplates the use of an antibody product or composition provided herein for the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer expresses LILRB2.

[0034] The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a part or all of an antibody product provided herein. The present disclosure provides an expression vector comprising the nucleic acid provided herein. The present disclosure provides a host cell comprising the expression vector provided herein.

[0035] The present disclosure provides a method for generating an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain, the method comprising: (a) growing the host cell provided herein under conditions such that the host cell expresses a protein comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region; and (b) purifying the protein comprising the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain.

[0036] The present disclosure provides a pharmaceutical composition comprising an antibody product provided herein and a pharmaceutically acceptable excipient. The pharmaceutical composition can be used to treat a subject suffering from cancer expressing LILRB2.

[0037] The present disclosure provides a method for detecting LILRB2 in a sample, tissue, or cell using the antibody products provided herein, the method comprising contacting the sample, the tissue, or the cell with the antibody products and detecting the antibody products.

[0038] The present disclosure provides a method for reducing the biological activity of LILRB2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody products or pharmaceutical compositions provided herein. In the method, the antibody products can mediate the depletion of at least one cancer cell expressing LILRB2.

[0039] The present disclosure provides a method for promoting an immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of the antibody products or pharmaceutical compositions provided herein.

[0040] The present disclosure provides a method for providing cancer immunotherapy to a subject in need thereof, wherein the cancer cells of the subject express LILRB2, the method comprising administering to the subject a therapeutically effective amount of the antibody products provided herein. The method can comprise administering an amount of the antibody products capable of effectively mediating cytotoxicity of the cancer through antibody-dependent cell cytotoxicity. The method can comprise administering an amount of the antibody products capable of effectively alleviating LILRB2-mediated T cell inhibition in the subject. The method can further comprise administering to the subject an amount of a PD-1 antagonist sufficient to alleviate PD-1 / PD-L1 axis-mediated T cell immunosuppression in the subject. The PD-1 antagonist can be a PD-1 antibody product.

[0041] The antibody products provided herein can be detectably labeled or can comprise conjugated toxins, drugs, receptors, enzymes, receptor ligands. The antibody products can comprise therapeutic agents or cytotoxic agents.

[0042] A pharmaceutical composition comprising the antibody products provided herein and a physiologically acceptable carrier or excipient, the pharmaceutical composition can reduce or prevent the binding of LILRB2 to its ligand and / or reduce or prevent signal transduction mediated by LILRB2. The ligand can be human leukocyte antigen A, human leukocyte antigen B, human leukocyte antigen C, human leukocyte antigen G, angiopoietin-like protein 2, angiopoietin-like protein 5, or a combination thereof. The ligand is expressed on the surface of myeloid cells or tumor cells.

[0043] The present disclosure provides methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject an effective amount of a pharmaceutical composition provided herein, wherein the subject has a cancer comprising cells expressing or overexpressing a ligand of LILRB2. In the methods, the antibody product or an antigen-binding fragment thereof increases the immune response, delays or arrests tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages to alter their activity, reduces tumor-associated macrophage-mediated immunosuppression, reduces or reverses T cell inhibition, or any combination thereof. The cancer or tumor may comprise macrophages expressing LILRB2. The methods may further comprise administering to the subject a second therapeutic agent. The second therapeutic agent may be an immune checkpoint inhibitor.

[0044] The following figures and detailed description, including examples, illustrate various non-limiting aspects of the subject matter contemplated herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Aspects of the present disclosure are illustrated by the following figures.

[0046] Figure 1A -B: Binding of B2A-IgG1 and B2A-IgG4 chimeras to human and cynomolgus monkey LILRB2 Fc on HEK293 cells expressing LILRB2, detected by ELISA.

[0047] Figure 2 : Blocking of LILRB2 binding to human ANGPTL-2, -5 by B2A-IgG4, detected by ELISA.

[0048] Figure 3A -C: Binding curves of human anti-LILRB2 chimeric antibodies to human monocytes and M0 and M2c macrophages.

[0049] Figure 4 : Enhanced IFN-γ secretion by LPS-stimulated PBMCs after treatment with anti-LILRB2 chimeric antibodies B2A-IgG1 and B2A-IgG4.

[0050] Figure 5 : Enhanced TNF-α secretion by human M0 macrophages after treatment with chimeric B2A-IgG1 and B2A-IgG4 antibodies in a CD40 ligand assay.

[0051] Figure 6A -B: Rescue of CD8+ T cell proliferation by chimeric LILRB2 antibodies in an M2c and CD8+ T cell co-culture assay, and rescue of IFN-γ and perforin responses by chimeric LILRB2 antibodies in an M2c and CD8+ T cell co-culture assay.

[0052] Figure 7 : The chimeric B2A-IgG1 LILRB2 antibody rescues IFN-γ release by exhausted T cells from M2c-mediated immunosuppression.

[0053] Figure 8 : Pharmacokinetic characteristics of chimeric B2A-IgG4 and B2A-IgG1 in humanized FcRn mice.

[0054] Figure 9A -B: Antitumor efficacy of chimeric B2A-IgG4 in humanized NSG-SGM3 mice and subcutaneous SK-MEL-5 human melanoma tumor model.

[0055] Figure 10 : Binding curves of humanized LILRB2 variants to monocytes, M0, and M2c macrophages.

[0056] Figure 11 : Humanized LILRB2 antibodies bind to multiple human myeloid cells including classical, intermediate, and non-classical monocytes and myeloid dendritic cells.

[0057] Figure 12 : Humanized LILRB2 variants bind to human neutrophils.

[0058] Figure 13 : The humanized anti-LILRB2 variant B2H1-55 does not trigger neutrophil activation in the whole blood of healthy subjects.

[0059] Figure 14 : Humanized LILRB2 antibodies trigger IFN-γ secretion by LPS-stimulated PBMCs.

[0060] Figure 15 : Rescue of CD8+ T cell proliferation and cytokine responses with humanized LILRB2 antibodies in M2c and CD8+ T cell proliferation assays.

[0061] Figure 16 : Humanized LILRB2 variants prevent the development of immunosuppressive macrophages.

[0062] Figure 17A -B: Relief of M2c-mediated immunosuppression in M2c / CD4+ T cell co-cultures with the selected humanized variant B2H1-55.

[0063] Figure 18 : Humanized LILRB2 antibodies rescue IFN-γ release by exhausted T cells from M2c-mediated immunosuppression.

[0064] Figure 19: Humanized anti-LILRB2 variant B2H1-55 in combination with a PD-1 antibody rescues IFN-γ release by exhausted T cells from M2c-mediated immunosuppression.

[0065] Figure 20 : Humanized anti-LILRB2 variant elicits minimal cytokine responses in whole blood of healthy subjects.

[0066] Figure 21A -B: Humanized anti-LILRB2 variant does not mediate ADCC of human monocytes and induces ADCC on HEK293 expressing human LILRB2.

[0067] Figure 22 : Pharmacokinetic characteristics of IgG1 humanized anti-LILRB2 variant in humanized FcRn mice.

[0068] Figure 23 : Humanized anti-LILRB2 variant inhibits LPS-mediated IL-10 secretion by human PBMC. Detailed Description

[0069] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0070] Antibody Product

[0071] The present disclosure provides antibody products that specifically bind to human LILRB2.

[0072] "PD-1 antagonist" means any compound or biomolecule that blocks the binding of PD-1 expressed on immune cells (T cells, B cells or NKT cells) to PD-L1 expressed on cancer cells, and preferably also blocks the binding of PD-1 expressed by immune cells to PD-L2 expressed on cancer cells. Alternative names or synonyms are given for PD-1 and its ligands. For PD-1: PDCD1, PD1, CD279 and SLEB2; for PD-L1: PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H; and for PD-L2: PDCD1L2, PDL2, B7-DC, Btdc and CD273. In any method of treatment, drug and use in a method of treatment, drug and use that has been disclosed in which a human individual is to be treated, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD-1. The human PD-1 amino acid sequence can be found in NCBI locus number: NP 005009. The human PD-L1 and PD-L2 amino acid sequences can each be found in NCBI locus numbers: NP 054862 and NP 079515.

[0073] "LILRB2 antagonist" means any compound or biomolecule that blocks the binding of LILRB2 to HLA-G, HLA-A, HLA-B, HLA-F, or angiopoietin-like protein (ANGPTL, such as ANGPTL2 or ANGPTL5). Alternative names or synonyms for LILRB2 and its ligands include, but are not limited to: ILT4 for ILT4, ILT-4, MIR10, MIR-10, LIR2, LIR-2, CD85D; MHC-G or major histocompatibility complex class I G for HLA-G; major histocompatibility complex class I A for HLA-A; AS, B-4901, major histocompatibility complex class I B for HLA-B; CDA12, HLA-CDA12 or major histocompatibility complex class I F for HLA-F; angiopoietin-3, ANG3, ANGPT3, ARP1, UNQ162, angiopoietin-like 2 for ANGPTL2; ARP4, HF ARP, PGAR, UNQ171, angiopoietin-like 54 for ANGPTL5; and CDT6. In any method of treatment, drug, and use in the methods of treatment, drugs, and uses that have been disclosed in which a human individual is to be treated, the LILRB2 antagonist blocks the binding of human LILRB2 to human HLA-G, HLA-A, HLA-B, HLA-C, HLA-F, ANGPTL2, or ANGPTL5. The human LILRB2 precursor amino acid sequence can be found in NCBI locus number: AAB88119.1. The human HLA-G, HLA-A, HLA-B, HLA-C, and HLA-F precursor amino acid sequences can each be found in NCBI locus numbers: P17693.1, P04439.2, P01889.3, P10321-1, and P30511.3. The human ANGPTL2 and ANGPTL5 precursor amino acid sequences can each be found in NCBI locus numbers Q9UKU9-1 and Q86XS5.

[0074] The protein sequence of human LILRB2 (NM_005874.5) is shown in SEQ ID NO:47. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is present in a gene cluster at chromosomal region 19q13.4. The encoded protein belongs to the class B subfamily of LIR receptors and contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits the stimulation of the immune response. The receptor is thought to control inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity. Multiple transcript variants encoding different isoforms of this gene have been found. The nucleotide sequence encoding human cDNA of LILRB2 (NM_005874.5) is shown in SEQ ID NO:53.

[0075] The terms "polypeptide" and "protein" are used interchangeably herein in a conventional manner to refer to a molecule formed of amino acids. A polypeptide is not limited to a specific length. Peptides are included in polypeptides unless otherwise specifically stated. This term does not refer to nor exclude post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art, both naturally occurring and non-naturally occurring. Polypeptides of interest in the context of the antibodies of the present disclosure include, but are not limited to, polypeptide fragments containing CDRs that are capable of binding to LILRB2 proteins expressed by myeloid cells or cancer cells.

[0076] The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. The length of the fragment is about 5 to 500 amino acids. For example, the length of the fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of LILRB2 antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of heavy or light chains, a portion of an antibody chain, or its variable region including only two CDRs, etc.

[0077] The term "isolated protein" as used herein means a subject protein that (1) is free of at least some of the other proteins normally found, (2) is substantially free of other proteins from the same source, (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) is operably associated (by covalent or non-covalent interactions) with a polypeptide that is not naturally associated with it, or (6) does not exist in nature. Genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof, of synthetic origin can encode such isolated proteins. Preferably, the isolated protein is substantially free of proteins or polypeptides or other contaminants that are present in its natural environment and that would interfere with its therapeutic, diagnostic, prophylactic, research, or other uses.

[0078] A "variant" of a polypeptide (such as an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0079] A "derivative" of a polypeptide is a polypeptide (such as an antibody) that has been chemically modified in some way other than by the insertions, deletions, or substitutions of variants, e.g., by conjugation with another chemical moiety.

[0080] The term "antibody" generally includes immunoglobulins, which comprise one or more polypeptide chains and have immunological functionality including specific binding to an antigen. In humans, antibodies typically comprise four linked polypeptide chains, i.e., a "tetramer", which includes two identical "heavy" chains and two identical smaller "light" chains. Each of the two heavy chains is linked to one of the light chains and the two are linked in parallel to each other. The linkages give the antibody a generally Y-shaped structure such that the linked portions of the heavy chains form the "legs" of the Y and each light chain (and the portion of the heavy chain to which each light chain is linked) forms an "arm" of the Y. Each arm of the antibody contains an antigen-binding site such that a typical antibody can bind to two of the antigen. In humans, there are five basic types or classes of antibodies, which are distinguished according to the structure of the heavy region and its functional purpose: IgG, IgA, IgE, IgD, IgM. In humans, some classes of intact antibodies may differ from the typical tetrameric "Y" structural unit, such as circulating IgM antibodies, which contain five such units linked in a roughly circular array at their base. More details regarding antibody structure and function are provided elsewhere herein.

[0081] In a typical antibody, each pair or conjugate in the tetrameric unit comprises a full-length "light" chain (about 25 kDa) and a full-length "heavy" chain (about 50 - 70 kDa). Each individual immunoglobulin chain is composed of a number of "immunoglobulin domains", each immunoglobulin domain consisting of about 90 to 110 amino acids and expressing a characteristic folding pattern. These domains are the basic units that make up the antibody polypeptide chain. The amino-terminal portion of each chain typically includes a variable domain responsible for antigen recognition. The carboxyl-terminal portion is more conserved evolutionarily than the amino-terminal of the chain and is referred to as the "constant region" or "C region".

[0082] The term "heavy chain" includes full-length immunoglobulin heavy chains and fragments thereof having sufficient variable domain sequence to confer binding specificity, used alone or in combination with light chain variable domains. Heavy chains are typically classified as mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, and these define the isotypes of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM and IgM2. IgA subtypes include IgA1 and IgA2. In humans, IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically contains one or more domains that can be responsible for effector functions. The number of heavy chain constant region domains will depend on the isotype. For example, full-length IgG heavy chains each contain three C region domains called CH1, CH2, and CH3, with CH3 closest to the carboxyl terminus. The antibody products provided can have any of these isotypes and subtypes. For example, the LILRB2 antibody product can be a full antibody of the IgG1 or IgG4 subtype.

[0083] The term "light chain" includes full-length immunoglobulin light chains and fragments thereof having sufficient variable domain sequence to confer binding specificity, used alone or in combination with heavy chain variable domains. Human light chains are typically classified as kappa (κ) or lambda (λ) light chains. Full-length light chains include an amino-terminal variable domain (VL) and a carboxyl-terminal constant domain (CL).

[0084] In light and heavy chains, the variable and constant regions are naturally joined by a "J" region of about twelve or more amino acids, and the heavy chain further includes a "D" region of about ten more amino acids. See, e.g., Fundamental Immunology, 2nd Ed., Chapter 7 (Paul ed.) 1989, New York: Raven Press.

[0085] The variable domains of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) joined by three hypervariable regions (more commonly referred to as "complementary determining regions" or CDRs). The CDRs of the two chains from each of the heavy / light chain pairs mentioned above are generally aligned by the framework regions to form a structure that specifically binds to a particular epitope (e.g., LILRB2) on a target protein. From the N-terminus to the C-terminus, both the naturally occurring light and heavy chain variable regions generally conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised for assigning numbers to the amino acids occupying positions in each of these domains.

[0086] The current art utilizes various numbering schemes that have different definitions of CDR length and position. For example, the Kabat numbering scheme is based on sequence alignment and uses the "variability parameter" at a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most frequently occurring amino acid at that position) to predict CDRs [Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., US Dept. of Health and Human Services (PHS), NIH, NIH Publication No. 91-3242 (1991)]. On the other hand, the Chothia numbering scheme is a structure-based numbering scheme in which antibody crystal structures are aligned to define loop structures as CDRs [Chothia and Lesk, J Mol Biol. 1987 196:901-17; Chothia et al., Nature. 1989 342:878-83]. The Martin numbering scheme focuses on the structural alignment of different framework regions with unconventional lengths [Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains". In: Kontermann and Dübel, eds., Antibody Engineering. Springer; Berlin, Germany: 2014. pp. 33-51]. The ImMunoGeneTics (IMGT) numbering scheme is a standardized numbering system based on the alignment of sequences from a complete reference gene database including the entire immunoglobulin superfamily [Lefranc et al., Dev Comp Immunol. 2003 27(1):55–77; (www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html)]. The Honneger numbering scheme (AHo) is based on the structural alignment of the 3D structure of the variable region and uses structurally conserved Cα positions to infer framework and CDR lengths [Honegger et al., J Mol Biol. 2001 309(3):657–70]. One of ordinary skill in the art understands that the definition of a CDR will vary based on the method used.

[0087] Some of the antibody products provided have structures commonly associated with naturally occurring antibodies. Thus, the term "antibody product" includes intact antibodies of any class or subclass or fragments thereof that can compete with an intact antibody for specific binding to a target antigen, and includes chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies and other forms. Notably, intact antibodies will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains, and V NAR domains from sharks. Antibody products can be derived from a single source only, or can be "chimeric", i.e., different parts of the antibody can be derived from two different antibodies. For example, the complementarity determining regions conferring antibody binding specificity can be derived from rat or murine, while the framework regions of the variable regions are derived from a different species source, such as human. In other chimeric forms, the light and heavy variable domains (optionally with constant domains) can be derived from one species and one or more constant domains from another species. See, e.g., U.S. Patent No. 11,352,444. The provided antibody products can be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody product" includes antibodies of other isotypes, derivatives, variants, and fragments thereof, in addition to antibodies containing two full-length heavy chains and two full-length light chains (such as IgG antibodies). The provided antibody products include, but are not limited to, monoclonal antibodies, human antibodies, chimeric antibodies, and humanized antibodies. The provided immunologically functional antibody fragments include, but are not limited to, scFv, Fab, Fab', F(ab')2, and domain antibody products.

[0088] As used herein, an "immunofunctional fragment" (or simply "fragment") of an immunoglobulin refers to a portion of an antibody that contains a light chain or a heavy chain (or both), is capable of specifically binding to an antigen, but lacks at least some of the amino acids present in the full-length chain in the light chain or heavy chain (or both). Such fragments are biologically active because they specifically bind to the target antigen and can compete with the intact antibody for specific binding to a given epitope. Such fragments will retain at least one CDR present in the full-length light or heavy chain and can include a single heavy and / or light chain or portions thereof. These biologically active fragments can be produced by recombinant DNA techniques or can be generated by enzymatic or chemical cleavage of the intact antibody. Immunofunctional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid, or rabbit. Further contemplated is that the functional portion of the antibodies of the present invention (e.g., one or more CDRs) can be covalently linked to a second protein or to a small molecule to produce a therapeutic agent directed to a specific target in vivo that has bifunctional therapeutic properties or has an extended serum half-life.

[0089] A "Fab fragment" contains one light chain (VL + CL) and a portion of the heavy chain that includes the variable domain and the CH1 domain (VH + CH1). The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0090] The "Fc" region contains two heavy chain fragments, each of which contains the CH2 and CH3 domains of the antibody and, in some cases, a lesser amount of the hinge region. The two heavy chain fragments are held together by two or more disulfide bonds (usually located in the hinge region) and by hydrophobic interactions of the CH3 domains.

[0091] A "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain, the CH1 domain, and a region between the CH1 and CH2 domains such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0092] An "F(ab')2 fragment" contains two light chains and two heavy chains, each of which contains a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0093] The "Fv region" contains the variable domains from both the heavy and light chains but lacks the constant domains.

[0094] "Single-chain antibody" is an Fv molecule in which the variable domains of the heavy and light chains have been joined by a flexible linker to form a single polypeptide chain, which single polypeptide chain forms the antigen-binding region. Single-chain antibodies are discussed in detail, for example, in PCT Publication No. WO 88 / 01649 and U.S. Patents Nos. 4,946,778 and 5,260,203.

[0095] "Domain antibody" is an immunofunctional immunoglobulin fragment containing only the variable domain of the heavy chain or the variable domain of the light chain. In some cases, two or more VH domains are covalently joined to a peptide linker to produce a bivalent domain antibody. The two VH domains of the bivalent domain antibody can target the same or different antigens.

[0096] "Bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific (see below).

[0097] "Multispecific antibody" is an antibody that targets more than one antigen or epitope.

[0098] "Bispecific", "dual-specific" or "bifunctional" antibodies are hybrid antibodies having two different antigen-binding sites. Bispecific antibodies are one type of multispecific antibody and can be produced by a variety of methods, including but not limited to the fusion of hybridomas or the ligation of Fab' fragments. See, for example, Songsivilai and Lachmann, Clin Exp Immunol. 1990 79:315-21; Kostelny et al., J Immunol. 1992 148:1547-53. The two binding sites of a bispecific antibody will bind to two different epitopes, which two different epitopes can be located on the same or different protein targets. "Trispecific" antibodies have three different antigen-binding sites.

[0099] The term "neutralizing antibody" refers to an antibody that binds to a ligand, where the neutralizing antibody prevents the ligand from binding to its binding partner and interrupts the biological response resulting from the binding of the ligand to its binding partner. When assessing the binding and specificity of an antibody or an immunofunctional fragment thereof, the antibody or fragment will substantially inhibit the binding of the ligand to its binding partner when an excess of the antibody reduces the amount of the binding partner bound by the ligand by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as measured in an in vitro competitive binding assay). In the case of an antibody product that binds to LILRB2, the neutralizing antibody product will reduce the ability of LILRB2 to bind to one or more of its ligands, thereby inhibiting LILRB2 activity (e.g., as shown in the examples herein).

[0100] The term "competition", when used in the context of antibody products competing for the same epitope, means that the competition between antibodies is determined by an assay in which the antibody product being tested blocks or inhibits the specific binding of a reference antibody product to a common antigen (e.g., LILRB2 or a fragment thereof). A variety of types of competitive binding assays can be used, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay [e.g., Stahli et al., Methods Enzymol. 1983 9:242-53]; solid-phase direct biotin-avidin EIA [e.g., Kirkland et al., J Immunol. 1986 137:3614-9]; solid-phase direct labeled assay, solid-phase direct labeled sandwich assay [e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)]; solid-phase direct labeled RIA using I-125 [e.g., Morel et al., Molec Immunol. 1988 25:7-15]; solid-phase direct biotin-avidin EIA [e.g., Cheung et al., Virology. 1990 176:546-52]; and direct labeled RIA [Moldenhauer et al., Scand J Immunol. 1990 32:77-82]. Generally, such assays involve the use of a purified antigen bound to a solid surface or cells carrying any of these, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Generally, the test antibody is present in excess. Antibodies identified by a competition assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an epitope that is close enough to the epitope bound by the reference antibody to cause steric hindrance. Generally, when the competing antibody is present in excess, the competing antibody will inhibit the specific binding of the reference antibody to the common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, the binding is inhibited by at least 80%, 85%, 90%, 95% or 97% or more by a selective binding agent such as an antibody and, additionally, is capable of being used in an animal to generate antibodies capable of binding to the antigen. An antigen can have one or more epitopes capable of interacting with different antibodies.

[0101] The term "epitope" includes any determinant capable of specifically binding to an antibody or to a T cell receptor. An epitope is a region of an antigen that is bound by an antibody that specifically targets the antigen, and when the antigen is a protein, the epitope includes specific amino acids that directly contact the antibody. In most cases, epitopes are located on proteins, but in some cases can be located on other types of molecules, such as nucleic acids. Epitope determinants can include the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Typically, an antibody specific for a particular target antigen will preferably recognize the epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0102] When the dissociation constant (K d ) is less than 100 nM, the antibody product "specifically binds" to its target antigen. When K d is less than 10 nM, the antibody specifically binds to the antigen with "high affinity", and when K d is less than 0.5 nM, the antibody specifically binds to the antigen with "extremely high affinity". The K d of the antibody product can range from about 0.5 nM to about 500 nM. The K d of the antibody product can range from about 100 to about 500 nM. Those skilled in the art will recognize that specifically binding does not mean exclusive binding, but allows for a degree of non-specific binding, which is typical in biological reactions between groups that have an affinity for each other.

[0103] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two reagents and can be expressed as the equilibrium dissociation constant K D , i.e., the calculated ratio of the dissociation constant and the association constant between an antibody and its antigen (K off / K on ). Affinity can also be expressed as the association constant K A , which is the reciprocal of K D . The binding affinities of the antibody products disclosed herein, as measured by K D , for human LILRB2 and for human LILRB1 range from 10 -4 M or lower, or as low as 10 -16 M or lower (e.g., about 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10-12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 M or lower). The antibodies described herein can be less than or equal to 10 -4 M, less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to 10 -7 M or less than or equal to 10 -8 M of K D specifically binds to a human LILRB2 polypeptide or to a human LILRB1 polypeptide. Methods for determining the affinity of two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (SPR), biolayer interferometry (BLI), etc.

[0104] As used herein, when an antibody product forms a complex with an antigen that is relatively stable under physiological conditions, the antibody product is said to have "immunospecificity" or "specificity" for the antigen or to "specifically bind" to the antigen. The terms "preferably binds" or "specifically binds" mean that the affinity of the antibody or its fragment for the epitope is greater than the affinity of the epitope for an unrelated amino acid sequence and, if cross-reactive with other polypeptides containing the epitope, is non-toxic at the level of administration formulated for human use. Such affinity can be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold the affinity of the antibody product for an unrelated amino acid sequence. The term also applies, for example, to the situation where an antibody product is specific for a particular epitope carried by more than one antigen, in which case the antibody or its antigen-binding fragment carrying the antigen-binding domain will be able to specifically bind to the epitope present in different antigens.

[0105] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule in the molecules being compared. For these calculations, gaps (if any) in the alignment must be resolved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in the following documents: Computational Molecular Biology, (edited by Lesk), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (edited by Smith), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (edited by Griffin and Griffin), 1994, New Jersey: Humana Press; Sequence Analysis in Molecular Biology, (by von Heinje), 1987, New York: Academic Press; Sequence Analysis Primer, (edited by Gribskov and Devereux), 1991, New York: M. Stockton Press; and Carillo et al., SIAM J Applied Math. 1988 48(5):1073-82.

[0106] In calculating the percent identity, the sequences to be compared are aligned in a manner that maximizes the matches between the sequences. An exemplary computer program for determining percent identity is the GCG program package, which includes GAP (Devereux et al., Nucl Acid Res. 1984 12:387-95; Genetics Computer Group, University of Wisconsin, Madison, Wisc.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned to achieve the best match of their corresponding amino acids or nucleotides (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3 times the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix to be used; the "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (which is typically 1 / 10 times the gap opening penalty) and a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. Standard comparison matrices [e.g., Dayhoff et al., Atlas of Protein Sequence and Structure, 5:345-352 (1978) for the PAM 250 comparison matrix; Henikoff et al., Proc Natl Acad Sci USA. 1992 89:10915-9 for the BLOSUM 62 comparison matrix] can also be used by the algorithm.

[0107] The recommended parameters for using the GAP program to determine the percent identity of a polypeptide or nucleotide sequence are as follows: algorithm: Needleman et al., J Mol Biol 1970 48:443-53; comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra; gap penalty: 12 (but no penalty for terminal gaps); gap length penalty: 4; similarity threshold: 0.

[0108] Some alignment schemes for aligning two amino acid sequences may only match a short region in one of the two sequences, and this small aligned region may have a very high sequence identity, even though there is no obvious relationship between the two full-length sequences. Thus, if desired, the selected alignment method (the GAP program) can be adjusted to produce an alignment spanning at least 50 contiguous amino acids of the target polypeptide.

[0109] Other exemplary programs for comparing and aligning sequence pairs include, but are not limited to, ALIGN (Myers and Miller, Comput Appl Biosci. 1988 4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA 1988 85(8):2444-8; Pearson, Methods Enzymol. 1990 183:63-98); and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 25(17):3389-402), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 12(1Pt 1):387-95).

[0110] The term "amino acid" includes its normal meaning in the art. The twenty naturally occurring amino acids and their abbreviations follow conventional usage. See, Immunology--A Synthesis, 2nd ed. (edited by Golub and Gren), Sinauer Associates: Sunderland, Mass. (1991). Stereoisomers of the twenty conventional amino acids, such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other non-conventional amino acids (e.g., D-amino acids) can be suitable components. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, according to standard usage and convention, the left hand direction is the amino-terminal direction, and the right hand direction is the carboxyl-terminal direction.

[0111] Table 10 below lists the heavy and light chain variable domains of the LILRB2-specific antibody named B2A, as well as the corresponding full-length heavy chains (hIgG1 and hIgG4) and light chains (κ) provided herein. The IMGT and Kabat CDRs are presented in Table 11. The CDRs of the two variable domains are shown: IMGT is shown with double underlining; Kabat is shown in bold.

[0112] Table 10 - Variable Regions and Full Length

[0113]

[0114]

[0115] Table 11 - Complementary Determining Regions

[0116]

[0117] Prepare humanized variants of the B2A antibody as described in the examples. The variable domains of representative humanized heavy and light chains are presented in Table 12.

[0118] Table 12

[0119]

[0120] Those skilled in the art will recognize that antibody products (such as full - length intact antibodies and antibody fragments that bind LILRB2) can be prepared based on the heavy and light chain variable domains given in Tables 10 and 12 or based on the CDRs given in Table 11. For illustration, as described in the examples, in addition to the full - length antibody chains shown in Table 10, full - length IgG1 or IgG4 heavy chains containing the VH variable domains (SEQ ID NO: 6 - 10) shown in Table 12 were prepared, and said heavy chains were combined with κ light chains containing the variable domains (SEQ ID NO: 11 - 15) shown in Table 12. The full - length IgG1 heavy chain sequence is represented as SEQ ID NO: 28 - 32. The full - length IgG4 heavy chain sequence is represented as SEQ ID NO: 33 - 37. The full - length κ light chain sequence is represented as SEQ ID NO: 38 - 42. Antibody products that bind LILRB2 were prepared using all combinations of heavy and light chains, and some of said antibody products were tested in various non - clinical assays as described in the examples.

[0121] An antibody product can comprise a light chain variable domain that comprises a sequence of amino acids that differs from the sequence of the light chain variable domain described herein at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, wherein each such sequence difference is independently a deletion, insertion, or substitution of one amino acid. The light chain variable regions in some antibodies comprise a sequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of the light chain variable region in Table 10 or Table 12.

[0122] An antibody product can comprise a heavy chain variable domain that comprises a sequence of amino acids that differs from the sequence of the heavy chain variable domains provided herein at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, where each such sequence difference is independently a deletion, insertion, or substitution of one amino acid. The heavy chain variable regions in some antibodies comprise a sequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the heavy chain variable region in Table 10 or Table 12. Still other antibody products include variant forms of the variant light chains and variant heavy chains as just described.

[0123] The provided antibody products can include one, two, three, four, five, or all six CDRs. Some antibody products include both a light chain CDR3 and a heavy chain CDR3. Certain antibody products have variant forms of the CDRs, where one or more (i.e., 2, 3, 4, 5, or 6) of the CDRs each have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the CDR sequences. For example, the antibody product can include both a light chain CDR3 and a heavy chain CDR3, each of which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain CDR3 sequence and the heavy chain CDR3 sequence, respectively. The CDR sequences of the provided antibody products can also differ from the CDR sequences in Table 10 or Table 12 such that the amino acid sequence for any given CDR differs from the sequences listed in Table 10 or Table 12 by no more than one, two, three, four, or five amino acid residues. The differences from the listed sequences are conservative substitutions.

[0124] When an antibody product is said to bind to an epitope of LILRB2, it means that the antibody product specifically binds to a region of LILRB2 specified by certain residues (e.g., one or more specific segments of the LILRB2 protein). In binding to an LILRB2 epitope, the antibody does not necessarily contact every residue within the LILRB2 peptide containing the specified residues. Nor does every single amino acid substitution or deletion within the LILRB2 peptide necessarily significantly affect the binding affinity. The exact epitope specificity of an antibody can be determined in a number of ways. For example, one method involves testing a set of overlapping peptides of about fifteen amino acids that span the sequence of LILRB2 and differ by increments of a small number of amino acids (e.g., three amino acids). The peptides are immobilized within the wells of a microtiter plate. Immobilization can be achieved by biotinylating one end of the peptide. Optionally, different samples of the same peptide can be biotinylated at the N and C termini and immobilized in separate wells for comparison. This can be used to identify end-specific antibodies. Optionally, additional peptides capped at the specific amino acids of interest can be included. This method can be used to identify antibodies that are end-specific for internal segments of LILRB2. The antibody product is screened for specific binding to each of the various peptides. The epitope is defined as the segment of amino acids that is common to all of the peptides to which the antibody is shown to specifically bind.

[0125] Antibody products that compete with one of the exemplary antibodies for specific binding to LILRB2 are also provided. Such antibody products can also bind to the same epitope as one of the exemplary antibodies. Antibody products that are expected to compete with or bind to the same epitope as an exemplary antibody or fragment are shown to have similar functional properties. Exemplary antibody products include antibody products having heavy and light chains, variable domains, and CDRs provided in Tables 10s, 11, or 12. Competing antibody products can include antibody products that bind to the epitopes described in the section on antibodies as well as the above epitopes.

[0126] The provided antibody products include monoclonal antibodies that bind to LILRB2. Monoclonal antibodies can be produced using any technique known in the art, e.g., by immortalizing spleen cells harvested from transgenic animals after completion of an immunization protocol. Spleen cells can be immortalized using any technique known in the art, e.g., by fusing spleen cells with myeloma cells to produce hybridomas. Myeloma cells in the fusion protocol for producing hybridomas preferably do not produce antibodies, have a high fusion efficiency, and lack enzymes such that they cannot grow in certain selective media that support only the growth of the desired fused cells (hybridomas). Examples of suitable cell lines for mouse fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XXOBul; examples of cell lines for rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines that can be used for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0127] In some cases, hybridoma cell lines are produced by immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with an LILRB2 immunogen; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells with a myeloma cell line, thereby producing hybridoma cells; establishing a hybridoma cell line from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies that bind to the LILRB2 polypeptide. Such hybridoma cell lines and LILRB2 monoclonal antibodies produced therefrom are provided herein.

[0128] Monoclonal antibodies secreted by the hybridoma cell lines can be purified using any useful technique known in the art of antibodies. The hybridomas or monoclonal antibodies can be further screened to identify monoclonal antibodies having specific properties. Examples of such screening are provided in the following examples.

[0129] Chimeric antibodies and humanized antibodies based on the above sequences are also provided. Monoclonal antibodies used as therapeutic agents can be modified in a variety of ways prior to use. One example is a "chimeric" antibody, which is an antibody composed of protein segments from different antibodies, the protein segments covalently joined to produce a functional immunoglobulin light chain or heavy chain or an immunofunctional portion thereof. Typically, a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass. For methods related to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proceedings of the National Academy of Sciences of the United States of America 1985 81:6851-5. CDR grafting is described in, for example, U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101.

[0130] Typically, the goal in preparing a chimeric antibody is to produce a chimera in which the number of amino acids from the intended patient species is maximized. One example is a "CDR grafted" antibody, in which the antibody contains one or more complementarity determining regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass. For human use, the V region or selected CDRs of a rodent antibody are typically grafted into a human antibody, thereby replacing the naturally occurring V region or CDRs in the human antibody.

[0131] "Humanized" antibody products are provided. Typically, humanized antibodies are produced from monoclonal antibodies initially generated in non-human animals. Certain amino acid residues in such monoclonal antibodies (usually from the non-antigen recognition portion of the antibody) are modified to be homologous to the corresponding residues in a human antibody of the corresponding isotype. For example, humanization can be performed using various methods by replacing at least a portion of the rodent variable region with the corresponding region of a human antibody [e.g., U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., Nature 1986 321:522-5; Riechmann et al., Nature 1988 332:323-7; Verhoeyen et al., Science 1988 239:1534-6]. Constant regions from species other than human can be used with one or more human variable regions to produce hybrid antibodies.

[0132] Fully human antibodies are provided. Methods for preparing fully human antibodies (“fully human antibodies”) that are specific for a given antigen without exposing humans to the antigen are known. One way to achieve the production of fully human antibodies is the “humanization” of the murine humoral immune system. Introducing the human immunoglobulin (Ig) locus into a mouse in which the endogenous Ig genes have been inactivated is one way to generate fully human monoclonal antibodies (MAbs) in mice (i.e., animals that can be immunized with any desired antigen). The use of fully human antibodies can minimize immunogenic responses and allergic reactions that can sometimes result from the administration of murine or murine-derived monoclonal antibodies as therapeutic agents to humans.

[0133] Fully human antibodies can be generated by immunizing transgenic animals, usually mice, that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids and are optionally conjugated to a carrier, such as a hapten. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 1993 90:2551-5; Jakobovits et al., Nature 1993 362:255-8; and Brüggemann et al., Year Immunol. 1993 7:33-40. In one example of such methods, transgenic animals are generated by disabling the endogenous murine immunoglobulin heavy and light chain loci encoding therein, and inserting large fragments of human genomic DNA containing loci encoding human heavy and light chain proteins into the murine genome. The partially modified animals (which have less than a complete complement of human immunoglobulin loci) are then crossed to obtain animals with all of the desired immune system modifications. When an immunogen is administered, the antibodies produced by these transgenic animals are immunospecific for immunogens having human rather than murine amino acid sequences, including the variable regions. Additional details of such methods are described, e.g., in WO96 / 33735 and WO94 / 02602. Additional methods related to transgenic mice for the preparation of human antibodies are described in U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; described in PCT Publications WO 91 / 10741, WO 90 / 04036; and described in EPO Publication EP 546073B1. Transgenic mice, referred to herein as "HuMab" mice, contain human immunoglobulin gene miniloci that encode unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous γ and κ chain loci (Lonberg et al., Nature 1994 368:856-9). Thus, the mice described above exhibit reduced expression of murine IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgGκ monoclonal antibodies. Exemplary mice having the entire human immunoglobulin locus in their germline are the XenoMouse (Abgenix) and another is the VelociImmune mouse (Regeneron Pharmaceuticals).Others are RenMab mice and RenLite mice (Biocytogen), and more recently AlivaMab mice (Ablexis).

[0134] Using hybridoma technology, antigen-specific human monoclonal antibodies with the desired specificity can be generated and selected from transgenic mice such as the transgenic mice described above. Such antibodies can be cloned and expressed using suitable vectors and host cells, or the antibodies can be harvested from cultured hybridoma cells.

[0135] Fully human antibodies can also be derived from phage display libraries (such as those disclosed in the following references: Hoogenboom and Winter, Journal of Molecular Biology 1992 227(2):381-8; and Marks et al., Journal of Molecular Biology 1991 222:581-97. Phage display technology mimics immune selection by displaying an antibody library on the surface of filamentous phage, and phage are subsequently selected by binding of the phage to the selected antigen. One such technique is described in PCT Publication No. WO 99 / 10494, which describes the use of such methods to isolate high-affinity and functional antagonistic antibodies for the c-Mpl and MuSK receptors.

[0136] Single-chain antibodies are provided. Single-chain antibodies are formed by linking the heavy and light chain variable domain (Fv region) fragments (such as the fragments shown in Table 10 or Table 12) via an amino acid bridge (short peptide linker), thereby producing a single polypeptide chain. Such single-chain Fv (scFv) can be prepared by fusing the DNA encoding the peptide linker located between the DNAs encoding the two variable domain polypeptides (VL and VH). The resulting polypeptide can fold itself to form an antigen-binding monomer, or the polypeptide can form a multimer (e.g., dimer, trimer, or tetramer), depending on the length of the flexible linker between the two variable domains. Techniques developed for generating single-chain antibodies include those described in the following: U.S. Patent No. 4,946,778; Bird et al., Science 1988 242:423-6; Huston et al., Proceedings of the National Academy of Sciences of the United States of America 1988 85:5879-83; Ward et al., Nature 1989 334:544-6; and de Graaf et al., Methods MolBiol. 2002 178:379-87. "Diabodies" are dimers of scFV.

[0137] Antibodies belonging to a subclass provided herein can be changed to antibodies from different subclasses using subclass conversion methods. For example, the variable domains described in Table 10 or Table 12 can be linked to the constant domains of any desired Ig isotype. Such techniques allow the preparation of new antibodies that have the antigen-binding properties of a given antibody (the parental antibody) but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parental antibody. Recombinant DNA techniques can be employed. Cloned DNA encoding a specific antibody polypeptide can be used in such procedures, for example, DNA encoding the constant domains of an antibody of the desired isotype. See, e.g., Lantto et al., Methods in Molecular Biology 2002 178:303-16. Thus, the antibodies provided include the desired isotypes (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE, and IgD).

[0138] The antibody products provided can include one or more of the CDRs in any of the heavy chain variable domains exemplified herein, wherein such CDRs are determined according to IMGT, Kabat, or other methods: (i) a CDR-H1 having at least 80% sequence identity with the CDR-H1 of SEQ ID NO:1, 6, 7, 8, 9, or 10; (ii) a CDR-H2 having at least 80% sequence identity with the CDR-H2 of SEQ ID NO:SEQ ID NO:1, 6, 7, 8, 9, or 10; and (iii) a CDR-H3 having at least 80% sequence identity with the CDR-H3 of SEQ ID NO:1, 6, 7, 8, 9, or 10. The antibody products provided can include one or more of the CDRs in any of the light chain variable domains exemplified herein, wherein such CDRs are determined according to IMGT, Kabat, or other methods: (i) a CDR-L1 having at least 80% sequence identity with the CDR-L1 of SEQ ID NO:2, 11, 12, 13, 14, or 15; (ii) a CDR-L2 having at least 80% sequence identity with the CDR-L2 of SEQ ID NO:SEQ ID NO:2, 11, 12, 13, 14, or 15; and (iii) a CDR-L3 having at least 80% sequence identity with the CDR-L3 of SEQ ID NO:2, 11, 12, 13, 14, or 15. In some embodiments, the CDRs will have at least 85%, at least 90%, at least 95%, or at least 99% identity with the CDR sequences that have been determined. The antibody products can include one, two, three, four, five, or all six of the above CDRs, so long as they specifically bind to hLILRB2.

[0139] The provided antibody products can include one or more of the following exemplary heavy-chain IMGT CDRs: (i) CDR-H1 having at least 80% sequence identity with SEQ ID NO:16; (ii) CDR-H2 having at least 80% sequence identity with SEQ ID NO:17; and (iii) CDR-H3 having at least 80% sequence identity with SEQ ID NO:18. The provided antibody products can include one or more of the following light-chain CDRs: (i) CDR-L1 having at least 80% sequence identity with SEQ ID NO:19; (ii) CDR-L2 having at least 80% sequence identity with SEQ ID NO:20; and (iii) CDR-L3 having at least 80% sequence identity with SEQ ID NO:21. In some embodiments, the CDRs will have at least 85%, at least 90%, at least 95% or at least 99% identity with the specified CDR sequences. The antibody products can include one, two, three, four, five or all six of the above CDRs, so long as they specifically bind to hLILRB2.

[0140] The provided antibody products can include one or more of the following exemplary heavy-chain Kabat CDRs: (i) CDR-H1 having at least 80% sequence identity with SEQ ID NO:22; (ii) CDR-H2 having at least 80% sequence identity with SEQ ID NO:23; and (iii) CDR-H3 having at least 80% sequence identity with SEQ ID NO:24. The provided antibody products can include one or more of the following light-chain CDRs: (i) CDR-L1 having at least 80% sequence identity with SEQ ID NO:25; (ii) CDR-L2 having at least 80% sequence identity with SEQ ID NO:26; and (iii) CDR-L3 having at least 80% sequence identity with SEQ ID NO:27. In some embodiments, the CDRs will have at least 85%, at least 90%, at least 95% or at least 99% identity with the specified CDR sequences. The antibody products can include one, two, three, four, five or all six of the above CDRs, so long as they specifically bind to hLILRB2.

[0141] The provided antibody products can include: (a) a heavy chain variable region having 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 1, 6, 7, 8, 9 or 10; (b) a light chain variable region having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 2, 11, 12, 13, 14 or 15; or (c) the heavy chain variable region of (a) and the light chain variable region of (b).

[0142] The other provided antibody products compete with antibodies such as the antibodies described above for specific binding to the LILRB2 polypeptide. For example, the provided antibody products compete with an antibody consisting of two identical heavy chains and two identical light chains, wherein the heavy chain comprises SEQ ID NO: 1, 6, 7, 8, 9 or 10 and the light chain comprises SEQ ID NO: 2, 11, 12, 13, 14 or 15.

[0143] LILRB2 antibody products are provided that have a half-life of at least one day in vitro or in vivo (e.g., when administered to a human subject). The half-life of the antibody products can be at least three days. The half-life of the antibody products can be four days or longer. The half-life of the antibody products can be eight days or longer.

[0144] Variant

[0145] Variant forms of the LILRB2 antibody products disclosed herein are provided (e.g., variant forms of antibody products having the sequences listed in Tables 10 and 12). For example, the antibody products can have one or more conservative amino acid substitutions in one or more of the heavy or light chain variable regions or CDRs listed in Tables 10 and 12.

[0146] Naturally occurring amino acids can be divided into groups based on common side-chain characteristics: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues affecting strand orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions can involve exchanging one member of one of these groups for another member of the same group. Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid moieties.

[0147] Non-conservative substitutions can involve the exchange of a member from one of the above classes for a member from another class. Such substituted residues can be introduced into regions of an antibody product that are homologous to human antibodies or into non-homologous regions of the molecule.

[0148] When making such changes, the hydrophilicity index of the amino acids can be considered. The hydrophilic character of a protein is calculated by assigning a numerical value ("hydrophilicity index") to each amino acid and then averaging these values repeatedly along the peptide chain. A hydrophilicity index has been assigned to each amino acid according to its hydrophobic and charge characteristics. The amino acids are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0149] It has been appreciated in the art the importance of hydrophilic character in conferring biological functions of protein interactions [e.g., Kyte and Doolittle, Journal of Molecular Biology 1982 157:105-31]. It is known that certain amino acids can substitute for other amino acids having similar hydrophilicity indices or scores and still retain similar biological activity. When making changes based on the hydrophilicity index, substitutions of amino acids can be made whose hydrophilicity indices are within ±0.2. Substitutions of amino acids can also be made whose hydrophilicity indices are within ±0.1. Substitutions of amino acids can also be made whose hydrophilicity indices are within ±0.5.

[0150] It should also be understood in the art that substitutions of similar residues in an amino acid sequence can be made effectively on the basis of the relative hydrophilicity or hydrophobicity of the residues, particularly when the resulting biologically functional protein or peptide is intended for use as an immunological molecule, as in the case of the present invention. As determined by the hydrophilicity of adjacent amino acids of a protein, the maximum local average hydrophilic character of a protein can be related to its immunogenicity and antigen binding or immunogenicity, i.e., to the biological properties of the protein.

[0151] A variety of methods are known for estimating the hydrophilicity or hydrophobicity of amino acid residues in a protein. A comparative survey of such methods is given in Biswas et al., Journal of Chromatography A 1000(1 - 2):637–55. Hopp and Woods (Molecular Immunology 1983 20(4):483 - 9) assigned hydrophilicity values to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 0.1); glutamic acid (+3.0 ± 0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In this ranking system, more hydrophilic residues are assigned positive values and less hydrophilic residues are assigned negative values. When making changes based on similar hydrophilicity values, substitutions of amino acids having hydrophilicity values within ±0.2, or substitutions of amino acids having hydrophilicity values within ±0.1, or substitutions of amino acids having hydrophilicity values within ±0.5 are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions".

[0152] One of ordinary skill in the art will be able to use known techniques to determine suitable variants of the polypeptides as shown herein. One of ordinary skill in the art can identify suitable regions of a molecule that can be altered without destroying activity by targeting regions that are thought to be unimportant for activity. One of ordinary skill in the art will also be able to identify residues and portions of a molecule that are conserved among similar polypeptides. Conservative amino acid substitutions can be made even in regions that may be important for biological activity or for structure, without destroying biological activity or adversely affecting polypeptide structure.

[0153] Additionally, one of ordinary skill in the art can review structure - function studies to identify residues that are important for activity or structure in similar polypeptides. Given such comparisons, the importance of amino acid residues in a protein can be predicted, where the amino acid residues correspond to amino acid residues that are important for activity or structure in a similar protein. One of ordinary skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0154] Those skilled in the art can also analyze the three-dimensional structures in similar polypeptides and the amino acid sequences related to such structures. Given such information, those skilled in the art can predict the alignment of the amino acid residues of the antibody relative to its three-dimensional structure. Those skilled in the art can choose not to make radical changes to the amino acid residues predicted to be on the surface of the protein, as such residues may be involved in important interactions with other molecules. In addition, those skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using assays for LILRB2 activity (see the examples below), thereby generating information on which amino acids can be altered and which cannot. In other words, based on the information collected from such routine experiments, those skilled in the art can readily determine the amino acid positions at which additional substitutions, either alone or in combination with other mutations, should be avoided.

[0155] Substantial modifications in the function and / or biochemical properties of the antibody products described herein can be achieved by creating substitutions in the amino acid sequences of the heavy and light chains that are significantly different in terms of maintaining the roles of the following: (a) the structure of the molecular backbone in the substitution region, such as in a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. "Conservative amino acid substitutions" can involve replacing a natural amino acid residue with a canonical residue, which has little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any natural residue in the polypeptide can also be replaced with alanine, as previously described for alanine scanning mutagenesis.

[0156] One of ordinary skill in the art can effect amino acid substitutions (whether conservative or non-conservative) of a subject antibody by applying conventional techniques. Amino acid substitutions include, but are not limited to, substitutions that result in: (1) decreased susceptibility to proteolysis; (2) decreased susceptibility to oxidation; (3) altered binding affinity to form a protein complex; (4) altered ligand or antigen binding affinity; and / or (4) imparted or modified other physiochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence. Substitutions can be made in the portions of the antibody that are outside of domains that form intermolecular contacts. Conservative amino acid substitutions can be used that substantially do not alter the structural characteristics of the parental sequence (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes the parental or native antibody). Examples of polypeptide secondary and tertiary structures that are well recognized in the art are described in Proteins, Structures and Molecular Principles (edited by Creighton), 1984, New York: W.H. Freeman and Company; Introduction to Protein Structure (edited by Branden and Tooze), 1991, New York: Garland Publishing; and Thornton et al., Nature 1991 354(6349):105-6, each of which is incorporated herein by reference.

[0157] Glycosylation variants of antibody products are provided, in which the number and / or type of one or more glycosylation sites has been altered compared to the amino acid sequence of the parental polypeptide. Antibody product variants can contain a greater or lesser number of N-linked glycosylation sites than the native antibody. N-linked glycosylation sites are characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue other than proline. Substituting amino acid residues to create this sequence provides potential new sites for the addition of N-linked carbohydrate chains. Alternatively, substitutions that eliminate or alter this sequence will prevent the addition of N-linked carbohydrate chains that are present in the native polypeptide. For example, glycosylation can be reduced by deleting Asn or by substituting Asn with a different amino acid. For example, one or more new N-linked sites are created. Antibodies typically have N-linked glycosylation sites in the Fc region.

[0158] Additional antibody product variants include cysteine variants in which one or more cysteine residues in the parental or native amino acid sequence are deleted or replaced with another amino acid (e.g., serine). Cysteine variants are useful, especially when the antibody must refold into a biologically active conformation. Cysteine variants can have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions caused by unpaired cysteines.

[0159] Effector Function

[0160] Antibody structure affects the role of the antibody in the immune system and the effects that the antibody can induce or influence. See, e.g., Vidarsson et al., Front Immunol. 2014 5 (Chapter 5):1-17. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Generally, Fc-mediated functions involve binding of the Fc portion of the antibody through specialized receptor molecules, "Fc receptors" or "FcRs", expressed by the cells whose function is affected.

[0161] In some embodiments, IgG is considered the most versatile immunoglobulin because it performs all the functions of an immunoglobulin molecule. IgG is the major Ig in serum and the only class of Ig that crosses the placenta. IgG also fixes complement, but the IgG4 subclass does not. Macrophages, monocytes, polymorphonuclear leukocytes (PMNs), and some lymphocytes have receptors for the Fc region of IgG. Not all subclasses bind equally well: IgG2 and IgG4 do not bind to Fc receptors. In some cases, binding to Fc receptors on PMNs, monocytes, and macrophages results in the cells now better internalizing the antigen. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.

[0162] In certain embodiments, the FcR is a native sequence human FcR. Additionally, preferred FcRs are receptors that bind to IgG antibodies (γ (“gamma”) receptors) and include receptors of the FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) subclasses, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain.

[0163] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The antibody “arms” the cytotoxic cells and is required for such killing. Primary NK cells, which are used to mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In some embodiments, to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay is performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0164] Alternatively, or additionally, in some embodiments, the ADCC activity of a molecule of interest is evaluated in vivo (e.g., in an animal model).

[0165] In some embodiments, the antibodies of the present disclosure bind to surface membrane proteins of M2-like macrophages and are internalized by these macrophages. This internalization process is thought to be related to the observed alteration of the functional immunosuppressive properties of these cells, i.e., the cells differentiate from the M2 state to a subtly activated state without killing the cells or inhibiting their proliferation. In some embodiments, upon internalization, the antibody reduces the expression of immunosuppressive soluble factors while increasing the expression of soluble factors that stimulate or promote the activity or proliferation of T cells, including CD4+ helper T cells and cytotoxic lymphocytes.

[0166] For certain therapeutic applications, the internalization process is used to kill or reduce the activity or proliferation of target cells that express the LILRB2 protein. The number of internalized antibody molecules will be sufficient to kill the cells or inhibit their growth. In some cases, depending on the potency of the antibody or antibody conjugate, the uptake of a single antibody molecule into a cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins have a strong killing ability such that the internalization of one molecule of a toxin conjugated to an antibody is sufficient to kill the target cell.

[0167] In some embodiments, the LILRB2 antibodies or antigen-binding fragments provided herein are conjugated or linked to a therapeutic moiety, an imaging or detectable moiety, or an affinity tag. Methods for conjugating or linking polypeptides are well known in the art. The association (binding) between the compound and the label includes any manner known in the art, including but not limited to covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the antibody or its antigen-binding fragment is conjugated or recombinant engineered with an affinity tag (e.g., a purification tag). Affinity tags, such as polyhistidine (e.g., His6) tags are conventional in the art.

[0168] In some embodiments, the LILRB2 antibody or antigen-binding fragment further comprises a detectable moiety. Detection is performed, for example, in vitro, in vivo, or ex vivo. For example, in vitro assays for detecting and / or determining (quantifying, qualitatively, etc.) the hLILRB2 protein expressed by macrophages using an antibody or its antigen-binding fragment include, but are not limited to, for example, ELISA, RIA, and Western blot. In some embodiments, in vitro detection, diagnosis, or monitoring of the antigen of the antibody is performed by obtaining a sample (e.g., a blood sample) from a subject and testing the sample in, for example, a standard ELISA assay.

[0169] Derivative

[0170] This document also provides derivatives of the LILRB2 antibody products described herein. The derived antibody products can comprise any molecule or substance that confers desired properties to the antibody product, such as increasing the half-life in a particular use. The derived antibody products can comprise, for example, a detectable (or label) moiety (e.g., a radioactive, colorimetric, antigenic, or enzyme molecule), a detectable bead (such as a magnetic or electron-dense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or drug-active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). Examples of molecules that can be used to derivatize the antibody products include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of the antibody products can be prepared using techniques well known in the art. The antibody can be conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinylpyrrolidone), polyethylene glycol, polyethylene glycol homopolymers, poly(propylene oxide / ethylene oxide) copolymers, polyoxyethylated polyols, and polyvinyl alcohol).

[0171] Other derivatives include covalent or aggregate conjugates of the LILRB2 antibody product with other proteins or polypeptides, such as by expressing a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of the LILRB2 antibody product. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, such as the yeast α-factor leader, or a peptide such as an epitope tag. The fusion protein containing the LILRB2 antibody product can comprise a peptide (e.g., poly-His) added to facilitate the purification or identification of the LILRB2 antibody product. The LILRB2 antibody product can also be linked to a FLAG peptide, as described in Hopp et al., Bio / Technology 1988 6:1204-10 and U.S. Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), thus enabling rapid assay and easy purification of the expressed recombinant protein. Reagents for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, Mo.).

[0172] Oligomers containing one or more LILRB2 antibody products can be used as LILRB2 antagonists. The oligomers can be in the form of covalently or non-covalently linked dimers, trimers or higher forms. Consider using oligomers comprising two or more LILRB2 antibody products, one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0173] The oligomers can comprise multiple LILRB2 antibody products joined via covalent or non-covalent interactions between peptide moieties fused to the LILRB2 antibody polypeptides. Such peptides can be peptide linkers (spacers), or peptides having properties that promote oligomerization. Leucine zippers and certain antibody-derived polypeptides are peptides that can promote the oligomerization of LILRB2 antibody products linked thereto, as described in more detail below.

[0174] The oligomers can comprise two to four LILRB2 antibody products. The LILRB2 product moieties of the oligomers can be in any of the forms described above, e.g., variants or fragments. The oligomers comprise LILRB2 antibody products having LILRB2 binding activity.

[0175] The preparation of fusion proteins comprising heterologous polypeptides fused to different portions of antibody-derived polypeptides, including the Fc domain, has been described, for example, by Ashkenazi et al., Proceedings of the National Academy of Sciences of the United States of America 1991 88(23):10535-9; Byrn et al., Nature 1990 344(6267):677-670; and Hollenbaugh and Aruffo, Current Protocols in Immunology( Curr Protoc Immunol .) 2002 48(1):10.19A.1-10.19A.11.

[0176] Dimers are provided, which dimers comprise two fusion proteins produced by fusing the LILRB2-binding fragment of an LILRB2 antibody to the Fc region of the antibody. For example, dimers can be prepared by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion proteins to assemble very similar antibody molecules, thereby forming interchain disulfide bonds between the Fc portions to produce dimers.

[0177] As used herein, the term "Fc polypeptide" includes polypeptides in native and mutant protein forms derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing hinge regions that promote dimerization. Fusion proteins comprising an Fc portion (and thus formed oligomers) have the advantage of simple purification by affinity chromatography compared to protein A or protein G columns.

[0178] An exemplary Fc polypeptide described in PCT Publication No. WO 93 / 10151 and U.S. Patent Nos. 5,426,048 and 5,262,522 (each of which is hereby incorporated by reference) is a single-chain polypeptide extending from the N-terminal hinge region of a human IgG1 antibody to the natural C-terminus of the Fc region. Another exemplary Fc polypeptide is the Fc mutant protein described in U.S. Patent No. 5,457,035 and in Baum et al., EMBO J. 1994 13:3992-4001 (1994). The amino acid sequence of this mutant protein is identical to the natural Fc sequence presented in PCT Publication No. WO 93 / 10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutant protein exhibits a reduced affinity for Fc receptors.

[0179] Alternatively, the oligomer is a fusion protein comprising a plurality of LILRB2 antibody polypeptides, with or without a peptide linker (spacer peptide). Among the suitable peptide linkers are those described in U.S. Patent Nos. 4,751,180 and 4,935,233.

[0180] Another method for preparing oligomeric LILRB2 antibody product derivatives involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of the protein in which they are present. Examples of leucine zipper domains suitable for generating soluble oligomeric proteins are described in PCT Publication No. WO 94 / 10308, and a leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., FEBS Lett. 1994 344:191-5. A modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., Semin Immunol. 1994 6:267-78. Generally, a recombinant fusion protein comprising an LILRB2 antibody fragment fused to a leucine zipper peptide is expressed in a suitable host cell, and the soluble oligomeric LILRB2 antibody product formed is recovered from the culture supernatant.

[0181] The LILRB2 antibody products described herein can also be derivatized or modified such that the products have a longer half-life compared to the underivatized or unmodified antibody. For example, the antibody products can contain point mutations to increase serum half-life, as described in PCT Publication No. WO 00 / 09560.

[0182] Nucleic Acids and Cells

[0183] Nucleic acids encoding one or more chains of the antibody products herein are provided, as well as polynucleotides sufficient to be used as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting the expression of polynucleotides, and complementary sequences of the above.

[0184] The nucleic acids provided encode the antibody products disclosed herein, the light chain variable regions as shown in Table 10 or Table 12 and / or the heavy chain variable regions as shown in Table 10 or Table 12. Due to the degeneracy of the genetic code, each of the polypeptide sequences listed in Table 10 or Table 12 also encodes other nucleic acid sequences in addition to the sequences listed in Table 10 or Table 12. The present disclosure provides each degenerate nucleotide sequence encoding each antibody product.

[0185] The term "polynucleotide" or "nucleic acid" means a single-stranded or double-stranded polymer. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of either nucleotide type. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioate, dithiophosphate, selenophosphate, diselenophosphate, phosphorothioanilate, phosphoraniladate, and phosphoroamidate. The term includes both single-stranded and double-stranded forms.

[0186] An "isolated nucleic acid molecule" means a DNA or RNA or some combination thereof of genomic, mRNA, cDNA or synthetic origin, which is not associated with all or part of the polynucleotide with which the isolated polynucleotide is naturally present, or is not linked to a polynucleotide to which it is not naturally linked. For the purposes of the present disclosure, it should be understood that a nucleic acid molecule "comprising" a particular nucleotide sequence does not encompass an entire chromosome. In addition to the specified sequence, an isolated nucleic acid molecule "comprising" the specified nucleic acid sequence may include coding sequences for up to ten or even up to twenty other proteins or portions thereof, or may include operably linked regulatory sequences controlling the expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0187] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5' direction. The direction of addition from 5' to 3' to a nascent RNA transcript is called the transcription direction; the sequence region at the 5' of the 5' end of the RNA transcript on the DNA strand having the same sequence as the RNA transcript is called the "upstream sequence"; the sequence region at the 3' of the 3' end of the RNA transcript on the DNA strand having the same sequence as the RNA transcript is called the "downstream sequence".

[0188] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of an encoding sequence to which it is linked. The nature of such control sequences can depend on the host organism. For example, control sequences for eukaryotes can include a promoter, a transcriptional enhancer sequence, and a transcriptional termination sequence that contain one or more recognition sites for transcription factors. A "control sequence" can include a leader sequence and / or a fusion partner sequence.

[0189] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein-coding information into a host cell.

[0190] The term "expression vector" or "expression construct" refers to a vector that is suitable for transforming a host cell and contains a nucleic acid sequence that directs and / or controls (in association with the host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of a coding region operably linked thereto.

[0191] As used herein, "operably linked" means that the components to which the term applies are in a relationship that permits them to perform their inherent functions under suitable conditions. For example, a control sequence in a vector that is "operably linked" to a protein-coding sequence is linked thereto such that the expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0192] The term "host cell" means a cell that has been transformed with a nucleic acid sequence or that is capable of being transformed with a nucleic acid sequence and thereby expressing a gene of interest. The term includes the progeny of the parental cell, whether or not the progeny are identical to the original parental cell in morphology or genetic constitution, as long as the gene of interest is present.

[0193] DNA encoding an antibody polypeptide (e.g., a heavy or light chain, variable domain only, or full length) can be isolated from the B cells of a mouse immunized with LILRB2 or an immunogenic fragment thereof. The DNA can be isolated by conventional procedures such as polymerase chain reaction (PCR). Another example of a known technique is phage display, by which nucleotide sequences encoding antibody polypeptides can be selected.

[0194] Nucleic acids that hybridize to other nucleic acids under specific hybridization conditions are provided. Methods for hybridizing nucleic acids are well known in the art. As defined herein, moderately stringent hybridization conditions use a prewashing solution (or other similar hybridization solution, such as a solution containing approximately 50% formamide, where the hybridization temperature is 42°C) with a hybridization buffer containing 5X sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), approximately 50% formamide, a hybridization temperature of 55°C, and a washing condition of 60°C in 0.5X SSC, 0.1% SDS. Stringent hybridization conditions hybridize in 6X SSC at 45°C, followed by one or more washes in 0.1X SSC, 0.2% SDS at 68°C. In addition, those skilled in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to each other generally remain hybridized to each other.

[0195] The basic parameters that affect the choice of hybridization conditions and guidance for designing appropriate conditions are shown by, for example, Sambrook, Fritsch, and Maniatis (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, edited by Ausubel et al., John Wiley & Sons, Inc., Sections 2.10 and 6.3 - 6.4), and can be readily determined by those of ordinary skill in the art based on, for example, the length and / or base composition of the DNA.

[0196] Changes can be introduced into the nucleic acid by mutation, thereby changing the amino acid sequence of the polypeptide encoded thereby (e.g., an antibody or antibody derivative). Any technique known in the art can be used to introduce the mutation. Site-directed mutagenesis protocols, for example, can be used to change one or more specific amino acid residues. Random mutagenesis protocols, for example, can be used to change one or more randomly selected residues. Regardless of how the mutants are prepared, the mutant polypeptides can be expressed and screened for the desired properties.

[0197] The polypeptide that is a component of the antibody product of interest is expressed in any suitable recombinant expression system.

[0198] An expression vector containing a nucleic acid encoding a LILRB2 antibody product is provided. Examples of vectors include, but are not limited to, plasmids, viral vectors, episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0199] Typically, expression vectors used in any host cell contain sequences for plasmid or virus maintenance and for cloning and expressing foreign nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" and generally include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple cloning region for insertion of a nucleic acid encoding a polypeptide to be expressed, and an optional marker element.

[0200] Optionally, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the coding sequence, an oligonucleotide sequence encoding polyHis (such as hexaHis), or another "tag" for which commercially available antibodies exist, such as FLAG, HA (for hemagglutinin of influenza virus), or myc. The tag is typically fused to the antibody protein upon expression and can be used as a means for affinity purification of the antibody from the host cell. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified antibody polypeptide by various means, such as cleavage using certain peptidases.

[0201] The flanking sequences in the expression vector can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences function in the host cell machinery and can be activated by the host cell machinery.

[0202] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the LILRB2 antibody product. Promoters are generally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate an increase in the level of transcription from DNA in response to some change in the culture conditions, such as the presence or absence of nutrients or a change in temperature, under their control. On the other hand, constitutive promoters initiate continuous gene product production; that is, there is little or no experimental control over gene expression. A large number of promoters recognized by various potential host cells are well known. A suitable promoter is operably linked to the DNA encoding the LILRB2 antibody product by removing the promoter from the source DNA by digestion with a restriction endonuclease or by amplifying the promoter by polymerase chain reaction and inserting the desired promoter sequence into the vector.

[0203] Suitable promoters for mammalian host cells are well known and include, but are not limited to, promoters obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters such as the heat shock promoter and the actin promoter.

[0204] Exemplary promoters that can be used in recombinant expression vectors include, but are not limited to: the SV40 early promoter region (Bemoist and Chambon, 1981, Nature, 290: 304-10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 1980, 22: 787-97); the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA, 1981, 78: 1444-5); the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 1982, 296: 39-42); prokaryotic expression vectors, such as the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA, 1978, 75: 3727-31); or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA, 1983, 80: 21-5).The following animal transcriptional control regions can also be used. These animal transcriptional control regions exhibit tissue specificity and have been used in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., Cell 1984 38:639-46; Ornitz et al., Cold Spring Harb Symp Quant Biol. 1986 50:399-409; MacDonald, Hepatology. 1987 7:425-515); the insulin gene control region that is active in pancreatic β cells (Hanahan, Nature 1985 315:115-22); the mouse mammary tumor virus control region that is active in testis, mammary gland, lymphocytes and mast cells (Leder et al., Cell 1986 45:485-95); the albumin gene control region that is active in the liver (Pinkert et al., Genes Dev 1987 1:268-76); the alpha-fetoprotein gene control region that is active in the liver (Krumlauf et al., Mol Cell Biol. 1985 5:1639-48; Hammer et al., Science 1987 235:53-8); the alpha1-antitrypsin gene control region that is active in the liver (Kelsey et al., Genes Dev 1987 1:161-71); the beta-globin gene control region that is active in myeloid cells (Mogram et al., Nature 1985 315:338-40; Kollias et al., Cell 1986 46:89-94); the myelin basic protein gene control region that is active in oligodendrocytes in the brain (Readhead et al., Cell 1987 48:703-12); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, Nature 1985 314:283-6); the gonadotropin-releasing hormone gene control region that is active in the hypothalamus (Mason et al., Science 1986 234:1372-8); and most specifically, the immunoglobulin gene control region that is active in lymphocytes (Grosschedl et al., Cell 1984 38:647-58; Adames et al., Nature 1985 318:533-8; Alexander et al., Mol Cell Biol. 1987 7:1436-44).

[0205] Enhancer sequences can be inserted into the vector to increase the transcription of the nucleic acid encoding the LILRB2 antibody product described herein in higher eukaryotes. A variety of enhancer sequences are known to be obtainable from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). Enhancer sequences from viruses can also be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer, and the adenovirus enhancer are exemplary enhancer elements for activating eukaryotic promoters. Although the enhancer can be spliced into the vector at a position located 5' or 3' of the nucleic acid molecule, the enhancer is typically placed at a site 5' of the promoter.

[0206] In an expression vector, the transcription termination sequence is typically located 3' at the end of the region encoding the polypeptide and is used to terminate transcription. The transcription termination sequence for expression in prokaryotic cells is typically a G-C rich fragment followed by a poly-T sequence. Although the sequence is readily cloned from a library or even commercially purchased as part of a vector, the sequence can also be easily synthesized using methods for nucleic acid synthesis (such as the methods described herein).

[0207] Selectable genes can be used to amplify the gene to be expressed. Amplification is a process in which a gene that cannot be present as a single copy and is expressed at a high enough level to allow the cell to survive and grow under certain selection conditions is tandemly repeated within the chromosome of successive generations of recombinant cells. Examples of suitable amplifiable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase. In the use of these markers, mammalian cell transformants are placed under selection pressure, where only the transformants uniquely adapt to survive by virtue of the selectable gene present in the vector. The selection pressure is applied by culturing the transformed cells under conditions of continuously increasing concentration of the selection agent in the medium, thereby allowing only those cells that have amplified the selectable gene to survive. In these cases, the DNA adjacent to the selectable gene (such as the DNA encoding the antibody) is co-amplified with the selectable gene. Thus, the amount of LILRB2 polypeptide synthesized from the amplified DNA is increased.

[0208] The ribosome binding site is generally necessary for the initiation of translation of the mRNA and is characterized by the Shine-Dalgarno sequence (for prokaryotes) or the Kozak sequence (for eukaryotes). The element is typically located 3' of the promoter and 5' of the coding sequence of the polypeptide to be expressed.

[0209] In some cases, such as when glycosylation is required in a eukaryotic host cell expression system, various pre-sequences can be manipulated to enhance glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be altered, or a precursor sequence can be added, which can also affect glycosylation. The final protein product can have one or more additional amino acids related to expression at the -1 position (relative to the first amino acid of the mature protein), and these amino acids may not have been completely removed. For example, the final protein product can have one or two amino acid residues attached to the amino terminus that are present at the peptidase cleavage site. Alternatively, if an enzyme cleaves at such a region within the mature polypeptide, the use of some enzyme cleavage sites can produce a slightly truncated but active form of the desired polypeptide.

[0210] When commercially available expression vectors lack some of the desired flanking sequences as described above, the vectors can be modified by ligating these sequences individually to the vector. After selecting and modifying the vector as needed, the nucleic acid molecule encoding the LILRB2 antibody product is inserted into an appropriate site in the vector.

[0211] The complete vector containing the sequence encoding the antibody product is inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the expression vector for the LILRB2 antibody product into the selected host cell can be accomplished by well-known methods, including methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofection, the DEAE-dextran method, or other known techniques. The selected method will vary in part with the type of host cell to be used. These methods and other suitable methods are well known to those skilled in the art.

[0212] Antibodies can be expressed in hybridoma cell lines or in cell lines other than hybridomas. Expression constructs encoding the antibodies can be used to transform mammalian, insect, or microbial host cells. Transformation can be carried out using any known method for introducing polynucleotides into host cells, including, for example, packaging the polynucleotide in a virus or phage and transducing the host cell with the construct by transfection procedures known in the art, as exemplified in U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The optimal transformation procedure to use will depend on the type of host cell to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, mixing of the nucleic acid with a positively charged lipid, and direct microinjection of the DNA into the cell nucleus.

[0213] When cultured under appropriate conditions, the transformed host cells synthesize LILRB2 antibody products, which can then be collected from the culture medium (if the host cells secrete the antibody products into the culture medium) or directly from the host cells producing the antibody products (if the antibody products are not secreted). The selection of appropriate host cells will depend on various factors, such as the desired expression level, the expected activity, or the necessary polypeptide modifications (such as glycosylation or phosphorylation), as well as the ease of folding into a bioactive molecule.

[0214] Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), human embryonic kidney cells (HEK) (e.g., HEK293), and many other cell lines. The optimal cell line for expressing a particular DNA construct can be selected by testing various cell lines to determine which cell lines have the highest level of expression and produce antibody products with the desired LILRB2 binding properties.

[0215] Nucleic acid molecules suitable as primers or hybridization probes for detecting nucleic acid sequences are also provided. The nucleic acid molecules can comprise only a portion of the nucleic acid sequence encoding the full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of the polypeptide (e.g., the LILRB2 binding portion).

[0216] Composition

[0217] Compositions comprising LILRB2 antibody products are also provided. Pharmaceutical compositions generally include one or more of a buffer, a pharmaceutically acceptable diluent, a carrier, a solubilizer, an emulsifier, and a preservative. The use of the above antibody products in the preparation of pharmaceutical compositions or drugs is also provided.

[0218] Acceptable formulation components for pharmaceutical preparations are preferably non-toxic to the recipient at the doses and concentrations employed. In addition to the antibody products provided herein, the compositions can contain components for modifying, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorptivity or permeability of the composition. Suitable materials for formulating pharmaceutical compositions include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as acetate, borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents, flavoring agents and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, such as polysorbates 20, polysorbates 80, etc., triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (See, Remington's Pharmaceutical Sciences, 23rd Edition, (edited by Adejare), 2020, Elsevier Academic Press).

[0219] The primary vehicle or carrier in a pharmaceutical composition can be aqueous or non-aqueous in nature. Suitable vehicles or carriers for such compositions include water for injection, saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Compositions comprising LILRB2 antibody products for storage can be prepared by mixing a selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or aqueous solution. Additionally, the LILRB2 antibody products can be formulated into lyophilized products using suitable excipients such as sucrose.

[0220] The formulation components are present in a concentration acceptable at the site of administration. It is advantageous to use a buffer to maintain the composition at physiological pH or slightly lower pH, generally in the pH range of about 4.0 to about 8.5, or alternatively about 5.0 to 8.0. The pharmaceutical composition can comprise a TRIS buffer with a pH of about 6.5 - 8.5 or an acetate buffer with a pH of about 4.0 - 5.5, and the buffer can further comprise sorbitol or a suitable alternative thereof.

[0221] Additional pharmaceutical compositions are in the form of sustained release or controlled release formulations. Techniques for formulating various other sustained release or controlled release means can be used, such as liposomal carriers, bioerodible microparticles or porous beads and depot injections (see, for example, PCT Publication No. WO 93 / 15722A1, which describes the controlled release of porous polymeric microparticles for delivering pharmaceutical compositions). Sustained release formulations can include semipermeable polymeric matrices in the form of shaped articles (e.g., membranes or microcapsules, polyesters, hydrogels, polylactic acid) (U.S. Patent Nos. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 1983 22:547 - 56), poly(2-hydroxyethyl methacrylate) (Langer et al., J Biomed Mater Res 1981 15:167 - 277) and Langer, Chem Tech 1982 12:98 - 105), ethylene vinyl acetate (Langer et al., ibid.) or poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, for example, Eppstein et al., Proc Natl Acad Sci USA 1985 82:3688 - 92; EPO Publication Nos. EP 036676; EP088046 and EP 143949.

[0222] Once the pharmaceutical composition has been formulated, the pharmaceutical composition can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized).

[0223] The components used to formulate the pharmaceutical composition preferably have a high degree of purity and are substantially free of potentially harmful contaminants (e.g., at least National Formulary (NF) grade, usually at least analytical grade, and more usually at least pharmaceutical grade). In addition, compositions for in vivo use are generally sterile. For a given compound that must be synthesized prior to use, the resulting product is generally substantially free of any potentially toxic agents, particularly any endotoxins that may be present in the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

[0224] Kits are provided for multi-dose or single-dose administration units. For example, the kits can each contain both a first container having the dried protein and a second container having an aqueous diluent, including, for example, single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes, lyophilized syringes, or needleless syringes).

[0225] The pharmaceutical composition can be delivered parenterally, typically by injection. The injection can be intravitreal, intraperitoneal, intraportal, intramuscular, intravenous, intrathecal, intracerebral (intraparenchymal), intraventricular, intraarterial, intralesional, perilesional, or subcutaneous. Eye drops can be used for intravitreal administration. In some cases, the injection can be localized to a particular bone or near a bone targeted for treatment. For parenteral administration, the antibody can be administered as a pyrogen-free, parenterally acceptable aqueous solution that contains the desired LILRB2 antibody product in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the LILRB2 antibody product is formulated into a properly preserved sterile isotonic solution.

[0226] A pharmaceutical composition comprising a subject LILRB2 antibody product can be administered by bolus injection or continuously by infusion, by an implantable device, a sustained release system, or other means for achieving delayed release. The pharmaceutical composition can also be administered locally via an implanted membrane, sponge, or another suitable material on which the desired molecule has been absorbed or encapsulated. When an implantable device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed release bolus, or continuous administration. The preparation can be formulated with agents such as injectable microspheres, bioerodible particles, polymeric compounds such as polylactic acid, polyglycolic acid, or copolymers (lactic / glycolic acid) (PLGA), beads, or liposomes, which can provide controlled or sustained release of the product, and then the product can be delivered via a depot injection. Formulations with hyaluronic acid have the effect of promoting circulation duration.

[0227] The subject compositions comprising LILRB2 antibody products can also be used ex vivo. In this case, cells, tissues, or organs removed from a patient are exposed to or cultured with the LILRB2 antibody product. The cultured cells can then be re-implanted into the patient or another patient, or used for other purposes.

[0228] Delivery of the LILRB2 antibody product can be accomplished by implanting certain cells that have been genetically engineered by the methods described herein to express and secrete the polypeptide. Such cells can be animal or human cells, and can be autologous, allogeneic, or xenogeneic, or can be immortalized. To reduce the chance of an immune response, the cells can be encapsulated to avoid infiltration by the surrounding tissue. The encapsulation material is typically a biocompatible, semi-permeable polymeric shell or membrane that allows release of one or more protein products, but prevents the cells from being destroyed by the patient's immune system or by other harmful agents from the surrounding tissue.

[0229] As used herein, "substantially pure" means that the described molecular species is the major species present, i.e., on a molar basis, the species is more abundant than any other individual species in the same mixture. A substantially pure molecule can be a composition in which the target species comprises at least 50% (on a molar basis) of all macromolecular species present. A substantially pure composition can comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. The target species can also be purified to substantial homogeneity, where contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

[0230] Dose

[0231] The provided pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatment.

[0232] As used herein, the terms “treatment,” “treating,” etc. refer to the administration of an agent or the performance of a procedure in order to obtain an effect. The effect is prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or the effect is therapeutic in terms of partially or completely curing a disease and / or symptoms of the disease. As used herein, “treatment” includes the treatment of a disease or disorder (e.g., cancer) in a mammal, specifically a human, and includes: (a) preventing the occurrence of a disease or symptoms of a disease in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., including a disease related to or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress. Treatment refers to any clinical indicator of successful treatment or improvement or prevention, including any objective or subjective parameter, such as, alleviation; remission; diminution of symptoms or making the disease condition more tolerable to the patient; slowing the rate of degeneration or debilitation; or making the terminal stages of deterioration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Thus, the term “treatment” includes the administration of a compound or agent of the present disclosure to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term “treatment effect” refers to the alleviation, elimination, or prevention of a disease, symptoms of a disease, or side effects of a disease in a subject. For example, a subject is “treated” for a disease or disorder if, after receiving a therapeutically effective amount of a combination of the LILRB2 antibody products provided herein, the patient exhibits one or more observable and / or measurable changes in the endpoints or symptoms of the disease condition.

[0233] An “effective response” according to the present disclosure is achieved when a subject experiences partial or complete remission or alleviation of the signs or symptoms of a disease, and in the case of treating cancer, specifically includes, but is not limited to, symptom improvement, delayed progression, cure, remission, extended survival, or other objective responses. The expected progression-free survival can be measured in months to years, depending on prognostic factors, including the number of recurrences, disease stage, and other factors. Extended survival includes, but is not limited to, a time period of at least 1 month (mo.), at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, etc. Overall survival can also be measured, e.g., in months to years. Alternatively, an effective response can be that the symptoms of the subject remain static.

[0234] A therapeutic agent is administered in a prophylactic method before the appearance of symptoms of an undesired disease or disorder, such that the disease or disorder is prevented, or alternatively, its progression is delayed. Thus, when used in combination with a prophylactic method, the term "therapeutically effective" means that, after treatment, a minority of subjects (on average) will develop the undesired disease or disorder or progression of symptom severity

[0235] The terms "recipient", "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject, specifically a human, who is the subject of a desired diagnosis, treatment, or therapy. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as laboratory, zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. A mammal can be a human

[0236] Typically, the toxicity and therapeutic efficacy of an antibody product can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, which include, for example, determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 . Compositions exhibiting a large therapeutic index are preferred

[0237] Data obtained from cell cultures and / or animal studies can be used to formulate a range of doses for humans. The dose of the active ingredient is generally within the range of circulating concentrations that include an ED 50 with very low or almost no toxicity. The dose can vary within this range depending on the dosage form used and the route of administration employed

[0238] The effective amount of a pharmaceutical composition comprising an LILRB2 antibody product to be used therapeutically or prophylactically will depend, for example, on the therapeutic context and the target. Those skilled in the art will understand that, accordingly, the appropriate dosage level for treatment will vary in part depending on the molecule being delivered, the indication for using the LILRB2 antibody, the route of administration, and the body size (weight, body surface, or organ size) and / or condition (age and general health status) of the patient. The clinician can titrate the dose and modify the route of administration to obtain the optimal therapeutic effect. Depending on the above factors, typical doses range from about 1 mg / kg to up to about 1600 mg / kg or more. The dose range can be from 1 mg / kg to about 200 mg / kg; or from 1 mg / kg to about 1200 mg / kg; or from 1 μg / kg to about 1600 mg / kg

[0239] The dosing frequency will depend on the pharmacokinetic parameters of the LILRB2 antibody product in the formulation. For example, the clinician will administer the composition until a dose is reached that achieves the desired effect. Thus, the composition can be administered as a single dose, or as two or more doses over time (the doses can contain the same amount of the desired molecule), or as a continuous infusion via an implant device or catheter. The treatment can be continuous or intermittent over time. Further refinement of the appropriate dose is routinely performed by those of ordinary skill in the art and is within the scope of their routine tasks. The appropriate dose can be determined by using appropriate dose-response data. An exemplary dosing schedule is every two to three weeks.

[0240] To treat a disease condition by targeting LILRB2, a composition comprising the subject LILRB2 antibody product is administered to a patient at a dose and for a time sufficient to induce a sustained improvement in at least one indicator reflecting the severity of the disease. An improvement is considered "sustained" if the patient exhibits at least two improvements spaced at least one to seven days apart, or in some cases one to six weeks apart. The appropriate interval will depend to some extent on the disease condition being treated; determining the appropriate interval for determining whether an improvement is sustained is within the purview of the skilled physician. The degree of improvement is determined based on signs or symptoms, and a questionnaire administered to the patient, such as a quality of life questionnaire, can also be employed.

[0241] Various indicators reflecting the degree of the patient's disease can be evaluated to determine whether the amount and duration of treatment are sufficient. The baseline value of the selected one or more indicators is determined by examining the patient prior to administration of the first dose of the antibody. Preferably, the baseline examination is performed within about 60 days after administration of the first dose. If the antibody is administered for the treatment of acute symptoms, such as for treating a fracture, the first dose is administered as soon as possible after the injury occurs.

[0242] Improvement is induced by administering the subject LILRB2 antibody product until the patient exhibits an improvement in the selected one or more indicators relative to the baseline. In treating a chronic disease, this degree of improvement is achieved by repeatedly administering the drug over a period of at least one month or longer, e.g., one, two, or three months or longer or indefinitely. A period of one to six weeks, or even a single dose, is generally sufficient to treat an acute condition. For an injury or acute condition, a single dose may be sufficient.

[0243] Although the degree of the patient's disease may improve after treatment based on one or more indicators, the treatment can be continued indefinitely at the same level or at a reduced dose or frequency. Once treatment is reduced or stopped, if the symptoms reappear, then treatment can be resumed at the original level.

[0244] Method of Use

[0245] The LILRB2 antibody products disclosed herein have multiple uses. For example, some of the antibodies and fragments can be used in specific binding assays, affinity purification of LILRB2 or its ligands, and screening assays for identifying other antagonists of LILRB2 activity. The antibody products can be used to treat various diseases associated with the activity of LILRB2.

[0246] The LILRB2 antibody products can be used to detect LILRB2 in biological samples. Such uses allow for the identification of cells or tissues that produce the protein, or as a diagnosis for detecting pathological conditions of overproduction or underproduction of LILRB2. Strong expression of LILRB2 by macrophages, osteoclasts, and other myeloid cells can be a marker of the activity of these cells, and detecting LILRB2 expression on myeloid cells can be used as a marker for diseases or disorders characterized by the cell types in question. Similarly, detecting LILRB2 expression by cancer cells can be used to identify subjects whose cancer may be amenable to treatment with the therapeutic LILRB2 antibody product methods disclosed herein.

[0247] Accordingly, methods for detecting cell activity in biological samples, such as samples of in vitro culture media or tissue samples from a subject, or in vivo in a subject, are provided, wherein the method comprises contacting cells expressing LILRB2 with the LILRB2 antibody products provided herein. The LILRB2 antibody products can be conjugated to a detectable moiety, and the method comprises directly detecting the moiety. The method can comprise indirectly detecting the binding of the LILRB2 antibody product to the cells by a detectable moiety bound to the antibody. For example, an IgG antibody conjugated to a detectable moiety can be used to bind to the LILRB2 antibody presented as an IgG isotype. The cells can be tumor cells. The cells can be myeloid cells (e.g., monocytes, dendritic cells, macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, immunosuppressive macrophages, or M2-like macrophages) or osteoclasts.

[0248] The provided antibody products can also be used in methods for screening for molecules that bind to LILRB2. For example, various competitive screening methods can be used. In some methods, the LILRB2 molecule or a fragment thereof to which the LILRB2 antibody product binds is contacted with the antibody product disclosed herein along with another molecule (i.e., a candidate molecule). A decrease in the binding between the antibody product and LILRB2 indicates that the candidate molecule binds to LILRB2. The binding of the antibody product can be detected using a variety of methods, such as ELISA. Detection of the binding between the LILRB2 antibody product and LILRB2 can be simplified by detectably labeling the antibody. In some methods, molecules that exhibit binding in the initial screening are further analyzed to determine whether the molecule inhibits or modulates LILRB2 activity.

[0249] The LILRB2 antibody products provided herein can be used to treat human diseases including cancer.

[0250] The LILRB2 antibody products described herein can be used to treat cancer alone or in combination with another anti-cancer therapeutic agent. The cancer to be treated is a cancer in which cancer cells are known to express LILRB2 or belong to a type previously observed to express LILRB2. Certain cancers belonging to the EGFR mutant have been found to express LILRB2 at a higher level, and thus treatment with the antibodies disclosed herein is contemplated. In contrast, the expression of LILRB2 in cancer cells has also been found to be negatively correlated with the expression of PD-L1. Thus, anti-LILRB2 treatment as described herein is also contemplated in the absence of PD-L1 expression by cancer cells. This treatment is indicated when therapeutic intervention in the PD-1 / PD-L1 axis is ineffective or expected to be ineffective.

[0251] Provided herein is a method for treating a patient with cancer, wherein the LILRB2 antibody product mediates the killing of cancer cells.

[0252] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and hematogenous tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. The term "cancer" further encompasses both primary cancer and metastatic cancer.

[0253] Examples of cancers that can be treated by the methods and compositions provided herein include, but are not limited to, bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastrointestinal cancer, rectal cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nasopharyngeal cancer of the oral cavity, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tongue cancer, and uterine cancer.

[0254] Cancers to be treated include, for example, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, Hodgkin's disease; Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, hepatocellular carcinoma, head and neck cancer, kidney cancer, melanoma, malignant mesothelioma, nasopharyngeal cancer, neuroblastoma, glioblastoma, pancreatic cancer, multiple myeloma, prostate cancer, small cell lung cancer, non-small cell lung cancer, and metastatic cancer. Cancers to be treated include, for example, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell carcinoma, gastric cancer, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid carcinoma, cutaneous squamous cell carcinoma, or ovarian cancer.

[0255] In addition, cancer can specifically have the following histological types, although not limited to these types: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous gland carcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant androblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant liposarcoma; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant cellular blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma;Stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant stromal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligoastrocytoma; primitive neuroectoderm; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides or cutaneous T cell lymphoma; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myelosarcoma; and hairy cell leukemia.

[0256] For example, a method for treating a subject having melanoma is provided. As used herein, "melanoma" refers to a condition characterized by the growth of tumors arising from the melanocyte system of the skin and other organs. Most melanocytes are present in the skin, but are also present in the meninges, digestive tract, lymph nodes, and eyes. When melanoma occurs in the skin, it is referred to as cutaneous melanoma. Melanoma can also occur in the eyes and is referred to as ocular or intraocular melanoma. Melanoma rarely occurs in the meninges, digestive tract, lymph nodes, or other areas where melanocytes are found.

[0257] Cancer cells treated with the methods provided herein can express LILRB2. Cancer cells can overexpress LILRB2.

[0258] One way by which LILRB2 antibody products can mediate cancer cell killing is through antibody-dependent cell cytotoxicity (“ADCC”). ADCC is the process by which an antibody coats a target cell (such as a cancer cell or a bacterial cell) and recruits effector cells to induce the death of the target cell via a non-phagocytic mechanism.

[0259] As described above, in a method of treatment, the LILRB2 antibody products provided herein can be used as a single therapy or in combination therapy. “Combination” refers to the administration of one therapeutic agent before, during, or after the administration of another therapeutic agent to a subject.

[0260] In combination therapy for cancer, the LILRB2 antibody products are used in combination with one or more other anti-cancer modalities for treating cancer in a subject. The anti-cancer modality can be chemotherapy or a biomolecule. The anti-cancer modality can be an immunotherapeutic molecule. The immunotherapy can be a checkpoint inhibitor. The checkpoint inhibitor can be a PD-1 antagonist. The checkpoint inhibitor can be a PD-L1 antagonist.

[0261] Provided herein are methods of treating a patient having cancer, the method comprising administering to the patient a combination of a LILRB2 antibody product as described herein and an immunotherapy.

[0262] As an example of a combination with an immunotherapy, provided are methods of treating a patient having cancer, the method comprising administering to the patient a therapeutically effective amount of a LILRB2 antibody product and one or more immune checkpoint inhibitors.

[0263] The term “immune checkpoint inhibitor” or “checkpoint inhibitor” generally refers to an agent that modulates an immune checkpoint protein (“checkpoint protein”). A checkpoint inhibitor can effect a complete or partial reduction, inhibition, interference with the activity of a checkpoint protein, or other changes to the structure of a checkpoint protein that alter the binding of the checkpoint protein to a ligand, and / or affect a pathway associated with the activity of a checkpoint protein, e.g., by acting as an antagonist of a checkpoint protein or a ligand of a checkpoint protein. An immune checkpoint inhibitor can be a compound, such as an antibody or other protein, that binds and antagonizes human programmed cell death protein 1 (PD-1; also known as PDCD1, CD279) or programmed cell death ligand 1 (PD-L1; also known as BZ-H1, CD274). Such immune checkpoint inhibitors are referred to as PD-1 antagonists and PD-L1 antagonists, respectively.

[0264] As disclosed herein, in certain environments and states, checkpoint proteins can interfere with T cell-mediated cancer cell killing. Checkpoint inhibitors can reverse the interference of checkpoint proteins, but in certain types of cancer (e.g., certain solid tumors), the interference with checkpoint proteins is insufficient. The present disclosure contemplates combining the antibody products provided herein with another checkpoint inhibitor to alleviate macrophage-mediated T cell exhaustion and stimulate T cell effector function.

[0265] Checkpoint inhibitors can inhibit one or more checkpoint proteins. Non-limiting examples of checkpoint proteins include: PD-1, CD28, CTLA-4, ICOS, TMIGD2, 4-1BB, BTLA, CD160, LIGHT, LAG3, OX40, CD27, CD40L, CD47, GITR, DNAM-1, TIGIT, CD96, PVRIG 2B4, TIM-3, galectin-9, CEACAM1, SIRPα, DC-SIGN, CD200R, DR3, CDCHK1, CHK2, A2aR or B-7 family proteins.

[0266] Checkpoint inhibitors can interact with ligands of checkpoint proteins. For example, by non-limiting examples, ligands of checkpoint proteins include: PD-L1, PD-L2, ICOS ligand, VISTA, 4-1BBL, herpesvirus entry mediator (HVEM), tumor necrosis factor receptor superfamily member 14 or TNFRSF14, MHC class I, MHC class II, PVR, OX-40L, CD70, CD40, GITRL, CD155, CD48, GAL9; HMGB1, CEASAM-1, phosphatidylserine (PtdSer), IDO, TDO, CD47, BTN2A1, CD200, TL1A, CD112, CD155, MHCII, LSECtin, CHK1, CHK2, A2aR or B-7 family ligands (e.g., CD80 (B7-1), CD86 (B7-2), B7-H3, B7-H4, B7-H7 (HHLA2), etc.).

[0267] Checkpoint inhibitors can be antagonists. For example, checkpoint inhibitors can antagonize checkpoint proteins. Checkpoint inhibitors can be antagonists of ligands of checkpoint proteins. Antagonists can be biomolecules, such as biotherapeutic agents. Checkpoint inhibitors can be antibodies or antigen-binding portions thereof, such as monoclonal antibodies, humanized antibodies, fully human antibodies, fusion proteins or combinations thereof. Checkpoint inhibitors can be small molecules. Checkpoint inhibitors can be rationally designed peptides. Checkpoint inhibitors can be cells or cell preparations (e.g., cells expressing checkpoint inhibitors).

[0268] Checkpoint inhibitors can inhibit PD-1. Programmed cell death 1 (PD-1) is a key checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. Among other things, PD-1 restricts the activity of T cells in peripheral tissues during the inflammatory response to infection. In addition, as a checkpoint protein, PD-1 blockade can enhance T cell proliferation and cytokine production in response to stimulation by specific antigen targets or allogeneic cells in a mixed lymphocyte reaction.

[0269] Without being bound by theory, it is believed that blocking PD-1 in combination with an LILRB2 antibody product as disclosed herein alleviates macrophage-mediated T cell inhibition / depletion, increases T cell proliferation and cytokine production, and improves immune cell effector function. PD-1 blockade can be accomplished by a variety of mechanisms. For example, PD-1 blockade can be achieved by blocking the binding of PD-1 to its ligand. PD-1 can be blocked with checkpoint inhibitors that act as PD-1 antagonists. For example, a PD-1 antagonist can be a PD-1 antibody (e.g., nivolumab, pembrolizumab, etc.). A PD-1 antagonist can be a small molecule [e.g., INCB-086550 (Incyte) or small molecules disclosed in, for example, Wu et al., Acta Pharmacol Sin. 2021 42:1-9; Jiao et al., Curr Pharm Des. 2018 24(41):4911-20; and Liu et al., Cancer Cell Int. 2021 21(1):239]. A PD-1 antagonist can be or can comprise a rationally designed peptide (e.g., APi2568). A PD-1 antagonist can be or can comprise a cell or cell preparation (e.g., a cell expressing a PD-1 binder, e.g., a PD-1 antibody, e.g., HerinCAR-PD1).

[0270] Exemplary PD-1 antibodies suitable for the methods include, but are not limited to, nivolumab (ONO-4538, BMS-936558, MDX1106, ; Bristol-Myers Squibb), pembrolizumab (MK-3475, ; Merck), cemiplimab (e.g., cemiplimab-rwlc (Libtayo TM ; Regeneron)), dostarlimab (e.g., dostarlimab-gxly (Jemperli TM; GlaxoSmithKline, pimivalimab (IgG4) (JTX-4014; Zeus Therapeutics), spartalizumab (IgG4) (PDR001; Novartis), camrelizumab (SHR1210; Jiangsu HengRui Medicine), sintilimab (IBI308; Innovent and Eli Lilly), tislelizumab (BGBA317; BeiGene), toripalimab (JS 001; Shanghai Junshi Bioscience), INCMGA00012 (MGA012; Incyte and MacroGenics), AMP-224 (PD-L2 / Ig fusion; AstraZeneca / MedImmune and GlaxoSmithKline), AMP-514 (IgG4κ) (MEDI0680; AstraZeneca), balstilimab (AGEN2034; Agenus) and / or a PD-1 binding domain of any of them.

[0271] Another exemplary PD-1 antagonist is a rationally designed peptide, such as APi2568, which contains a B cell epitope (amino acids 92-110 from PD-1), said epitope being linked via a 4-amino acid linker to a promiscuous T cell epitope (amino acid residues 288-302 from the measles virus fusion protein), and combined with water for injection (WFI) to form the drug IMU-201, which becomes PD1-Vaxx when emulsified with the excipient Montanide ISA 720VG.

[0272] Another exemplary PD-1 antagonist is a cell expressing a PD-1 antibody, such as a CAR-T cell expressing a PD-1 antibody (e.g., HerinCAR-PD1 cells).

[0273] In some embodiments, the checkpoint inhibitor is a PD-L1 antagonist, such as a PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: avelumab, durvalumab, atezolizumab, envafolimab, cosibelimab, LY3300054, CA-170, BMS-936559, and PD-L1 binding fragments or combinations thereof. In some embodiments, the PD-L1 antagonist comprises AUNP-12, BMS-986189, a PD-L1 binding domain comprising the CDRs of an antibody selected from the group consisting of: avelumab, durvalumab, atezolizumab, envafolimab, cosibelimab (CK-301), LY3300054, CA-170, and BMS-936559, and active fragments or combinations thereof.

[0274] Checkpoint inhibitors can inhibit cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or its ligand. CTLA-4 antibodies bind to CTLA-4 and block the interaction of CTLA-4 with its ligands CD80 / CD86, which are expressed on antigen-presenting cells. Thus, CTLA-4 inhibitors that block the interaction of CTLA-4 with its ligand can block the negative downregulation of the immune response triggered by the interaction of these molecules. Thus, checkpoint inhibitors can be CTLA-4 antagonists, such as those described in U.S. Patent Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. In addition, exemplary CTLA-4 antibodies include: ipilimumab (10D1, MDX-D010, Yervoy TM ; Bristol-Myers Squibb Company), tremelimumab (ticilimumab, CP-675,206; MedImmune), and quavonlimab (MK-1308; Merck). CTLA-4 antagonists can comprise the CTLA-4 binding domain or fragments thereof of any CTLA-4 antagonist. CTLA-4 antagonists can comprise small molecules (see, e.g., Wang et al., Biochim Biophys Acta Rev Cancer. 2019 1871(2):199-224).

[0275] Lymphocyte activation gene 3 (LAG-3, also known as CD223) is a CD4-related transmembrane protein that competitively binds to MHC II and serves as a co-inhibitory checkpoint for T cell activation [e.g., Goldberg and Drake, Curr Top Microbiol Immunol. 2011 344:269-78]. Checkpoint inhibitors can be LAG3 antagonists. LAG3 antagonists can be LAG-3 binding proteins (e.g., antibodies) or proteins that bind to LAG3 ligands. Non-limiting examples of LAG-3 antibodies include: LAG525 (IMP701, Novartis / PrimaBiomed), MK-4280 (Merck Sharp & Dohme), REGN3767 (Regeneron Pharmaceuticals), relatlimab (BMS-986016, Bristol-Myers Squibb), and BI 754111 (Boehringer Ingelheim).

[0276] T cell immunoglobulin mucin 3 (TIM-3, also known as hepatitis A virus cellular receptor (HAVCR2)) is a type I glycoprotein receptor that binds to the S-type lectin galectin-9 (Gal-9). TIM-3 is a ligand that is widely expressed in lymphocytes, liver, small intestine, thymus, kidney, spleen, lung, muscle, reticulocytes, and brain tissue. Binding of the TIM-3 receptor to Gal-9 triggers downstream signaling to negatively regulate T cell survival and function. A checkpoint inhibitor can be an agent that inhibits TIM-3. A checkpoint inhibitor can be a TIM-3 antagonist, such as a TIM3 antibody or an antibody against a TIM-3 ligand. A TIM-3 antagonist can comprise the TIM-3 binding domain of any TIM-3 antagonist or a fragment thereof. Non-limiting examples of TIM-3 antagonists include: TSR-022 (AnaptysBio / Tesaro, Inc.) and MGB453 (Novartis). Additional exemplary TIM-3 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Patent Nos. 9,103,832, 8,552,156, 8,647,623, 8,841,418; U.S. Patent Publications 2016 / 0200815, 2015 / 0284468, 2014 / 0134639, 2014 / 0044728, 2012 / 0189617, 2015 / 0086574, 2013 / 0022623; and PCT Publications WO2016 / 068802, WO2016 / 068803, WO2016 / 071448, WO2011 / 155607, and WO2013 / 006490.

[0277] T cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on lymphocytes. TIGIT interacts with CD155 expressed on antigen-presenting cells or tumor cells to downregulate T cell and natural killer (NK) cell functions. Checkpoint inhibitors can be TIGIT antagonists. TIGIT antagonists can bind to TIGIT or to TIGIT ligands. TIGIT antagonists can be TIGIT antibodies or antibodies against TIGIT ligands. TIGIT antagonists comprise the TIGIT-binding domain or a fragment thereof of any TIGIT antagonist. Non-limiting examples of TIGIT antagonists include: Tiragolumab (MTIG7192A; RG6058) (Genentech / Roche), AB154 (Arcus Bioscience), Vibostolimab (MK-7684) (Merck), BMS-985207 (Bristol Myers Squibb), ASP8374 (Astellas Pharma; Potenza Therapeutics), and ASP8374 (Astellas Pharma; Potenza Therapeutics).

[0278] Exemplary anti-CD27 agonists include MK-5890 (Merck).

[0279] Exemplary ICOS antibodies include Vopratelimab (JTX-2011; Zeus).

[0280] In some embodiments, the LILRB2 antibody product and the immune checkpoint inhibitor are co-formulated. In some embodiments, the LILRB2 antibody product and the immune checkpoint inhibitor are in separate formulations. In some embodiments, the LILRB2 antibody is administered in combination with co-formulations of PD-1 antagonists (such as PD-1 antibodies) and CTLA-4 antagonists (such as CTLA-4 antibodies), controlling the dose of each component to provide a safe and effective treatment for the subject. In some embodiments, the LILRB2 antibody is administered in combination with the co-formulation of Pembrolizumab / Zivolimab (MK-1308A; Merck).

[0281] Also provided are methods of treating cancer, the methods comprising administering a therapeutically effective amount of the LILRB2 antibody products provided herein and a colony stimulating factor 1 (CSF1) antagonist. Colony stimulating factor 1 receptor (CSF1R) inhibitors are being developed for cancer therapy. Canarile et al., J Immunother Cancer. 2017 5(a):53. For example, pexidartinib (PLX-3397) has been shown to alter the distribution of tumor-associated macrophages in the tumor microenvironment and promote the enrichment of macrophages with an M1-like phenotype. The CSF1 antagonist can be a CSF1 antibody or a CSF1R inhibitor. Such CSF1 antagonists include, for example, pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, emactuzumab, AMG820, IMC-CS4, MCS110, PD-0360324, and cabiralizumab.

[0282] Also provided are methods of treating cancer, the methods comprising administering a therapeutically effective amount of the LILRB2 antibody products provided herein and an anti-CD40 antibody antagonist.

[0283] Also provided are methods of treating cancer, the methods comprising administering a therapeutically effective amount of the LILRB2 antibody products provided herein and an inhibitory anti-CD47 antibody.

[0284] Also provided are methods of treating cancer, the methods comprising administering a therapeutically effective amount of the LILRB2 antibody products provided herein and an effective amount of a class II histone deacetylase (HDAC) inhibitor, such as TMP195.

[0285] Also provided are methods of treating cancer, the methods comprising administering a therapeutically effective amount of the LILRB2 antibody products provided herein and a TLR7 or TLR8 agonist. The toll-like receptors TLR7 and TLR8 appear to be involved in macrophage polarization in the tumor microenvironment. Agonists of one or both of these receptors can promote the functional orientation of tumor-associated macrophages toward an M1-like phenotype. β-Cyclodextrin nanoparticles loaded with resiquimod have been reported to have antitumor effects, which can be enhanced in the presence of a PD-1 antagonist. Rodell et al., Nat Biomed Eng. 2018 2:578–88. Such TLR7 / TLR8 agonists include, for example, resiquimod (B848), motolimod (VTX-2337), and imiquimod.

[0286] Also provided are methods of treating a disease condition of a subject, wherein the disease condition is characterized by or mediated by LILRB2 expression by myeloid cells. The myeloid cells can be macrophages. The myeloid cells can be osteoclasts or osteoclast precursors. The methods comprise administering to the subject a therapeutically effective amount of an LILRB2 antibody product disclosed herein.

[0287] Also provided are methods of treating or preventing a bone metabolism disorder in a subject. The bone metabolism disorder can be osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or cancer metastasis to bone, giant cell tumor, osteopenia, tooth loss caused by periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, or osteogenesis imperfecta. The bone metabolism disorder can be osteoporosis. The osteoporosis can be postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis caused by the use of therapeutic agents such as steroids or immunosuppressants, or osteoporosis associated with rheumatoid arthritis.

[0288] Other Terms

[0289] As used herein, unless the context clearly indicates otherwise, the singular forms "a," "and," and "the" include plural referents. Thus, for example, reference to "an antibody" includes a plurality of antibodies.

[0290] As used herein, unless the context clearly indicates otherwise, all numerical values or numerical ranges include the whole integers within such ranges or covering such ranges and fractions of values or integers within such ranges or covering such ranges. Thus, for example, reference to a range of 90 - 100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another example, reference to a range of 1 - 5,000 - fold includes 1 - fold, 2 - fold, 3 - fold, 4 - fold, 5 - fold, 6 - fold, 7 - fold, 8 - fold, 9 - fold, 10 - fold, 11 - fold, 12 - fold, 13 - fold, 14 - fold, 15 - fold, 16 - fold, 17 - fold, 18 - fold, 19 - fold, or 20 - fold, etc., and 1.1 - fold, 1.2 - fold, 1.3 - fold, 1.4 - fold, or 1.5 - fold, etc., 2.1 - fold, 2.2 - fold, 2.3 - fold, 2.4 - fold, or 2.5 - fold, etc., and so on.

[0291] As used herein, "about" a number refers to a range that includes the recited number and ranges from 10% less than the recited number to 10% more than the recited number. An "about" range refers to a range that is 10% less than the lower limit of the recited range and extends to 10% more than the upper limit of the recited range.

[0292] As used herein, "can", "can be", "may", or "may be" all denote something that is envisioned by the inventors as being functional and useful as part of the subject matter provided.

[0293] Examples

[0294] While the following examples describe specific embodiments, those skilled in the art will envision variations and modifications. Accordingly, only those limitations that appear in the claims apply to the invention.

[0295] To generate anti-human LILRB2 antibodies from rabbit B cells, rabbits were immunized with human LILRB2 protein. B cells from the immunized rabbits were cultured at clonal density and the supernatant was evaluated for IgG antibodies for (a) binding to human and cynomolgus LILRB2 detected by enzyme-linked immunosorbent assay (ELISA), (b) blocking the binding of LILRB2 to HLA-G, (c) binding to cells expressing LILRB2, and (d) lack of binding to other LILR (A / B) family members. The variable regions from the positive hits were sequenced, cloned, and expressed as recombinant rabbit / human IgG4 and IgG1 Fc chimeras.

[0296] Top clones were selected based on activity in a panel of functional and phenotypic assays using primary human macrophages and T cells and anti-tumor activity in xenograft models. The selected clones were humanized by computer methods and the ability of the humanized antibodies to rescue T cell functional activity (activation and proliferation) from M2c macrophage-mediated immunosuppression was screened in vitro.

[0297] Example 1: Immunization, Cloning, and Initial Screening

[0298] The antibody designated B2A is among the antibodies cloned from B cells derived from rabbits immunized with human LILRB2 protein. Briefly, two female New Zealand white rabbits were immunized with purified human LILRB2 extracellular domain (ECD)–rabbit Fc fusion protein (SEQ ID NO:50) (OncoResponse) using standard immunization methods. Boosters were administered to the rabbits on days 21, 42, and 73 after the primary immunization. Pre-immune bleeds and test bleeds were evaluated for specific antibody titers by indirect ELISA. On day 83 after the primary immunization, heparinized whole blood was collected for rabbit monoclonal antibody development. B cells from the peripheral blood of both rabbits were collected after the last booster and then isolated, purified, and cultured at clonal density using proprietary methods. Biopanning was performed using human LILRB2 ECD–human Fc fusion protein (SEQ ID NO:51) (OncoResponse). B cell culture supernatants from 80 96-well plates were transferred to ELISA plates coated with human Fc LILRB2 ECD fusion protein. Indirect ELISA was performed using a secondary anti-rabbit IgG antibody (ImmunoPrecise). To recover antibodies specific for the target antigen, negative screening of positive candidates was performed against an irrelevant human Fc fusion protein (ImmunoPrecise). The ability of B cell supernatants to block the binding of LILRB2-Fc (R&D Systems, catalog number 2078-T4) to HLA-G was also evaluated. The top 96 responsive wells were stored in standard RNA lysis buffer (ImmunoPrecise) for antibody RNA isolation and recombinant plasmid DNA generation.

[0299] The rabbit antibody heavy and light (κ) chain variable regions were cloned into separate mammalian expression vectors containing the human IgG heavy and κ constant regions for the top-ranked positive clones. Recombinant monoclonal antibodies were generated using transfected HEK-293 cells, purified using standard methods, and evaluated in several biochemical and cell-based functional assays.

[0300] Example 2: Binding of Chimeric Antibodies to LILRB2 Detected by ELISA (Human and Cynomolgus Monkey)

[0301] To confirm binding to human and cynomolgus monkey LILRB2, the binding of antibodies in the supernatants was evaluated by enzyme-linked immunosorbent assay (ELISA). Recombinant LILRB2 protein (human: SEQ ID NO:48; cynomolgus monkey) was diluted to 2 μg / mL in PBS and added at 25 μL / well to a 384-well high-binding ELISA plate (Greiner Bio-One Microlon TM) and incubated overnight at 4°C. The plates were washed three times with wash buffer (0.05% in PBS 20) using a microplate washer and then blocked for 1 hour at room temperature (RT) with 90 μL / well of ELISA Blocking Buffer A (1% BSA in PBS). After blocking, 25 μL / well of LILRB2 antibody or isotype control was added to the plates and incubated for 1 hour at room temperature. After primary antibody binding, the plates were washed three times with wash buffer using a microplate washer. The secondary detection antibody (HRP-goat anti-rabbit Fab fragment) was diluted 1:3000 in assay diluent (1% BSA in PBS) and 25 μL / well was added to the plates and incubated for 1 hour at room temperature in the dark. After incubation, the plates were washed four times with wash buffer using a microplate washer. After removal of the final wash, 25 μL / well of 1-Step TM Ultra TMB-ELISA substrate solution (Thermo Fisher, catalog number 34028) was added and the plates were incubated for 5 - 10 minutes at room temperature in the dark. After development, the reaction was terminated by adding 25 μL / well of 0.3 M HCl and the plates were read at 450 nm using an EnVision (Perkin Elmer) microplate reader. The EC 50 value was calculated based on the log concentration of the primary antibody at the optical density at 450 nm. The binding of anti-LILRB2 B2A-IgG1 to human and cynomolgus monkey LILRB2 is presented in Figure 1a, showing binding to human LILRB2 with an EC 50 of 3.81 ng / mL but no binding to cynomolgus monkey LILRB2.

[0302] Example 3: Preparation of cells expressing human LILRB2

[0303] Generate cells stably expressing human LILRB2. Human embryonic kidney cells (HEK293T / 17; ATCC, CRL-11268) were cultured in DMEM medium (Gibco, cat. no. 11965-084) supplemented with 10% fetal bovine serum (FBS; HyClone, cat. no. SH30396.03; heat inactivated before use) according to ATCC guidelines. Pre-packaged lentiviral particles containing the puromycin-selectable lentiviral vector backbone were purchased from G&P Biosciences: without the gene of interest (negative control; cat. no. LTV0001) or with human LILRB2 (SEQ ID NO:52) (cat. no. LTV2992). HEK293T cells (5 x 10 4 ) were transduced with human LILRB2 lentiviral particles at a multiplicity of infection (MOI) of 10 for 24 hours in the presence of 8 μg / mL polybrene infection / transfection reagent (Millipore, cat. no. TR-1003-G) at 37 °C, 5% CO2. Then the virus-containing medium was removed, and the cells were allowed to recover in fresh medium for two days before selection in medium containing 0.75 μg / mL puromycin (Thermo Fisher Scientific, cat. no. A1113803). Stable LILRB2 expression was selected based on puromycin as a selection marker, and the cell population expressing LILRB2 was isolated and cultured. Expression of LILRB2 in the puromycin-resistant cell population was confirmed by flow cytometry using a BDFACSymphony TM cell analyzer and a LILRB2-specific antibody. In addition to LILRB2 transduction, cells were transduced with negative control lentiviral particles using the same protocol to generate a stable puromycin-resistant control cell line (containing the lentiviral vector backbone without gene insert) for assays.

[0304] Once target expression was confirmed, the cells were amplified. The selected cell polyclonal pool was stored at 4 - 5 million cells / mL / vial, and a master stock was prepared. Storage of the cells involved pelleting the cells from the culture by centrifugation, removing the medium, and resuspending the cells first in DMEM + 10% FBS at 4 °C, and then adding an equal volume of 2X ATCC-recommended freezing medium (DMEM + 10% FBS plus 10% DMSO) such that the cells were present at a density of 4 - 5 million / mL in 1X freezing medium (DMEM + 10% FBS plus 5% DMSO). Aliquots of the cell suspension were placed in a 4 °C Mr.Frosty TM alcohol-based slow freezing system and immediately stored at -80 °C. These frozen cell aliquots were stored at -80 °C for one day and then transferred to the liquid nitrogen vapor phase tank.

[0305] For determination, the master stock vial was thawed in a 37 °C water bath for one minute and centrifuged to pellet the cells. The cells were resuspended in DMEM + 10% FBS and the expression of the target protein was examined by flow cytometry. These cells were amplified in culture to prepare 30 working stocks and the expression of the target protein was examined by flow cytometry. These cells were amplified in culture to prepare 30 working stocks.

[0306] Example 4: Binding of LILRB2 antibody to cells expressing human LILRB2

[0307] To confirm the binding of the LILRB2 antibody, a flow cytometry binding assay (Example 3) was performed using HEK293T cells stably expressing human LILRB2. The modified HEK293T cells were incubated in blocking buffer B (FACS buffer (PBS, 1% FBS + 0.05% NaN3 (Ricca Chemical, cat. no. 7144.8 - 16, diluted), 2 mM EDTA) containing 10% FBS for 30 minutes at 4 °C to block non - specific binding. Titrations of the LILRB2 antibody conjugated to AF647 (Thermo Fisher Scientific, cat. no. 20106) and an isotype control antibody (Tumor Response, Inc.) were added directly on top of the HEK293T cells in blocking buffer B and then incubated for 1 hour at 4 °C. The cells were washed 2 times in FACS buffer and then incubated for 30 minutes with AF647 goat anti - human IgG Fc (Jackson ImmunoResearch, cat. no. 109 - 605 - 098). The cells were washed 2 times in FACS buffer and then stained with Zombie Violet fixable viability dye (BioLegend, cat. no. 423114) for 10 minutes at room temperature, washed with FACS buffer and resuspended in 100 μL of FACS buffer for acquisition on a BD FACSymphony TM or BD FACSCanto TM II flow cytometer (BD Biosciences). The mean fluorescence intensity (MFI) binding of the LILRB2 antibody was gated on live cells using FlowJO software (10.5.3, FlowJO, LLC) and GraphPad Prism for EC 50 binding calculations. The B2A - IgG1 and B2A - IgG4 chimeras bound to HEK293T cells expressing LILRB2 with equivalent EC 50 values of 0.21 nM and 0.30 nM, respectively. Figure 1BA representative example is shown.

[0308] Example 5: The LILRB2 antibody does not bind to other LILRB or LILRA family members

[0309] To determine that the anti-LILRB2 antibody specifically binds to LILRB2 and not to other LILRB and LILRA family members, cross-reactivity assays were performed. The cross-reactivity of the LILRB2 antibody with other LILRB proteins was evaluated by binding of the antibody to transiently transfected HEK293-6E cells, measured by flow cytometry. Binding to HEK293-6E cells transiently transfected with human LILRB-1, -2, -3, -4, and 5 plasmids (Origene, catalog numbers RC219949, RC217935, RC211228, RC220932, RC206516) or to cells transiently transfected with human LILRA-1, -2, -4, -5, and -6 plasmids (Origene, catalog numbers RC210808, RC205626, RC220452, RC212310, RC212965) was evaluated. AF647 mouse anti-human IgG-Fc secondary antibody (Jackson ImmunoResearch, catalog number 109-605-098) was used to detect the human anti-LILRB2 antibody bound to the cells. Mouse primary antibodies against LILRA and LILRB targets and corresponding isotype controls were used to confirm specific binding of all tested targets (R&D Systems), while AF647 F(ab′)2 fragment goat anti-mouse antibody (Jackson ImmunoResearch, catalog number 115-606-062) was used to detect the bound positive control antibody. The binding of B2A-IgG1 and B2A-IgG4 to other LILRA and LILRB family members is presented in Tables 1A and 1B. No binding of B2A-IgG4 or B2A-IgG1 to LILRB-1, -3, -4, or -5 was observed, nor was any antibody binding to LILRA-1, -2, -4, -5, or -6 observed, thus confirming the specificity of these antibodies.

[0310] Tables 1A and 1B - Flow cytometry binding of B2A-IgG1 and B2A-IgG4 to other LILR family members with selected chimeric clones

[0311]

[0312]

[0313] Example 6: The LILRB2 antibody blocks the binding of LILRB2-Fc to ANGPTL-2 and -5

[0314] Angiopoietins and angiopoietin-like (ANGPTL) proteins are secreted glycoproteins that play roles in angiogenesis, lipid metabolism, hematopoietic stem cell expansion, and inflammation. Angiopoietins signal through the tyrosine kinase receptors Tie1 or Tie2, while ANGPTL proteins are considered orphan ligands. ANGPTL-1, -2, -5, and -7 have been shown to bind to human leukocyte immunoglobulin-like receptor B2 (LILRB2). To determine whether anti-LILRB2 antibodies block the binding of ANGPTL-2 and -5 to human LILRB2, a blocking ELISA assay was performed. The proteins were purchased from R&D Systems (catalog numbers 9795-AN-050, 6675-AN-025 / CF) and reconstituted in PBS or water. A 384-well plate was coated with ANGPTL-2 or ANGPTL-5 at a concentration of 5 μg / mL and sealed and incubated overnight at 4°C. After incubation, the plate was washed with wash buffer and then blocked with Blocking Buffer C (3% BSA in PBS diluted + 0.05% 20) for 2 hours at 37°C. Anti-LILRB2 antibodies were incubated with LILRB2-Fc protein for 1 hour at room temperature. Blocking Buffer C was removed from the plate, and then the anti-LILRB2 antibody titration / LILRB2-Fc mixture was incubated in the wells overnight at 4°C. The plate was washed with wash buffer, and then goat anti-human IgG Fc biotinylated secondary antibody was diluted in assay buffer and added to the wells. The secondary antibody was incubated on the wells for 2 hours at room temperature and then washed with wash buffer. Streptavidin-HRP was diluted in assay buffer according to the manufacturer's instructions and incubated on the wells for 30 minutes at room temperature in the dark. The plate was washed with wash buffer, and then the manufacturer's reagent was added at a 1:1 dilution and incubated on the wells for 7.5 minutes at room temperature. Stop solution was added, and the absorbance was measured at 450 nm. As Figure 2 shown, B2A-IgG4 blocked the binding of LILRB2-Fc to ANGPTL-2 and -5 in a dose-dependent manner.

[0315] Example 7A: LILRB2 antibody B2A-IgG4 blocks the binding of LILRB2-Fc to HLA-G

[0316] Tumor-associated HLA-G is an HLA class I ligand for LILRB2. Binding of HLA-G to LILRB2 induces immunosuppressive signals in myeloid cells expressing LILRB2. To determine whether anti-LILRB2 antibodies block the HLA-G-LILRB2 interaction and prevent induction of inhibitory signals, a blocking ELISA assay was performed in a dose titration. A 384-well plate was coated with monomeric HLA-G at a concentration of 5 μg / mL (Fred Hutchinson Cancer Research Center, catalog number bHLA-G), diluted in PBS, and then the plate was incubated for 1 hour at room temperature. The plate was rinsed three times with wash buffer and then blocked with blocking buffer A for 30 minutes at room temperature. During the blocking incubation step, LILRB2-Fc protein and antibody titrations were prepared in blocking buffer A and then the LILRB2-Fc protein was incubated with the antibody titrations for 30 minutes at room temperature to allow binding before addition to the plate. The plate was rinsed three times with wash buffer and then 25 μL of the protein / antibody mixture was added to each well and incubated for 1 hour at room temperature. After antibody incubation, the plate was washed three times with wash buffer. The anti-human IgG Fc-HRP secondary antibody was diluted and added at 25 μL / well. The plate was incubated for 1 hour at room temperature in the dark. After secondary antibody incubation, the plate was washed four times with wash buffer. 25 μL / well of UltraPure TMB was added for development and incubated for 5 minutes at room temperature. The reaction was terminated using 0.3 M HCl (25 μL / well). Absorbance was read at 450 nM using Envision. The IC 50 for HLA-G blockade by clone B2A-IgG4 was 0.066 μg / mL.

[0317] Example 7B: LILRB2 antibody B2A-IgG4 blocks the binding of LILRB2-Fc to HLA-G expressed on tumor cells

[0318] Binding of LILRB2 on macrophages to HLA-G on cancer cells enhances the immunosuppressive function of myeloid cells. The ability of LILRB2 antibodies to block HLA-G binding on cancer cells is critical for the efficacy of anti-LILRB2 antibodies. The binding of LILRB2 antibodies to recombinant LILRB2-Fc protein and HLA-G expressed on 721.221B lymphoma cells was evaluated by flow cytometry.

[0319] Dilute the recombinant human LILRB2-Fc-Avitag protein (Acro Biosystems, catalog number LI2-H82F5) to 30 μg / mL in FACS buffer (PBS containing 2 mM EDTA + 1% FBS + 0.05% NaN3), and combine it in equal volumes with anti-LILRB2 antibodies at concentrations of 40, 20, 10, or 5 μg / mL, then incubate at 4°C for 1 hour. Wash the B cells (721.221) with PBS and count them, then resuspend them at a cell density of 1.3x 10 6 / mL (50x10 3 cells / well) in Fc blocking solution in FACS buffer (10% NGS (Sigma, catalog number G6767) in PBS, 2.5% FBS, 1% anti-CD32 (BD Biosciences, catalog number LI2-H82F5), 2 mM EDTA, 0.05% NaN3), then incubate at room temperature for 30 minutes. Add the cells (50 μL) to the wells pre-incubated with LILRB2-Fc-Avitag protein / antibody to achieve a final concentration of 7.5 μg / mL LILRB2-Fc-Avitag protein and 20, 10, 5, or 2.5 μg / mL antibody, then incubate at room temperature for 1 hour. Then wash the cells with FACS buffer (100 μL / well), and centrifuge the plate at 450x g for 5 minutes to pellet. Remove the supernatant, and perform a second wash using 250 μL of FACS buffer per well, then centrifuge again at 450x g for 5 minutes to pellet.

[0320] Add streptavidin-PE (Biolegend, catalog number 405204) diluted at 1:250 (75 μL / well) and incubate the cell / antibody mixture in the dark for 30 minutes at room temperature. Then, wash the cells twice in PBS containing 1 mM EDTA at a volume of 200 μL / well and then centrifuge at 450 x g for 5 minutes to pellet the cells. Resuspend the cells in 50 μL of cell viability dye (Zombie violet, Biolegend, catalog number 423114) diluted at 1:2000 and incubate in the dark for 10 minutes at room temperature. Perform a final wash with 200 μL / well of FACS buffer and then pellet the cells by centrifuging at 450 x g for 5 minutes. Remove the supernatant and resuspend the cells in 100 μL of FACS buffer for flow cytometry analysis as described in Example 4. The B2A-IgG4 clone blocked the binding of LILRB2-Fc to 721.221 cells expressing HLA-G in a dose-dependent manner, while the IgG4 isotype control did not affect the binding of LILRB2-Fc to 721.221 cells. Complete inhibition of binding was observed at concentrations of 20 and 10 μg / mL by the B2A-IgG4 clone, and partial blockade of 82% and 32% (normalized) was observed at concentrations of 5 and 2.5 μg / mL, respectively.

[0321] Example 8: Binning of LILRB2 antibodies

[0322] In the blocking flow cytometry assay, anti-LILRB2 antibodies were evaluated using HEK293T-LILRB2 cells (Example 3) stably expressing LILRB2 and biotin-conjugated reference LILRB2 antibodies prepared according to the literature: B2Comp1 (PCT Publication WO2021 / 138079 A1, designated as MK-4830 therein) and B2Comp2 (US Patent Publication US2019 / 0194327A1, designated as J-19.h1 therein). The variable domains of these antibodies were cloned into IgG4 and λ (B2Comp1) or κ (B2Comp2) constant regions. LILRB2 flow cytometry binning classifies antibodies into different bins based on their ability to block the binding of the reference antibody to HEK293T-LILRB2 cells. Briefly, HEK293T-LILRB2 cells were blocked with blocking buffer B for 20 minutes at 4°C. After incubation with blocking buffer B, "cold" blocking anti-LILRB2 antibodies in FACS buffer were added to the HEK293T-LILRB2 cells to achieve final concentrations of 0.1, 1, or 10 μg / mL, and then incubated for 1 hour at room temperature. Then, "hot" biotinylated reference antibody was added to achieve a final assay concentration of 5 ng / mL. After incubation for 45 minutes at room temperature, the cells were washed in FACS buffer and incubated with streptavidin-APC (Invitrogen, catalog number S32357) for 45 minutes at room temperature to quantify the binding of the reference to HEK293T-LILRB2 cells. The cells were then washed in FACS buffer and stained with the cell viability dye eFluor TM 780 (e780; Thermo Fisher Scientific, catalog number 65-0865-14) for 15 minutes. The cells were washed with FACS buffer and resuspended in 100 μL of FACS buffer for acquisition by flow cytometry as described in Example 4.

[0323] The chimeric anti-LILRB2 antibodies were binned into 4 different categories:

[0324] Bin 1: Antibodies that block the binding of B2Comp1 and B2Comp2 to HEK293T-LILRB2 cells and share epitopes with both B2Comp1 and B2Comp2

[0325] Bin 2: Antibodies that block B2Comp1 but not the binding of B2Comp2 to HEK293T-LILRB2 cells and share epitopes with B2Comp1

[0326] Bin 3: Antibodies that neither block the binding of B2Comp1 nor B2Comp2 to HEK293T-LILRB2 cells

[0327] Bin 4: An antibody that truly blocks B2Comp2 but not the binding of B2Comp1 to HEK293T-LILRB2 cells and shares an epitope with B2Comp2

[0328] The binning data are shown in Table 2. B2A-IgG4 at 0.1, 1, and 10 μg / mL did not block the binding of one of the control LILRB2 antibodies to HEK293T-LILRB2 cells, indicating that B2A-IgG4 does not share an epitope with B2Comp1 or B2Comp2 (Bin 3). This classifies the anti-LILRB2 antibodies described herein as having new epitopes that are not shared with some other published anti-LILRB2 antibodies.

[0329] Table 2 – B2A-IgG4 did not block the binding of reference antibodies

[0330]

[0331] Example 9: Isolation and differentiation of human primary cells

[0332] Evaluating anti-LILRB2 antibodies in immunological assays requires the isolation of human T cells and monocytes, as well as the differentiation of monocytes into immunosuppressive macrophages. Various techniques are known in the art, such as the techniques for the cells used in these examples. Isolation products are collected from a subject, and autologous monocytes and T cells are isolated using the described technique or another technique commonly used in the art. Briefly, human monocytes and T cells are isolated from white blood cells (WBCs) according to standard techniques. (LeukoPak, No. 4510-01 Full LeukoPak, BloodWorks Northwest, Seattle, WA). Peripheral blood mononuclear cells (PBMCs) are purified from LeukoPaks by standard density gradient centrifugation ( Premium 1.073 or 1.077, GE Healthcare, No. 17-5449-52 or Cytiva No. 17144003). The supernatant is discarded, and the pellet is resuspended in 20 mL of EasySep TM buffer (STEMCELL Technologies, No. 20144) for counting PBMCs and for further isolation of monocytes and T cells. Monocytes are isolated using the EasySep TM Human Monocyte Isolation Kit (STEMCELL Technologies, No. 19359) according to the manufacturer's instructions. Using EasySep TMHuman CD3+, CD4+ and CD8+ T Cell Isolation Kits (StemCell Technologies, catalog numbers 19051, 17952, and 17953 respectively) were used to isolate total CD3+, CD4+ and CD8+ T cells. These negative selection kits use antibodies to label unwanted cell types for removal, allowing the desired target cells to be isolated from the sample intact.

[0333] Example 10: Macrophage Generation

[0334] Macrophages can be generated from PBMC-derived monocytes using conventional techniques, as described below.

[0335] Generation of M0 macrophages: On day 0, monocytes isolated from an individual subject (as described in Example 9) were plated at 25 - 50 x 10 TM cells / 100 μL / well in M0 medium (90% X-VIVO 3 15 + 10% FBS + 100 ng / mL human M-CSF (PeproTech, catalog number 300-25)) in 96-well culture plates (Costar, catalog number 09-761-175). The cells were incubated at 37°C and 5% CO2 for 5 to 6 days to generate M0 macrophages.

[0336] Generation of immunosuppressive M2c macrophages: On day 5 of culture, M0 macrophages were polarized to M2c macrophages by gently aspirating the medium from each plate and replacing it with 100 μL / well of M2c medium (M0 medium + 20 ng / mL human IL-10 (PeproTech, catalog number 200-10)). The cells were incubated at 37°C and 5% CO2 for 2 days. On days 7 - 8 of culture, M2c macrophages were ready for co-culture assay setup. M2c macrophages were isolated from the plates by incubation with macrophage isolation solution DXF (PromoCell) and washed in PBS before downstream assays.

[0337] Example 11: Generation of Exhausted T Cells

[0338] Exhausted T cells are indicators of the immunosuppressive tumor microenvironment and contribute to cancer immune escape. To mimic exhausted T cells in the TME, exhausted T cells were generated by repeated stimulation. Exhausted T cells with a blast-like morphology were generated from human PBMC by repeated (3 times) phytohemagglutinin (PHA) stimulation. The cells were counted and plated at 1 x 10 6Cells were incubated at 1 cell / mL in T cell blast medium (90% IMDM (Thermo Fisher Scientific (Gibco), catalog number 12440053) + 10% human serum + 2 μg / mL PHA-L (Sigma-Aldrich (Roche), catalog number 11249738001) + 4 ng / mL recombinant human IL-2 (R&D Systems, catalog number 202-IL)). Cells were split at a ratio of 1:2 or 1:3 every 3 to 4 days and cultured for a total of 10 days (split 2 times within 10 days; a total of 3 PHA stimulations). Fresh T cell blast medium was added to the cells at each split. Cells were harvested on day 10 and set up for co-culture assays or frozen for future use. The exhausted T cell phenotype was confirmed by the expression of PD-1, TIM-3, and TIGIT, as well as the transcription factor Eomes (data not shown).

[0339] Example 12: Binding of LILRB2 antibody to human monocytes and M0 and M2c macrophages

[0340] The ability of anti-LILRB2 antibody to bind to myeloid cell subsets expressing LILRB2, including monocytes, M0 macrophages, and M2c macrophages, was evaluated. M2c macrophages were used as a surrogate for tumor-associated macrophages, an inhibitory macrophage commonly found in the tumor microenvironment. Frozen monocytes were removed from the stock solution in gaseous liquid nitrogen and thawed by gently vortexing in a water bath at 37 °C, then resuspended in X-VIVO TM 15 medium containing 10% FBS. PBMCs were placed in a 15 mL conical tube and centrifuged at 300 x g for 5 minutes, then the supernatant was removed. The cells were resuspended at a cell density of 2.5 x 10 5 cells / mL in assay medium (X-VIVO TM 15 + 10% FBS). The cells were plated at 100 μL / well (25K cells / well) into a 96-well flat-bottom plate. M0 and M2c macrophages were generated as described in Example 10 and collected from the flask by incubating in macrophage dissociation solution DXF for 15 minutes at room temperature, then removed from the culture flask into X-VIVO TM 15 medium. After centrifugation, the cells were resuspended in FACS blocking buffer D (FACS buffer + 10% FBS + 0.5 mg / mL human IgG1) and then incubated at 4 °C for 30 minutes. 25 μL of cells in FACS blocking buffer D were added at 2.5 x 10 4Cells per well were transferred to a 384-well plate, and 25 μL of titrated AF647-conjugated LILRB2 antibody or AF647-conjugated IgG1 isotype control was added directly to each well at 2-fold the final assay concentration. The cells were incubated with the antibody at 4 °C for 1 hour. The cells were washed twice with FACS buffer, stained with Zombie UV live / dead viability dye (BioLegend, catalog number 423107) (1:500 dilution) in the dark for 15 minutes at room temperature, washed with FACS buffer, and resuspended in 200 μL of FACS buffer for acquisition by flow cytometry as described in Example 4. GraphPad Prism was used for EC50 binding calculations. B2A-IgG1 and B2A-IgG4 bind to human monocytes and M0 and M2c macrophages (Figure 3).

[0341] Example 13: LILRB2 Antibody Enhances IFN-γ Response in LPS-Stimulated PBMCs

[0342] To determine whether LILRB2 blockade enhances innate immune responses and thereby enhances IFN-γ secretion to alleviate the immunosuppressive TME, the effects of B2A-IgG1 and IgG4 chimeras on IFN-γ secretion by PBMCs in response to lipopolysaccharide (LPS) were evaluated. Frozen PBMCs were removed from storage in liquid nitrogen vapor phase and thawed by gentle vortexing in a water bath at 37 °C and then resuspended in assay medium. The PBMCs were placed in 15 mL conical tubes and centrifuged at 300 x g for 5 minutes, and then the supernatant was removed. The cells were resuspended in assay medium at 3 x 10 6 cells / mL. The cells were plated at 100 μL / well (300K cells / well) into 96-well flat-bottom plates. This cell density was optimized for the average level of IFN-γ response for each subject. Fewer cells were plated per well if the subject was known to produce a higher cytokine response. The cells were allowed to sit for 1 hour at 37 °C, 5% CO2.

[0343] Dilute the LILRB2 antibody to 4-fold concentration (final concentration of 1 μg / mL) in assay medium and add 50 μL / well of the diluted antibody. For wells not receiving antibody, add 50 μL / well of assay medium. Incubate the cells with the antibody diluent for 2 hours at 37 °C, 5% CO2 prior to LPS stimulation. Dilute LPS to 4-fold concentration (final concentration of 1 μg / mL in the assay) in assay medium and add 50 μL / well to the cell / antibody mixture, except for subject samples known to have a high IFN-γ response, for which the final concentration of LPS is 0.1 μg / mL. For control wells (no LPS), add 50 μL / well of assay medium. Incubate the cells at 37 °C, 5% CO2 with a final volume of 200 μL / well for 24 hours. Harvest the supernatant (150 μL) after 24 hours of incubation and freeze at -80 °C or directly test for IFN-γ secretion by ELISA (R&D Systems). The anti-LILRB2 antibody promotes a pro-inflammatory phenotype by showing enhanced IFNγ secretion from PBMCs stimulated with LPS. Both B2A-IgG1 and B2A-IgG4 elicited similar IFN-γ secretion curves, shown in Figure 4 three representative subjects in

[0344] Example 14: LILRB2 antibody enhances TNF-α secretion in CD40-activated macrophages.

[0345] CD40L expressed on T cells is the primary molecule responsible for activating macrophages in the TME by binding via cell-cell contact to CD40 expressed on macrophages. The CD40 / CD40L interaction is important for activating macrophages to act as effector cells mediating inflammation in T cell-mediated inflammatory processes. An assay was developed to evaluate the inflammatory cytokines produced by macrophages treated with LILRB2 antibody in the absence of T cells. A HEK293 cell line (CrownBio, catalog number C2041) modified to express CD40L was used to mimic the binding and subsequent stimulation by activated T cells. M0 macrophages were generated from monocytes (25x10 3 / well) as described in Example 10. Remove the medium from the wells and add 100 μL / well of fresh assay medium. Add the LILRB2 antibodies (B2A-IgG1 and B2A-IgG4) at 5-fold concentration at 50 μL / well and incubate on the macrophages for 2 hours at 37 °C, 5% CO2. During the 2-hour incubation, harvest the HEK293 cells expressing CD40L from the flask and irradiate with 40 Gy. After pre-incubation with the LILRB2 antibody, add the HEK293 cells expressing CD40L (5x10 3), thereby making the final well volume in the 96-well plate 250 μL / well, and incubating overnight at 37 °C, 5% CO2. After incubation, 200 μL of supernatant was harvested from the wells and frozen at -80 °C or immediately assayed for TNF-α secretion by HTRF (CisBio, catalog number 62HTNFAPET) according to the manufacturer's instructions. Both B2A-IgG1 and B2A-IgG4 showed a substantial enhancement of TNF-α secretion by macrophages activated by CD40L, as shown in four different subjects ( Figure 5 ).

[0346] Example 15: LILRB2 antibody alleviates M2c-mediated immunosuppression in M2c / CD8+ or M2c / CD4+ T cell co-cultures

[0347] Crosstalk between immunosuppressive myeloid cells expressing LILRB2 and T cells results in T cell exhaustion and a lack of anti-tumor immune response in the TME. This crosstalk can be modeled by in vitro co-cultures of autologous monocyte-derived M2c macrophages and anti-CD3-stimulated CD8+ or CD4+ T cells. Reduction of immunosuppression can be evaluated by quantifying T cell proliferation and IFN-γ and perforin secretion as surrogates for T cell activation and anti-tumor activity. After polarizing M0 macrophages to M2c macrophages as described in Example 10, the supernatant was removed from the macrophages in the 96-well culture plate and replaced with 100 μL of assay medium containing OKT3 (Biolegend catalog number 317326) at a final concentration of 0.25 or 0.63 μg / mL. LILRB2 antibody (dose titrated from 20–0.0015 μg / mL) was added at a volume of 50 μL / well, and then the plate was incubated at 37 °C, 5% CO2 for 1–2 hours. When M2c macrophages were incubated with anti-bodies, autologous CD4+ or CD8+ T cells were isolated from PBMCs as described in Example 9. As described in Example 7B, isolated CD8+ or CD4+ T cells were labeled with CellTrace violet (Thermo Fisher Scientific, catalog number C34557). Excess CellTrace TM was washed away with pre-warmed assay medium, and the labeled CD8+ or CD4+ T cells were resuspended in assay medium, and then M2c:CD4+ or M2c:CD8+ T cells at a 1:1 ratio were added to the M2c / antibody preparation at a volume of 100 μL at 5 x 10 5 cells / mL. The cells were then incubated at 37 °C, 5% CO2 for 72 hours.

[0348] Transfer the supernatant containing T cells to a V-bottom 96-well plate, centrifuge to pellet the T cells, and collect the culture supernatant, which is frozen at -80 °C for quantification of human IFN-γ and perforin levels by MSD-ELISA (Meso Scale Discovery, custom U plex assay). Stain the T cell pellet with e780 viability dye for 10 minutes in the dark at room temperature, wash with 150 μL of FACS buffer, and resuspend in 100 μL of FACS buffer for acquisition on a BD FACSymphony TM or FACSCanto TM flow cytometer (BD Biosciences). Analyze the percentage and total count of proliferating CD8+ or CD4+ T cells using FlowJO software and report as the percentage of CellTrace+ dividing cells or the total count of CellTrace+ dividing cells.

[0349] In these M2c / T cell co-culture assays, the chimeric antibody clones mitigate M2c-macrophage-mediated immunosuppression, as measured by restoration of CD8+ T cell proliferation and IFN-γ secretion and perforin release ( Figure 6A and 6B ).

[0350] Example 16: LILRB2 antibody rescues IFN-γ responses of exhausted T cells in co-cultures with M2c macrophages

[0351] One hallmark of an ineffective anti-cancer immune response is T cell exhaustion in the tumor microenvironment. Exhausted T cells are T cells with reduced cytokine expression and effector function. Reversing T cell exhaustion and restoring anti-tumor potential represent a promising strategy for treating cancer. The ability of the LILRB2 antibody to rescue exhausted T cells from immunosuppression by LILRB2 was evaluated by an assay measuring macrophage-mediated immunosuppression using exhausted T cells and M2c cells in co-cultures.

[0352] T Cell Blasts and M2c Co-Culture Assay

[0353] To measure the ability of the LILRB2 antibody to rescue T cell blasts from M2c-mediated immunosuppression, the medium from M2c macrophages was removed and replaced with assay medium containing LILRB2 or isotype control antibody and incubated at 37 °C, 5% CO2 for 2 hours. OKT3 antibody at a final concentration of 0.25 μg / mL was added to the wells and incubated at 37 °C, 5% CO2 for 30 minutes. Finally, T cell blasts (Example 11) were added to the M2c / LILRB2 antibody mixture + OKT3 at a 1:1 ratio and incubated at 37 °C, 5% CO2 for 72 hours. IFN-γ levels were quantified by ELISA (R&D Systems or Mesoscale) from supernatants collected 72 hours after OKT3 stimulation. B2A-IgG1 rescued the IFN-γ response of exhausted T cells from M2c macrophage-mediated immunosuppression ( Figure 7 ). B2A-IgG4 was not active in this assay (data not shown).

[0354] Example 17: Pharmacokinetic characteristics of chimeric LILRB2 antibodies in humanized FcRn mice

[0355] Therapeutic monoclonal antibodies for cancer treatment generally belong to the immunoglobulin G (IgG) subclass. The half-life of monoclonal antibodies tested in mice is not relevant to that observed in humans because the binding affinity of human IgG for the neonatal Fc receptor (FcRn) of mice is higher than that for human FcRn. FcRn is a major histocompatibility complex (MHC) class I-like heterodimer containing an Fc-binding domain and β2-microglobulin (β2m). FcRn binds to the Fc portion of IgG in the acidic environment of lysosomes to prevent its degradation, and the IgG antibody bound by FcRn is recycled back to the extracellular surface where IgG dissociates from FcRn and re-enters the circulation, which in turn results in an extended half-life of these IgG monoclonal antibodies.

[0356] Create different FcRn transgenic mice to knock out murine FcRn and express human FcRn. Several groups have demonstrated that in these humanized FcRn mice, the PK of human therapeutic antibodies is related to human PK and shows comparable PK in non-human primates (Petkova SB et al., Int Immunol. 2006 18(12):1759-69; Tam SH et al., MAbs. 2013 5(3):397–405; Wang W et al., Drug Metab Dispos. 2011 39(9):1469-77; Roopenian DC et al., Methods Mol Biol. 2010 602:93-104; Avery LB et al., MAbs. 2016 8(6):1064–1078; Proetzel G et al., Methods. 2014 65(1):148–153). For example, the Tg32 and Tg276 mouse strains have been engineered on a C57BL / 6 background. These mice are immunocompetent and have all murine immune cells, lacking only murine FcRn, and express human FcRn in these models.

[0357] Female homozygous Tg32 FcRn mice were purchased at 6 - 8 weeks of age (Jackson Laboratory, stock number 014565) and housed in micro-isolation cages under specific pathogen-free conditions in a vivarium at Bloodworks Northwest. All procedures were conducted in accordance with the institutional guidelines of the Bloodworks Northwest Institutional Animal Care and Use Committee protocol number 5390-01. Mice were identified using ear tags. All mice were acclimated for at least 5 days prior to the start of antibody administration. On the day of dosing, the initial body weight of each mouse was recorded. The mice were divided into two LILRB2 antibody treatment groups: B2A-IgG4 and B2A-IgG1, 12 mice / group. Twelve mice from each treatment group were divided into three blood sampling groups of 4 mice each and received a single intraperitoneal (IP) dose of 20 mg / kg of either B2A-IgG4 or B2A-IgG1 antibody. Blood was sampled from each group at 3 alternating time points as follows: Group 1: 0.25, 4, 96 hours; Group 2: 1, 24, 168 hours; Group 3: 2, 48, 240 hours.

[0358] Blood Draw and Serum Preparation

[0359] Blood was collected via retro-orbital bleeding (at 0.25, 1, and 2 hours), submandibular bleeding (at 4, 24, and 48 hours), and terminal cardiac puncture (at 96, 168, and 240 hours). Whole blood was allowed to clot for at least 30 minutes at room temperature. Clots were removed by centrifugation (2000 x g) for 10 minutes at 4°C. Serum was aliquoted into 4 new microtubes and frozen at -80°C until analysis.

[0360] ELISA Assay for Determining Anti-LILRB2 Antibody Concentration in Mouse Serum

[0361] Human LILRB2 capture ELISA was performed to determine the antibody concentration in each group. Recombinant human LILRB2-HIS-tagged protein was diluted to 2 μg / mL in PBS and added to 384-well high-binding ELISA plates (Greiner Bio-One Microlon TM ) at 25 μL / well and incubated overnight at 4°C. The plates were washed four times with wash buffer using a microplate washer and then blocked with 90 μL / well of blocking buffer E (3% BSA in PBS) for 1 hour at room temperature. Stock solutions of B2A-IgG1 and B2A-IgG4 antibodies were diluted in assay buffer (0.05% BSA in TBS-T (0.05% in Tris-buffered saline, pH 7.4)) to prepare the highest standard concentration of 40 ng / mL. 15-point, 2-fold serial dilutions of the standards were diluted in 0.001 to 0.002% mouse serum. Serum samples from anti-LILRB2-treated mice were also diluted in assay buffer. Different dilutions were tested according to the time points of serum collection and the administered antibodies. Each standard dilution and mouse serum dilution was assayed in duplicate wells. After blocking, 25 μL / well of the diluted standards and diluted mouse serum were added to the assay plates and incubated for 1 hour at room temperature. After primary antibody binding, the plates were washed five times with wash buffer using a microplate washer. The secondary detection antibody (HRP-goat anti-human IgG Fc specific) was diluted to 1:5000 in assay diluent (1% BSA in PBS) and 25 μL was added to each well of the plates and incubated for 1 hour at room temperature in the dark. The plates were washed five times with wash buffer using a microplate washer. After removing the final wash solution, 25 μL / well of 1-Step TM Ultra TMB-ELISA substrate solution was added and the plates were incubated in the dark for 5 - 10 minutes at room temperature. After development, the reaction was terminated by adding 25 μL / well of 0.3 M HCl and the plates were read at 450 nm using an EnVision (PerkinElmer) microplate reader.

[0362] PK Analysis

[0363] At time points of 0.25 and up to 240 hours after anti-LILRB2 antibody administration, anti-LILRB2 antibody serum levels were determined according to different blood draw volumes. The mean B2A-IgG4 and B2A-IgG1 antibody serum levels from 4 mice at each time point were generated to produce a composite PK curve. The non-compartmental PK parameters after IP injection were performed on the mean serum concentration curve over time using the Microsoft Excel PK Solver 2.0 add-in, and the PK parameters were calculated using the PK Solver software (Table 4). Antibody serum exposures were plotted using GraphPad Prism for Windows (GraphPad Software). The half-lives of B2A-IgG1 and B2A-IgG4 antibodies were shown to be 7.0 days and 9.9 days in humanized FcRn mice (Table 3 and Figure 8 ).

[0364] Table 3 - B2A-IgG4 and B2A-IgG1 have typical PK curves in humanized FcRn mice

[0365] Parameter Unit B2A-IgG4 B2A-IgG1 Dose mg / kg 20 20 <![CDATA[t 1 / 2 > Hour 238 168 <![CDATA[t 1 / 2 > Day 9.90 7.01 <![CDATA[T max > Hour 4 4 <![CDATA[C max > μg / mL 183 189 <![CDATA[AUC (0-t) > μg / mL*h 24791 27797 <![CDATA[AUC (0-inf) > μg / mL*h 46028 44238

[0366] Example 18: LILRB2 antibody inhibits tumor growth in humanized NSG-SGM3 mice bearing subcutaneous human SK-MEL-5 melanoma

[0367] The anti-tumor efficacy of B2A-IgG4 was tested in an in vivo humanized tumor model. Female humanized NSG-SGM3 mice were purchased from Jackson Laboratories (JAX West). The immune systems of triple-transgenic NSG-SGM3 mice expressing human IL-3, GM-CSF (CSF2), and SCF (KITLG) (strain No. 013062) were reconstituted by intravenous injection of human UBC CD34+ hematopoietic stem cells into irradiated 3-week-old NSG-SGM3 mice. Engraftment of human CD45+ cells was monitored weekly by Jackson Laboratories to assess humanization in peripheral blood, and only animals with a minimum engraftment of 25% human CD45+ cells were received from Jackson Laboratories and participated in the study.

[0368] In vivo tumor xenograft model and anti-LILRB2 antibody administration:

[0369] Female humanized NSG-SGM3 were housed in micro-isolation cages under specific pathogen-free conditions in the vivarium of the Northwest Blood Center. All procedures were conducted in accordance with the institutional guidelines of the Northwest Blood Center's IACUC protocol number 5390-02. Mice were identified using ear tags. All mice were acclimated for at least 5 days before the start of the study. 2 x 10 in 100 μL PBS (R&D Systems, No. 3632-005-02) with 20% Matrigel 6SK-MEL-5 human melanoma cells (HLA class A *02:01; ATCC) were subcutaneously (SC) inoculated into the right flanks of mice.

[0370] Tumor size was measured twice a week using digital calipers. Tumor volume was calculated: Tumor volume (mm 3 ) = (L x W 2 / 2), where L is the largest dimension and W is the smallest dimension. When the average tumor size reached approximately 50 mm 3 , mice were randomly divided into groups based on both tumor size and engraftment of human CD45+ cells, with each group containing 8 - 9 mice. On the day of randomization (day 9), mice were administered 20 mg / kg B2A-IgG4 or IgG4 isotype control intraperitoneally (IP), and this was repeated every 7 days (i.e., days 9, 16, 23, 30, and 37 after tumor inoculation) (arrows in Figure 9a). Mice were sacrificed on day 41 for final tumor weight measurement (Figure 9b).

[0371] The following formula was used to calculate the mean tumor growth inhibition (TGI) for all days of data collection up to day 27 (Table 5).

[0372]

[0373] GraphPad Prism software was used with parametric RM (repeated measures) two-way ANOVA and Geisser-Greenhouse correction to confirm statistical differences in tumor volume. The standard error of the mean of daily tumor volumes was calculated. P-values were considered significant as follows: *P < 0.05; **P < 0.01 and ***P < 0.001.

[0374] Compared to the group treated with IgG4 control, mice treated with anti-LILRB2 B2A-IgG4 achieved 79% tumor growth inhibition (TGI) and 33% tumor regression by day 41, indicating the ability of B2A-IgG4 to delay tumor growth (Table 5 and Figure 9a).

[0375] B2A-IgG4 achieved anti-tumor activity in this humanized NSG-SGM3 human SK-MEL-5 melanoma mouse model ( Figure 9A and 9B and Table 4), while the IgG4 control antibody showed a lack of anti-tumor activity in the same tumor model ( Figure 9A and 9B ).

[0376] Table 4 - In vivo tumor growth inhibition (TGI) and tumor regression with B2A-IgG4 treatment in the SK-MEL-5 tumor model in humanized NSG-SGM3 mice

[0377]

[0378] Example 19: Antibody Humanization

[0379] The identified rabbit / human chimeric B2A-IgG4 mAb was selected for humanization. Humanization was performed in silico using a proprietary method (Fusion Antibodies, Belfast, Northern Ireland). In this system, a model of the parental variable domain was generated to enable structure-guided humanization. The sequence was aligned with a set of human germline sequences selected for preferred manufacturability characteristics, and proprietary CDRx TM The humanization platform grafted non-human amino acids onto the human sequence. The first round of humanization of the clone met EC 50 standards, so only one round of humanization was performed.

[0380] Five heavy chains and five light chains for B2A-IgG4 were generated. The humanized amino acid sequences were submitted to GenScript. GenScript used its proprietary method to back-translate and codon-optimize each variable region sequence for mammalian cell expression. Genes encoding the signal sequence plus variable region were synthesized at GenScript and cloned into the pTT5 vector backbone, with the human constant IgG4 region for the heavy chain and the human kappa constant region for the light chain. The resulting plasmid vectors (one for the light chain and one for the heavy chain) were transiently co-transfected into HEK293-6E cells (National Research Council of Canada (NRC)), and the conditioned medium was harvested after seven days. The recombinant antibodies were purified by protein A affinity chromatography. These affinity-purified antibodies were then used to generate binding data.

[0381] Success was defined as achieving a binding constant within two-fold of the parental mAb, and in this case, the rabbit variable region fused to the human IgG4 constant region was used as the parental chimeric mAb to determine EC 50 .

[0382] Twenty-five IgG4 variants were expressed as a combinatorial library of five light chains and five heavy chains in mammalian cell culture. In addition, B2A-IgG4 was included as a transfection control. The conditioned medium from these transient transfections was assayed for human IgG concentration. Plate-based ELISA was performed (see Example 2), where LILRB2-His was immobilized and the binding of humanized mAb variants was detected via an anti-human IgG4 HRP-labeled secondary antibody. The parental rabbit / human chimeric clone transfection was used as a benchmark EC 50 positive control. Antibody titers and EC 50The values are shown in Table 5.

[0383] Table 5 - Antibody Humanization: Binding EC of Several Humanized Variants 50 Superior to the parental chimeric

[0384] Variant mAb Titer [μg / mL] <![CDATA[EC 50 [ng / mL]]]> B2H4-54 231 2.25 B2H4-25 438 4.56 B2H4-24 492 3.23 B2H4-45 511 3.61 B2H4-41 313 3.76 B2H4-51 218 3.85 B2H4-11 476 4.20 B2H4-13 785 4.50 B2H4-25 438 4.56 B2H4-44 348 4.60 B2H4-35 649 4.67 B2H4-43 406 4.96 B2H4-52 431 5.37 Parental B2A-IgG4 212 2.74

[0385] Example 20: Binding of Humanized Variant Antibodies to LILRB2 and to Cells Expressing LILRB2 by ELISA

[0386] The selected humanized variant antibodies were purified and their ability to bind to human LILRB2 was tested by ELISA using the method described in Example 2, and their ability to bind to HEK293T cells expressing human LILRB2 was tested using the method described in Example 4. Representative data for the IgG4 variants are shown in Table 6. The humanized variants of B2A-IgG4 showed similar binding to recombinant and cell-expressed LILRB2 compared to the parental chimeric antibody.

[0387] Table 6 - EC 50 ELISA LILRB2 Binding and Binding to Cells Expressing HEK293T-LILRB2

[0388]

[0389] Example 21: Humanized LILRB2 Antibodies Do Not Bind to Other LILRB or LILRA Family Members

[0390] The ability of the selected humanized variant antibodies to bind to other LILRB or LILRA family members was tested using the method of Example 5 and commercially available positive control antibodies. The humanized LILRB2 antibody variants did not bind to any members of the LILRA family or to any additional LILRB family members. Representative data are given in Tables 7A and 7B.

[0391] Tables 7A and 7B – Binding of Humanized Variants to LILRB Family Members

[0392]

[0393]

[0394] Example 22: Humanized LILRB2 Variant Antibodies Block the Binding of LILRB2-Fc to HLA-G

[0395] Using the method described in Example 7, the ability of the humanized IgG4 variant antibody to block the binding of human LILRB2-Fc to monomeric HLA-G was selected by ELISA assay. The humanized variant blocked the binding of LILRB2-Fc to HLA-G. Representative data are given in Table 9.

[0396] Table 8 – Binding of humanized variants blocking LILRB2-Fc to HLA-G

[0397] Variant <![CDATA[Experiment IHLA-GIC 50 [μg / mL]]]> <![CDATA[Experiment II HLA-G IC 50 [μg / mL]]]> B2H4-54 4.04 4.09 B2H4-55 6.89 7.17 B2H4-24 7.01 7.50 B2H4-45 6.52 7.12 B2H4-41 5.78 6.76 B2H4-51 8.11 9.05 B2H4-11 7.31 7.99 B2H4-13 5.85 5.97 B2H4-25 7.64 8.29 B2H4-44 7.04 7.29 B2H4-35 5.16 5.14

[0398] Example 23: Binding of the humanized variant B2H1-55 antibody to monocytes and M0 and M2c macrophages

[0399] According to the method described in Example 12, the binding of the humanized variant B2H1-55 to monocytes, M0, and M2c macrophages was tested. Data compiled from 15 subjects (monocytes), 7 subjects (M0 macrophages), and 10 subjects (M2c macrophages) are shown in Figure 10 , where a dose titration of AF647-conjugated B2H1-55 was tested. The humanized variant B2H1-55 showed dose-dependent binding to monocytes, M0, and M2c macrophages, and the mean EC 50 were 35.6, 74.8, and 24.3 ng / mL, respectively.

[0400] Example 24: The humanized LILRB2 antibody B2H1-55 binds to a subset of myeloid cells but not to lymphocytes. LILRB2 is mainly expressed on myeloid cells in whole blood. To determine that the LILRB2 antibody does not bind to whole blood lymphocytes and to test the binding of the humanized antibody B2H1-55 to the whole blood myeloid cell population, a whole blood immunophenotyping assay was performed. Whole blood from healthy subjects was purchased (Northwestern Blood Bank). Tubes containing the blood were gently inverted to evenly distribute the plasma and cells, and then the blood was thoroughly mixed with the blocking mixture (10% FBS + 500 μg / mL human IgG1 myeloma plasma (Athens Research) + 0.05% NaN3), pipetted into 96-well deep plates, and then incubated at 4°C for 1 hour. The cells were resuspended at the 30-minute mark during incubation with the blocking mixture. Antibody titrations were performed in FACS buffer and added to the blood at final concentrations of 10, 1, and 0.1 μg / mL. The mixture was incubated with the primary antibody in the dark at 4°C for 45 minutes and then placed at room temperature for 15 minutes, for a total incubation time of 1 hour. The RBC lysis buffer was added to the blood mixture with 1X RBC lysis buffer (BD Pharm Lyse TM; BD Biosciences, cat. no. 555899) The red blood cells (RBCs) were lysed by three consecutive RBC lysis steps, pipetted up and down to mix thoroughly, and then incubated for 10 minutes at room temperature in the dark. After incubation, the cell plates were sealed, centrifuged at 200 x g for 5 minutes, and then the supernatant was aspirated from the wells. PBS (1X) was added to terminate RBC lysis, and then centrifuged at 200 x g for 5 minutes, and the supernatant was removed. The cells were then transferred to a 96-well V-bottom plate. Viability staining was performed by adding viability dye (Zombie violet, BioLegend, cat. no. 423114) at a 1:500 dilution and incubating for 20 minutes at room temperature in the dark. The cells were resuspended in FACS buffer and centrifuged to remove the viability dye, and the supernatant was removed. The cells were then resuspended in FACS Blocking Buffer D and incubated for 15 minutes at room temperature. The flow cytometry antibody mixture (fluorophore-conjugated BV421 anti-human CD3 (BioLegend, cat. no. 300434), BV711 anti-human CD4 (BioLegend, cat. no. 300558), APC / Cy7 anti-human CD8 (BioLegend, cat. no. 344714), PE anti-human CD11c (BioLegend, cat. no. 337206), BUV496 anti-human CD14 (BD Biosciences, cat. no. 741200), BUV805 anti-human CD15 (BD Biosciences cat. no. 742057), BV786 anti-human CD16 (BD Biosciences cat. no. 563690), BV605 anti-human CD19 (BioLegend, cat. no. 302244), FITC anti-human CD56 (BioLegend, cat. no. 318304), and PE / Cy7 anti-human HLA-DR (BioLegend, cat. no. 307616)) was added directly on top of the blocking buffer in the wells, and then incubated for 30 minutes at room temperature in the dark. After incubation, FACS buffer was added to the wells for washing, and then the plate was centrifuged at 350 x g for 5 minutes and resuspended in 350 μL of FACS buffer for acquisition on a FACSymphony TM cytometer. Data from three subjects are shown in Figure 11 , where binding of B2H1-55 at two concentrations was measured compared to an hIgG1 isotype control. The humanized LILRB2 antibody B2H1-55 binds only to myeloid cells, including classical, non-classical, and intermediate monocytes, myeloid dendritic cells, and neutrophils. B2H1-55 does not interact with human T cells, B cells, or NK cells.

[0401] Example 25: Binding of Humanized Variant B2H1-55 to Neutrophils

[0402] It has been reported that human neutrophils express LILRB2. To determine whether the humanized anti-LILRB2 variant binds to and activates human neutrophils, the binding of the humanized antibody to LILRB2 expressed on neutrophils in whole blood from healthy donors (Northwest Blood Center, Seattle, WA) was evaluated. Neutrophils were directly isolated from whole blood by immunomagnetic negative selection according to the manufacturer's instructions (StemCell Technologies, catalog number 19666). After neutrophil isolation, the neutrophils were resuspended in blocking buffer G (FACS buffer + 10% FBS + 2 mM EDTA + 0.05% NaN3 + 500 μg / mL IgG myeloma plasma), and then plated at 5 x 10 5 cells / mL in a 96-well plate. The cells were incubated in blocking buffer G for 30 minutes at 4°C. B2H1-55 internally conjugated to AF647 (Alexa Fluor TM 647 NHS ester, Thermo Fisher Scientific, catalog number A20106) was titrated at final concentrations of 10, 1, and 0.1 μg / mL in FACS buffer. The diluted antibody was added to the neutrophils at a 1:1 dilution, and then incubated for 120 minutes at 4°C in the dark. After antibody incubation, the cells were washed with FACS buffer and then centrifuged at 450 x g for 5 minutes to pellet the cells. The cells were resuspended in viability dye and incubated for 10 minutes at room temperature in the dark. After viability staining, the cells were washed with FACS buffer and then centrifuged at 450 x g for 5 minutes. The cells were resuspended in FACS buffer and analyzed by flow cytometry using a BD FACSymphony TM cell analyzer. Representative data are shown in Figure 12 , where dose-dependent binding of B2H1-55 to neutrophils was observed. The data shown are for six subjects from two independent experiments.

[0403] Example 26: Humanized LILRB2 Antibody Does Not Induce Neutrophil Activation

[0404] Test the potential of the humanized variant B2H1-55 to activate human neutrophils in whole blood from healthy subjects. Titrations of the soluble B2H1-55 humanized variant, IgG1 isotype control, and a commercial positive control (BioLegend, anti-CD16 clone 3G8) were added to whole blood and incubated for 2 hours in a 96-well plate at 37 °C. The final concentrations of the antibodies in whole blood were 100, 50, 10, and 0.1 μg / ml. After incubation with the antibodies, the cells were washed with FACS buffer and then centrifuged at 300 x g for 5 minutes to pellet the cells, and the supernatant was discarded. The whole blood cells were then stained at 4 °C for 20 minutes with fluorochrome-conjugated BV421 anti-human CD11b (BioLegend, cat. no. 301324), AF647 anti-human CD62L (BioLegend, cat. no. 304818), FITC anti-human CD66b (BioLegend, cat. no. 305104), and BUV395 anti-human CD15 (BD Biosciences, cat. no. 740318). The cells were washed with FACS buffer and then centrifuged at 300 x g for 5 minutes to pellet the cells, and the supernatant was discarded. RBC lysis was performed by adding 200 μL of 1X lysing solution (BD Biosciences, cat. no. 349202, 1:10 dilution) to all wells, gently mixing by pipetting up and down, and incubating at room temperature in the dark for 3-5 minutes. The plate was immediately centrifuged at 200 x g for 5 minutes, and the supernatant was discarded to stop cell lysis. The cells were washed 5 minutes with FACS buffer at 300 x g, resuspended in FACS buffer, and evaluated by flow cytometry using a BD FACSymphony TM cytometer. Changes in neutrophil frequency (CD66b+CD15+)( Figure 13 PMNs in) and surface density of activation markers (increase in CD11b and decrease in CD62L) were analyzed using FlowJo software.

[0405] Representative data from 2 healthy subjects are shown in Figure 13 , where the humanized variant B2H1-55 was compared to an anti-CD16 positive control antibody (clone 3G8) and an isotype control antibody. B2H1-55 did not induce activation of the cells after binding to neutrophils, as shown by the unchanged CD11b expression and retention of surface CD62L. The effects associated with the positive control antibody ( Figure 13 ) were consistent with the reported activity of anti-CD16 antibodies.

[0406] Example 27: Humanized LILRB2 antibody enhances IFN-γ response in LPS-stimulated PBMCs

[0407] To determine whether the humanized variants enhance the innate immune response of human monocytes by blocking the interaction of LILRB2 with its HLA ligands, the effects of the variants on IFN-γ production and IL-10 secretion by LPS-stimulated human PBMCs were evaluated. The ability of the humanized variants to enhance IFN-γ secretion and IL-10 secretion by LPS-stimulated PBMCs was tested according to the method described in Example 13. Similar to IFN-γ, IL-10 (R&D Systems) was quantified in the supernatants collected 24 hours after LPS stimulation. The humanized variants induced a pro-inflammatory innate Th1-like phenotype, whereupon stimulation with LPS, PBMCs exhibited enhanced IFN-γ secretion and reduced IL-10 production. Representative IFN-γ data are shown in Figure 14 , where three humanized variants were compared to the B2A-IgG1 parent, the benchmark antibody B2Comp1, and the hIgG1 isotype control antibody. The data shown are for two representative subjects at two antibody concentrations. The humanized LILRB2 variants enhanced IFN-γ secretion from LPS-stimulated PBMCs. As Figure 23 shown in A-C, for the representative humanized variants, the humanized anti-LILRB2 variants inhibited LPS-mediated IL-10 release by human PBMCs in a dose-dependent manner.

[0408] The combination of the humanized anti-LILRB2 variants with Toll-like receptor 2 (TLR) ligands was also tested, such as heat-killed Listeria monocytogenes (HKLM, InvivoGen, catalog number: Tlrl-hklm, 2.5 x 10 7 cells / mL) and Pam3CSK4 (InvivoGen, catalog number: Tlrl-pms, 100 ng / mL). The humanized anti-LILRB2 variants enhanced IFN-γ secretion and reduced IL-10 release by PBMCs treated with HKLM (2.5 x 10 7 cells / mL) or Pam3CSK4 (100 ng / mL) (data not shown).

[0409] Example 28: Humanized LILRB2 antibody alleviates M2c-mediated immunosuppression in the M2c / T cell co-culture assay

[0410] The ability of the humanized variants to restore the proliferation, IFN-γ secretion, and perforin release by CD8+ T cells co-cultured with immunosuppressive M2c macrophages was tested according to the method described in Example 15. Representative data are shown in Figure 15Similar to the LILRB2 parental chimera B2A-IgG1, the humanized LILRB2 variant alleviates M2c-mediated immunosuppression by enhancing CD8+ T cell proliferation, IFNγ secretion, and perforin release in a dose-dependent manner.

[0411] Example 29: Humanized LILRB2 antibody prevents the development of immunosuppressive macrophages

[0412] To determine whether anti-LILRB2 antibodies might interfere with the generation of tumor-associated macrophages, an M2c / CD8+ T cell co-culture assay was performed as described in Example 15, with a "pre-protocol" step added during M0 to M2c polarization. For the pre-protocol step, M0 macrophages were polarized into M2c macrophages for 2 days ("during polarization") in the presence of anti-LILRB2 antibody or isotype control. After 2 days of M0 to M2c macrophage polarization, the antibody was washed off before co-culturing with CD8+ T cells for 72 hours, and no treatment was added to the M2c macrophages after the "post-polarization" step. The humanized variant B2H1-55 restored the proliferation of CD8+ T cells when added as a pre-protocol treatment, indicating that the M2c-mediated immunosuppressive effect on these cells had been alleviated. Representative data are shown in Figure 16 .

[0413] Example 30: LILRB2 antibody alleviates M2c macrophage-mediated immunosuppression in M2c / CD4+ T cell co-cultures

[0414] Using the method described in Example 15, the ability of the humanized variant B2H1-55 to restore the proliferation and IFN-γ and perforin secretion of CD4+ T cells co-cultured with immunosuppressive M2c macrophages was tested. B2H1-55 and IgG1 isotype control were tested at 5 or 10 μg / mL. Representative data are shown in Figure 17A and 17B . The humanized variant B2H1-55 alleviates M2c-mediated inhibition, as shown by rescuing CD4+ T cell proliferation ( Figure 17A ) and IFN-γ and perforin release ( Figure 17B ).

[0415] Example 31: Humanized variants rescue IFN-γ responses by exhausted T cells from M2c-mediated immunosuppression

[0416] Using the method described in Example 16, the ability of selected humanized variant antibodies to rescue IFN-γ responses by exhausted T cells was tested. Figure 18Representative data for IFN-γ assays are given. Similar to the results shown with B2A-IgG1, treatment with the humanized variant alleviates M2c macrophage-mediated immunosuppression, as shown by IFN-γ secretion by depleted T cells.

[0417] Example 32: Treatment with a humanized LILRB2 antibody in combination with a PD-1 antibody alleviates immunosuppression of M2c-mediated depleted T cells

[0418] To determine whether LILRB2 blockade enhances the efficacy of anti-PD-1 antibodies, the combination of the humanized variant and a PD-1 antibody was evaluated in the above M2c / depleted T cell co-culture assay. The experiment followed the method in Example 16, adding depleted T cells treated with anti-PD-1 or isotype control.

[0419] The medium was removed from the wells of M2c macrophages and replaced with assay medium containing LILRB2 or isotype control antibody and incubated at 37 °C, 5% CO2 for 2 hours. The final concentration of the anti-LILRB2 antibody or IgG1 isotype was 12 to 333 ng / mL. After the initial 2-hour pretreatment, 50 μL / well of anti-human CD3 clone OKT3 (final assay concentration 0.250 μg / mL) was added to the assay medium and the plate was incubated at 37 °C for 30 minutes, after which depleted T cells were added.

[0420] The depleted T cells were pre-incubated for 2 hours with a PD-1 (Pem-hIgG4 S228P) antibody (InvivoGen, cat. no. hpd1pe-mab14) or IgG4 isotype control, as Figure 19 shown. The final concentration of the anti-PD-1 antibody was 1 μg / mL. Supernatants were collected 72 hours after addition of T cells and IFN-γ levels were determined by ELISA (R&D Systems or Meso Scale). The results are shown in Figure 19 . LILRB2 blockade enhances the efficacy of anti-PD-1 antibodies and rescues depleted T cells from M2c-mediated immunosuppression, as shown by enhanced IFN-γ secretion.

[0421] Example 33: Humanized LILRB2 antibody induces modest cytokine secretion in whole blood

[0422] Immune-modulating therapeutic antibodies carry a risk of cytokine release syndrome, a rapid systemic inflammatory response characterized by the secretion of inflammatory cytokines by immune cells. Antibody target binding can induce cytokines either by directly activating lymphocytes and myeloid cells or by interacting with Fcγ receptors on myeloid cells and NK cells. Whole blood in vitro cytokine release assays are a standard assay for assessing the risk of cytokine release syndrome mediated by therapeutic antibodies. Whether treatment with a humanized LILRB2 variant triggers the release of inflammatory cytokines was evaluated in whole blood from healthy study subjects.

[0423] The LILRB2 antibody and control antibody were diluted to a 10-fold final concentration in PBS in a dilution plate. The diluted LILRB2 antibody (25 μL / well) was transferred to a 96-well plate. Whole blood from healthy subjects was purchased (Northwest Blood Center, Seattle, WA). Whole blood (225 μL / well) was added to the LILRB2 antibody without mixing to avoid cell lysis. The blood / antibody mixture was incubated at 37 °C, 5% CO2 for 24 - 48 hours. After incubation, the plates were centrifuged at 350 x g for 5 minutes to pellet the cells. Plasma (65 μL) was harvested from the surface of each well and immediately evaluated for IL-6, TNF-α, IFN-γ, and IL-1β cytokine secretion by MSD-ELISA according to the manufacturer's instructions (Mesoscale Discovery). The release of IL-1β, IL-6, IFN-γ, and TNF-α in response to treatment with the humanized variant was compared to cytokine induction by the corresponding human IgG1 isotype, untreated control, and anti-CD52 positive control antibody (alemtuzumab, CAS 216503-57-0, BOC Sciences catalog number B0084-305393). Representative data from 3 healthy subjects are shown in Figure 20 . In all tested subjects, treatment with the LILRB2 humanized variant did not trigger the release of TNF-α or IL-1β (data not shown). In one study subject, minimal IFN-γ secretion was observed after treatment with the LILRB2 humanized variants B2H1-55 and B2H1-52 ( Figure 20 subject Y in). In two subjects, minimal IL-6 secretion was observed after treatment with the LILRB2 humanized variant at the highest dose of 150 μg / mL, indicating that these antibodies did not induce cytokine release syndrome in whole blood. Data for one representative subject (subject X) are shown in Figure 20, which describes the IL-6 response sometimes observed. Treatment with the humanized LILRB2 variant did not trigger the release of IL-6, TNF-α, IFN-γ or IL-1β cytokine secretion in the whole blood of 6 out of 8 healthy subjects. The observed cytokine levels were similar to those induced by the control group LILRB2 antibody B2Comp1 (data not shown) (IgG4 heavy chain (SEQ ID NO:43), λ light chain (SEQ ID NO:44)), and lower than those induced by the CD52 antibody positive control.

[0424] Example 34: The humanized antibody variant did not induce ADCC in human monocytes or HEK293 cells expressing LILRB2

[0425] Test the ability of the humanized variant to induce antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells against human monocytes or HEK293T cells expressing human LILRB2. Cytotoxic activity was quantified by flow cytometry using a widely used quantitative method for ADCC (Yamashita M, 2016 Scientific Reports, 6:19772, DOI:10.1038 / srep19772). Monocytes were isolated from cryopreserved PBMCs of healthy subjects as described in Example 9. Using the EasySep TM Human NK Cell Isolation Kit (StemCell Technologies, catalog number 17955), NK cells were isolated from cryopreserved PBMCs of healthy subjects.

[0426] For the NK cell and human monocyte ADCC assays, the isolated monocytes (target cells) were plated at 25 μL / well (10 4 monocytes / well) in a 96-well low-adhesion plate in assay medium and combined with 25 μL of 4-fold diluted anti-LILRB2 antibody or isotype control in medium to achieve a final assay concentration of 10 μg / mL. The target cells and antibodies were incubated at 37 °C for 1 hour. Subsequently, autologous NK cells (effector cells) (50 μL / well) were added to the target cells to achieve a final effector:target ratio of 8:1 in effector + target + antibody with a final volume of 100 μL / well. The cells were incubated at 37 °C and 5% CO2 for 4 hours.

[0427] For the NK cell and HEK293T-LILRB2 ADCC assays, as described in Example 14, with CellTrace TMPurple dye-labeled HEK293T cells expressing hLILRB2 (target cells) or cells transfected with a mimic (negative control target cells). K562 human CML line lacks HLA class I and does not express human LILRB2 and was included as a positive control target for NK cell killing ability. K562 tumors were labeled with CellTrace TM TM Purple (CTV), similar to HEK293T cells. Excess CTV was washed away with pre-warmed assay medium, and the labeled HEK293T-LILRB2 or K562 was resuspended in assay medium and then plated at 25 μL / well (10 3 target cells / well) in a 96-well low-adhesion plate and combined with 25 μL of 4-fold diluted anti-LILRB2 antibody or isotype control in medium to achieve a final assay concentration of 10 μg / mL. The target cells and antibodies were incubated at 37 °C for 1 hour. Subsequently, autologous NK cells (effector cells) (50 μL / well) were added to the target cells to achieve a final effector:target ratio of 8:1 in effector + target + antibody with a final volume of 100 μL / well. The cells were incubated at 37 °C and 5% CO2 for 4 hours.

[0428] Detection of NK cell killing

[0429] The cells were gently pipetted up and down to detach from the low-adhesion plate. The cells were transferred to a 96-well V-bottom plate, centrifuged at 300 x g for 5 minutes, the supernatant was discarded, and the cells were prepared for flow cytometry staining. For NK and monocyte ADCC assays, the cells were resuspended in FACS buffer containing 2 μL of human TruStain FcX TM (BioLegend, Cat. No. 422302), fluorescein-labeled anti-human CD16 (BioLegend, Cat. No. 302046), and anti-human CD14 (BD Biosciences, Cat. No. 563561) antibodies (for gating monocytes) and incubated at 4 °C for 20 minutes. The cells were washed in FACS buffer, centrifuged at 300 x g for 5 minutes, and the supernatant was discarded. The cells were resuspended in 50 μL / well of e780 viability dye (1:1000 dilution in PBS) and incubated at room temperature for 15 minutes. The cells were washed and resuspended in FACS buffer and then analyzed by flow cytometry using a BD FACSymphony TM cell analyzer and FlowJO software. NK killing was analyzed by gating and reporting the frequency of CD14+ e780-positive cells.

[0430] Similar to the NK / monocyte assay, HEK293T or K562 cells were gently pipetted up and down in the assay plate and transferred to a 96-well V-bottom plate for flow cytometry staining. The cells were resuspended in 50 μL / well of e780 (1:1000 dilution in PBS) and incubated for 15 minutes at room temperature. The cells were washed in FACS buffer, resuspended in FACS buffer, and analyzed by flow cytometry as described. NK killing was analyzed by gating and reporting the frequency of CTV+e780 positive cells.

[0431] Figure 21A Pooled data from NK / monocyte ADCC assays from 2 - 10 healthy subjects are shown, where hIgG1 isotype control and B2H1-55 antibody were tested in 10 healthy subjects, and B2H1-11, B2H1-52, B2H1-35, and B2A-IgG1 were tested in two healthy subjects. Figure 21B NK / HEK293T-LILRB2 assays for three representative subjects are shown. Compared to the IgG1 isotype control, the parental B2A-IgG1 and humanized variants did not induce ADCC of human monocytes but induced killing of HEK293T-LILRB2 cells (Figure 21).

[0432] Example 35: Pharmacokinetic Characteristics of Humanized Antibody Variants in Humanized FcRn Mice

[0433] According to the method written in Example 17, the pharmacokinetic characteristics of the humanized variants were evaluated in humanized FcRn mice. The mice were divided into 3 humanized LILRB2 antibody treatment groups: B2H1-55, B2H1-35, and B2H1-52, with 12 mice / group. Twelve mice from each treatment group were divided into three blood sampling groups, 4 mice / group, and received a single intraperitoneal (IP) dose of 10 mg / kg of the antibody. Blood was sampled from each group at 3 alternating time points as follows: Group 1: 0.25, 4, 96 hours; Group 2: 1, 24, 168 hours; Group 3: 2, 48, 240 hours. Serum collection and PK analysis were performed as described in Example 17. The PK characteristics of variants B2H1-55, B2H1-35, and B2H1-52 are shown in Table 9 and Figure 22 in. The humanized anti-LILRB2 variants showed a half-life range of 5 - 10 days in humanized FcRn mice, which is typical for IgG1 antibodies.

[0434] Table 9 – Pharmacokinetic Characteristics of Single-Dose Humanized LILRB2 Antibodies in Humanized FcRn Mice

[0435] Parameter Unit B2H1-55 B2H1-52 B2H1-35 Dose mg / kg 10 10 10 <![CDATA[t 1 / 2 > Hour 231 219 139.5 <![CDATA[t 1 / 2 > Day 9.6 9.12 5.81 <![CDATA[T max > Hour 2 2 2 <![CDATA[C max > μg / mL 107.7 99.5 117.9 <![CDATA[AUC (0-t) > μg / mL*h 15664 13909 16581 <![CDATA[AUC (0-inf) > μg / mL*h 29278 24667 22671

[0436] All documents mentioned in this application are hereby incorporated by reference in their entirety.

Claims

1. An antibody product that binds to human LILRB2, said antibody product comprising CDR-H1 as shown in SEQ ID NO:16, CDR-H2 as shown in SEQ ID NO:17, CDR-H3 as shown in SEQ ID NO:24, CDR-L1 as shown in SEQ ID NO:19, CDR-L2 as shown in SEQ ID NO:20, and CDR-L3 as shown in SEQ ID NO:

21.

2. The antibody product according to claim 1, which comprises CDR-H1 as shown in SEQ ID NO:16, CDR-H2 as shown in SEQ ID NO:17, CDR-H3 as shown in SEQ ID NO:18, CDR-L1 as shown in SEQ ID NO:19, CDR-L2 as shown in SEQ ID NO:20, and CDR-L3 as shown in SEQ ID NO:

21.

3. The antibody product according to claim 1, which comprises CDR-H1 as shown in SEQ ID NO:22, CDR-H2 as shown in SEQ ID NO:23, CDR-H3 as shown in SEQ ID NO:24, CDR-L1 as shown in SEQ ID NO:25, CDR-L2 as shown in SEQ ID NO:26, and CDR-L3 as shown in SEQ ID NO:

21.

4. The antibody product according to any one of claims 1 to 3, which comprises a heavy chain variable region, said heavy chain variable region comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:1, 6, 7, 8, 9, or 10; or (b) the amino acid sequence shown in SEQ ID NO:1, 6, 7, 8, 9, or 10.

5. The antibody product according to any one of claims 1 to 5, which comprises a light chain variable region, said light chain variable region comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:2, 11, 12, 13, 14, or 15; or (b) the amino acid sequence shown in SEQ ID NO:2, 11, 12, 13, 14, or 15.

6. The antibody product according to any one of claims 1 to 5, which comprises: (a) a heavy chain variable region that comprises SEQ ID NO:1; and a light chain variable region SEQ ID NO:2; (b) a heavy chain variable region SEQ ID NO:6; and a light chain variable region that comprises SEQ ID NO:11; (c) a heavy chain variable region that comprises SEQ ID NO:6; and a light chain variable region that comprises SEQ ID NO:12; (d) a heavy chain variable region that comprises SEQ ID NO:6; and a light chain variable region that comprises SEQ ID NO:13; (e) A heavy chain variable region comprising SEQ ID NO:6; and a light chain variable region comprising SEQ ID NO:14; (f) A heavy chain variable region comprising SEQ ID NO:6; and a light chain variable region comprising SEQ ID NO:15; (g) A heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:11; (h) A heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:12; (i) A heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:13; (j) A heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:14; (k) A heavy chain variable region comprising SEQ ID NO:7; and a light chain variable region comprising SEQ ID NO:15; (l) A heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region comprising SEQ ID NO:11; (m) A heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region comprising SEQ ID NO:12; (n) A heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region comprising SEQ ID NO:13; (o) A heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region comprising SEQ ID NO:14; (p) A heavy chain variable region comprising SEQ ID NO:8; and a light chain variable region comprising SEQ ID NO:15; (q) A heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:11; (r) A heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:12; (s) A heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:13; (t) A heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:14; (u) A heavy chain variable region comprising SEQ ID NO:9; and a light chain variable region comprising SEQ ID NO:15; (v) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:11; (w) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:12; (x) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:13; (y) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:14; or (z) A heavy chain variable region comprising SEQ ID NO:10; and a light chain variable region comprising SEQ ID NO:

15.

7. The antibody product according to any one of claims 1 to 6, which comprises a heavy chain comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH).

8. The antibody product according to any one of claims 1 to 7, which comprises a light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL).

9. The antibody product according to any one of claims 1 to 8, which comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH), and the light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL).

10. The antibody product according to any one of claims 1 to 9, which comprises an IgA, IgD, IgE, IgG or IgM heavy chain constant domain.

11. The antibody product according to claim 7 or claim 9, wherein the heavy chain constant domain is an IgG1 constant domain, an IgG2 constant domain or an IgG4 constant domain.

12. The antibody product according to claim 11, wherein the heavy chain constant domain is an IgG1 constant domain.

13. The antibody product according to any one of claims 1 to 12, which comprises a heavy chain amino acid sequence comprising: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:3, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32; or (b) the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO: 31 or SEQ ID NO:

32.

14. The antibody product according to any one of claims 1 to 11, wherein the heavy chain constant domain is an IgG4 constant domain.

15. The antibody product according to any one of claims 1 to 11 and 14, which comprises a heavy chain amino acid sequence, and the heavy chain amino acid sequence comprises: (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37; or (b) the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36 or 37.

16. An antibody product that binds to human LILRB2, and the antibody product comprises: (a) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:1; and a κ light chain having an amino acid sequence comprising SEQ ID NO:2; (b) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (c) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (d) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (e) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (f) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:6; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (g) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (h) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (i) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (j)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (k)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:7; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (l)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (m)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (n)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (o)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (p)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:8; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (q)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (r)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (s)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (t)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; (u)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:9; and a κ light chain having an amino acid sequence comprising SEQ ID NO:15; (v)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:11; (w)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:12; (x)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:13; (y)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:14; or (z)An IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:10; and a κ light chain having an amino acid sequence comprising SEQ ID NO:

15.

17. The antibody product according to any one of claims 1 to 9 and 11 to 16, which is an IgG1 or IgG4 antibody.

18. The antibody product according to any one of claims 1 to 17, which comprises a human light chain constant region comprising a κ domain or a fragment thereof.

19. The antibody product according to any one of claims 1 to 18, wherein the antibody product binds to human LILRB2.

20. The antibody product according to any one of claims 1 to 19, which specifically binds to human LILRB2 expressed by myeloid cells or cancer cells.

21. The antibody product according to any one of claims 1 to 20, wherein the antibody product specifically binds to human LILRB2 with a KD of 0.5 nM to 500 nM.

22. The antibody product according to any one of claims 1 to 20, wherein the antibody product binds to human immunosuppressive myeloid cells.

23. The antibody product according to claim 21, wherein the immunosuppressive myeloid cells are in the tumor microenvironment.

24. The antibody product according to claim 21, wherein the immunosuppressive myeloid cells are macrophages, myeloid dendritic cells or myeloid-derived suppressor cells.

25. The antibody product according to claim 21, wherein the immunosuppressive myeloid cells are M2a, M2b, M2c or M2d macrophages.

26. The antibody product according to any one of claims 1 to 24, wherein the antibody product binds specifically to human M2c macrophages with a K of 0.5 nM to 500 nM D ​ 27. The antibody product according to any one of claims 1 to 25, wherein the antibody product is a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody or a single-chain antibody.

28. The antibody product according to any one of claims 1 to 25, wherein the antibody product is a monospecific, bispecific, trispecific or multispecific antibody.

29. The antibody product according to any one of claims 1 to 27, which is bound by an Fc receptor expressed on immunosuppressive macrophages or other myeloid cells.

30. The antibody product according to claim 28, which binds to CD16 (FcγRIIIa), CD32 (FcγRII) or CD64 (FcγRI) expressed on immunosuppressive macrophages or other myeloid cells.

31. The antibody product according to claim 30, which binds to LILRB2 and binds to CD16 (FcγRIIIa), CD32 (FcγRII) or CD64 (FcγRI) expressed on the same myeloid cells.

32. The antibody product according to claim 30, which binds to LILRB2 on a first cell and binds to CD16 (FcγRIIIa), CD32 (FcγRII) or CD64 (FcγRI) expressed on a second cell.

33. A method of providing cancer immunotherapy to a subject in need thereof, wherein the cancer is associated with the presence of immunosuppressive macrophages, the method comprising administering to the subject a therapeutically effective amount of the antibody product according to any one of claims 1 to 32.

34. The method according to claim 33, wherein the antibody product binds to macrophages, and the binding of the antibody product to macrophages produces at least one of the following effects: (a) Promote the activation of CD4+ T cells, CD8+ T cells, NK cells or any combination thereof; (b) Promote the proliferation of CD4+ T cells, CD8+ T cells, NK cells or any combination thereof; (c) Prevent macrophages from polarizing into immunosuppressive macrophages; and (d) Enhance the innate anti-tumor response.

35. The method according to claim 33, wherein the activation of the CD4+ T cells, CD8+ T cells, NK cells or any combination thereof is measured as an increase in the level of IFN-γ, TNF-α or perforin or any combination thereof or a decrease in the level of IL-10 release.

36. The method according to any one of claims 33 to 35, wherein the binding of the antibody product to macrophages is not cytotoxic to the macrophages.

37. The method according to any one of claims 33 to 36, wherein the binding of the antibody product to macrophages produces at least one of the following effects: (a) Internalization of the antibody product by the macrophages; (b) Secretion of TNFα, IL-6, perforin or any combination thereof; (c) Reduction of IL-10 release; (d) Activation of CD4+ T cells, CD8+ T cells, NK cells or any combination thereof; (e) Proliferation of CD4+ T cells, CD8+ T cells, NK cells or any combination thereof; and (f) Promotion of tumor cell killing in the tumor microenvironment.

38. The method according to claim 37, wherein the binding produces two or more of (a) to (f); three or more of (a) to (f); four or more of (a) to (f); five or more of (a) to (f); or all of (a) to (f).

39. The method according to any one of claims 33 to 38, wherein the binding of the antibody product to macrophages increases the immune-stimulatory activity in the tumor microenvironment.

40. The method according to any one of claims 33 to 38, wherein the binding of the antibody product to macrophages reduces the immunosuppressive activity of the macrophages.

41. The method according to any one of claims 33 to 38, wherein the binding of the antibody product to macrophages reduces the pro-tumor activity of the macrophages.

42. The method according to any one of claims 33 to 38, wherein the binding of the antibody product promotes CD4+ T cell activation, CD4+ T cell proliferation, or both CD4+ T cell activation and proliferation.

43. The method according to any one of claims 33 to 38, wherein the binding of the antibody product promotes CD8+ T cell activation, CD8+ T cell proliferation, or both CD8+ T cell activation and proliferation.

44. The method according to any one of claims 33 to 38, wherein the binding of the antibody product promotes cytotoxic lymphocyte-mediated cancer cell killing.

45. The method according to any one of claims 33 to 38, wherein the binding of the antibody product promotes NK cell-mediated tumor cell killing.

46. The method according to any one of claims 33 to 46, wherein the binding of the antibody product to macrophages reduces the inhibition of cytotoxic T cell-mediated tumor cell killing in the tumor microenvironment.

47. The method according to any one of claims 33 to 46, wherein the cancer is a sarcoma, carcinoma, or hematogenous cancer.

48. The method according to claim 47, wherein the cancer is glioblastoma multiforme, head and neck cancer, clear cell renal cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, testicular germ cell cancer, gastric cancer, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid cancer, cutaneous squamous cell carcinoma, or ovarian cancer.

49. The method according to claim 47, wherein the cells of the cancer overexpress LILRB2.

50. The method according to any one of claims 33 to 49, further comprising administering to the subject an effective amount of an anti-cancer therapeutic agent.

51. The method according to claim 50, wherein the anti-cancer therapeutic agent comprises an immune checkpoint inhibitor.

52. The method according to claim 51, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

53. The method according to claim 52, wherein the effective amount of the PD-1 antagonist is an amount effective to relieve the immunosuppression of T cells.

54. The method according to claim 53, wherein the immunosuppression of the T cells comprises immunosuppression mediated by the interaction of the T cells with myeloid cells expressing PD-L1.

55. A composition comprising (a) an antibody product according to any one of claims 1 to 32, and (b) an excipient.

56. An article comprising the composition according to claim 55 and a container.

57. Use of an antibody product according to any one of claims 1 to 32 or a composition according to claim 53 for the preparation of a medicament for treating cancer in a subject in need thereof.

58. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain variable region of claim 4.

59. An isolated nucleic acid comprising a nucleotide sequence encoding the light chain variable region of claim 5.

60. An expression vector comprising the nucleic acid according to claim 58.

61. An expression vector comprising the nucleic acid according to claim 59.

62. An expression vector comprising the nucleic acids according to claims 58 and 59.

63. A host cell comprising the expression vector according to any one of claims 60 to 62.

64. A method for producing a protein comprising an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain, the method comprising: (a) growing the host cell according to claim 63 under conditions such that the host cell expresses the protein comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region; and (b) purifying the protein comprising the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain.

65. A method for producing an antibody product that binds to human LILRB2, the method comprising: (a) growing the host cell comprising the expression vector according to claim 62 under conditions such that the host cell expresses a protein comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, thereby producing the antibody product; and (b) purifying the antibody product.

66. A pharmaceutical composition comprising an antibody product according to any one of claims 1 to 32 and a pharmaceutically acceptable excipient.

67. The antibody product according to any one of claims 1 to 32 or the pharmaceutical composition according to claim 55 for treating a subject suffering from cancer expressing LILRB2.

68. Use of an antibody product according to any one of claims 1 to 32 for the preparation of a medicament for treating cancer expressing LILRB2.

69. A method for detecting LILRB2 in a sample, tissue or cell using an antibody product according to any one of claims 1 to 32, the method comprising contacting the sample, the tissue or the cell with the antibody product and detecting the antibody product.

70. A method for reducing the biological activity of LILRB2 in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antibody product according to any one of claims 1 to 32 or a pharmaceutical composition according to claim 55.

71. The method according to claim 70, wherein the antibody product mediates depletion of at least one cancer cell expressing LILRB2.

72. The method according to claim 70, wherein the subject has a tumor with a tumor microenvironment, and the anti-tumor immune response within the tumor microenvironment is increased.

73. A method of promoting an immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of the antibody product according to any one of claims 1 to 32 or the pharmaceutical composition according to claim 55.

74. A method of providing cancer immunotherapy to a subject in need thereof, wherein the cancer cells express LILRB2, the method comprising administering to the subject a therapeutically effective amount of the antibody product according to any one of claims 1 to 32.

75. The method according to 74, wherein administering the antibody product comprises administering an amount of the antibody product capable of effectively mediating cell killing of the cancer by antibody-dependent cytotoxicity.

76. The method according to claim 74, wherein administering the antibody product comprises administering an amount of the antibody product capable of effectively alleviating LILRB2-mediated T cell inhibition in the subject.

77. The method according to claim 74, further comprising administering to the subject an amount of a PD-1 antagonist sufficient to alleviate PD-1 / PD-L1 axis-mediated T cell immunosuppression in the subject.

78. The method according to claim 77, wherein the PD-1 antagonist is a PD-1 antibody product.

79. The antibody product according to any one of claims 1 to 32, wherein the antibody product is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

80. The antibody product according to any one of claims 79, wherein the antibody product comprises a therapeutic agent or a cytotoxic agent.

81. A pharmaceutical composition comprising the antibody product according to any one of claims 1 to 32 and a physiologically acceptable carrier or excipient, wherein the antibody product reduces or prevents the binding of LILRB2 to its ligand and / or reduces or prevents LILRB2-mediated signal transduction.

82. The pharmaceutical composition according to claim 81, wherein the ligand is human leukocyte antigen A, human leukocyte antigen B, human leukocyte antigen C, human leukocyte antigen G, angiopoietin-like protein 2, angiopoietin-like protein 5, or a combination thereof.

83. The pharmaceutical composition according to claim 81 or 82, wherein the ligand is expressed on the surface of myeloid cells or tumor cells.

84. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition according to any one of claims 81 to 83.

85. The method according to claim 84, wherein the subject has a cancer comprising cells expressing or overexpressing a ligand of LILRB2.

86. The method according to any one of claims 84 to 85, wherein the antibody product or its antigen-binding fragment increases the immune response, delays or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages to alter their activity, reduces tumor-associated macrophage-mediated immunosuppression, reduces or reverses T cell inhibition, or a combination thereof.

87. The method according to any one of claims 84 to 86, wherein the cancer or tumor comprises macrophages expressing LILRB2.

88. The method according to any one of claims 84 to 87, further comprising administering a second therapeutic agent to the subject.

89. The method according to claim 88, wherein the second therapeutic agent is an immune checkpoint inhibitor.

90. An antibody product that binds to human LILRB2, the antibody product comprising CDR-H1 shown in SEQ ID NO:22, CDR-H2 shown in SEQ ID NO:17, CDR-H3 shown in SEQ ID NO:24, CDR-L1 shown in SEQ ID NO:19, CDR-L2 shown in SEQ ID NO:20, and CDR-L3 shown in SEQ ID NO:

21.

91. The antibody product according to claim 90, the antibody product comprising CDR-H1 shown in SEQ ID NO:16, CDR-H2 shown in SEQ ID NO:23, CDR-H3 shown in SEQ ID NO:18, CDR-L1 shown in SEQ ID NO:25, CDR-L2 shown in SEQ ID NO:26, and CDR-L3 shown in SEQ ID NO:27.

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