LILRB2 Antibody Products and Methods

Abstract

The present disclosure relates to LILRB2 antibody products and methods of using the same. The antibody products specifically bind to LILRB2 on cells, such as myeloid cells or cancer cells. The antibody products can be used in methods of treating diseases, such as cancer immunotherapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 380,034, filed October 18, 2022, U.S. Provisional Patent Application No. 63 / 490,705, filed March 16, 2023, and U.S. Provisional Patent Application No. 63 / 514,057, filed July 17, 2023, all of which are incorporated by reference in their entireties.

[0002] Incorporation by reference of sequence listing This application contains as another part of the disclosure a sequence listing in computer readable format (Filename: 58365_Seqlisting.XML; dated October 13, 2023, 100,193 bytes), which is incorporated herein by reference in its entirety.

[0003] 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 for treating diseases, such as methods for treating cancer. [Background technology]

[0004] Cancer is characterized by the accumulation of growth-modifying genetic alterations, along with the ability to evade detection or elimination by the immune system. Cancer immunotherapy can overcome this immune tolerance, resulting in immune recognition and anti-tumor responses. However, tumors typically use multiple mechanisms to mediate immune evasion, such as the reduction or loss of tumor antigenicity, immune evasion, or the presence of cellular or molecular factors in the tumor microenvironment (TME) that inhibit the killing of cancer cells by CD8+ T cells.

[0005] The recent development of antibodies targeting immune checkpoints has transformed the way malignant solid tumors are treated, offering hope that cure or long-term remission may be achieved even in subjects with advanced disease. 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 the first-line treatment of metastatic disease in numerous tumor types, including lung, melanoma, triple-negative breast cancer, and head and neck cancer.

[0006] Despite this success, most patients treated with these CPIs either fail to respond or ultimately progress or relapse. Furthermore, CPIs have failed to improve outcomes in patients with tumors 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 (MDSCs) and tumor-associated macrophages (TAMs), are critical components of the TME and contribute to immune evasion by many solid tumors. MDSCs and TAMs suppress antitumor immune responses and promote a tumorigenic environment. High levels of tumor infiltration by MDSCs and TAMs generally predict an unfavorable prognosis in patients with solid tumors. Alleviating the immunosuppression of myeloid cells in the TME to improve T cell-mediated responses is a rational adjunct to CPI therapy.

[0007] Leukocyte immunoglobulin-like receptor B2 (LILRB2), also known as ILT4, LIR2, MIR-10, and CD85d, is an ITIM-containing immunosuppressive member of the leukocyte immunoglobulin-like receptor family that is expressed on myeloid lineage cells (monocytes, macrophages, dendritic cells, and granulocytes) but not on lymphocytes. In the TME, LILRB2 is found on myeloid-derived suppressor cells (MDSCs) and tumor-supporting tumor-associated macrophages (TAMs). LILRB2 appears to have a dual role in cancer biology: 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 immune suppression by myeloid cells and promote tumor immune evasion in the TME. Such ligands include the non-classical MHC class I molecule human leukocyte antigen G (HLA-G) and the 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. Furthermore, ANGPTL2 and ANGPTL5 promote lung cancer development and survival through tumor-expressed LILRB2 by SHP1 signaling LILRB2 expression in myeloid cells in the TME or HLA-G expression by tumors, which correlates with poor survival in multiple cancers.

[0008] Recent data indicate that blocking LILRB2 in combination with cytokines (e.g., IL-4, IL-10) or innate differentiation / polarization signals promotes the generation of anti-tumor myeloid cells in vitro. Furthermore, the combination of anti-LILRB2 antibodies with anti-PD-1 treatment enhanced tumor growth inhibition (TGI) in humanized tumor models.

[0009] Antibodies targeting and antagonizing 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). Initial clinical data using the first-in-class anti-LILRB2 antibody MK-4830 suggest that blocking LILRB2 overrides PD-1 resistance mechanisms in patients with advanced solid tumors. MK-4830 administered as monotherapy or in combination with pembrolizumab (anti-PD-1) was well tolerated and demonstrated dose-related evidence of target engagement and antitumor activity in patients lacking predictive biomarkers associated with response to anti-PD-1 monotherapy. The overall response rate for MK-4830 / pembrolizumab combination treatment was 24%. MK-4830 clinical trial data support the development of anti-LILRB2 antibodies in combination with CPI therapy.

[0010] PCT Publication No. WO 2021 / 138079 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.

[0011] There remains a need in the art for LILRB2 antibody products and methods for their therapeutic use. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2021 / 138079 Summary of the Invention

[0013] The present disclosure provides LILRB2 antibody products (also sometimes referred to herein as "anti-LILRB2 antibody products"), compositions comprising such antibody products, and methods for their production and use in the treatment of diseases, including cancer.

[0014] The LILRB2 antibody products provided herein may be intact antibodies or may include immunologically functional antibody fragments, including antibodies with naturally occurring or recombinant structures, 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 compositions thereof can be used to prevent or treat a variety of different disease states, including, but not limited to, cancer. The LILRB2 antibodies provided herein have a useful half-life and exhibit antitumor activity in humanized mouse models. The humanized LILRB2 antibodies provided herein restore effector function to activated and exhausted T cells from M2c-mediated immunosuppression and enhance the secretion of pro-inflammatory cytokines by M0 macrophages or LPS-stimulated PBMCs.

[0015] The LILRB2 antibody products provided herein are believed to bind to LILRB2 at an epitope distinct from that bound by antibodies currently in clinical use. In preclinical studies, the antibody products enhanced LPS-induced IFN-γ production by peripheral blood mononuclear cells (PBMCs), reduced IL-10 release, and alleviated the immunosuppression of pro-tumorigenic macrophages, inducing T cell proliferation and IFN-γ and perforin secretion by T cells. Furthermore, the antibody products inhibit the development of immunosuppressive macrophages. In contrast to comparable LILRB2 antibodies, representative antibody products have demonstrated the ability to restore the ability of exhausted T cells to secrete IFN-γ in the presence of pro-tumorigenic macrophages, and also significantly enhanced the activity of pembrolizumab in combination studies. Importantly, the chimeric antibody products demonstrated superior antitumor activity in humanized mouse tumor models, with significant tumor growth inhibition and tumor regression.

[0016] Without wishing to be bound by any particular theory, it is contemplated herein that the anti-immunosuppressive activity of the antibodies provided appears to result from two distinct but coordinated mechanisms: engagement of the variable region of LILRB2 and engagement of the heavy chain of the Fc receptor.

[0017] On the one hand, LILRB2 expressed in myeloid cells is thought to negatively regulate antitumor immunity through binding to HLA-G on tumor cells and MHC class 1 on myeloid cells. Binding of LILRB2 to these ligands promotes Ca transport across the plasma membrane of myeloid cells. 2+ LILRB2 promotes immunosuppressive myeloid cells in the tumor microenvironment through signal transduction inhibition, recruitment of Src homology domain 2 containing protein tyrosine phosphatase-1 (SHP1) and SHP2 phosphatases in myeloid cell membranes, and production of cytokines by myeloid cells that can prevent checkpoint inhibitor (CPI) therapy from enhancing antitumor T cell responses. Individually or collectively, these phenomena can result in reduced cancer cell killing. LILRB2 is also thought to further hinder tumor cell killing by competing with cytotoxic T lymphocytes for binding to MHC class I.

[0018] Activating Fc receptors (FcγRIA, FcγRIIA, and FcγRIIIA) on myeloid cells are known to play an essential role in promoting cell activation, differentiation, and induction of adaptive immune responses through the regulation of antigen presentation. These receptors signal via immunoreceptor tyrosine-based activation motifs via SRC family kinases and spleen tyrosine kinases, resulting in the transcriptional activation of several pro-inflammatory cytokines and chemokines that drive cell recruitment, migration, differentiation, and survival.

[0019] The antibodies disclosed herein appear to couple LILRB2 antagonism with Fc receptor-mediated activation. The antibodies bind to a unique epitope on LILRB2, blocking not only myeloid cell interactions with tumor cell HLA-G but also cis-interactions with MHC class I, thereby inactivating both inhibitory signals on myeloid cells. This action promotes the polarization of tumor-infiltrating myeloid cells toward an inflammatory phenotype and simultaneously frees MHC class I on these cells to engage receptors necessary for optimal cytotoxic T lymphocyte activation. Furthermore, the antibodies can enhance antitumor immunity by providing immunostimulatory signals through activating FcγRIIIA. This dual or co-engagement mechanism confers novel attributes to these antibodies for targeting myeloid cells to reverse CPI resistance, enhance tumor cell killing, and improve patient outcomes.

[0020] Preclinical data for representative antibody products demonstrate a typical human IgG PK profile in humanized FcRn mice. The antibody products do not cross-react with non-human primate LILRB2 and show no cross-reactivity to other inhibitory or activating LILRB2 family members. Treatment with representative antibody products did not induce proinflammatory cytokine release or neutrophil activation in human whole blood, and the representative antibody products did not deplete human monocytes or neutrophils, suggesting a tolerable safety profile.

[0021] The present disclosure provides an antibody product that binds to human LILRB2, the antibody product comprising CDR-H1 set forth in SEQ ID NO: 16, CDR-H2 set forth in SEQ ID NO: 17, CDR-H3 set forth in SEQ ID NO: 24, CDR-L1 set forth in SEQ ID NO: 19, CDR-L2 set forth in SEQ ID NO: 20, and CDR-L3 set forth in SEQ ID NO: 21. The antibody product may comprise CDR-H1 set forth in SEQ ID NO: 16, CDR-H2 set forth in SEQ ID NO: 17, CDR-H3 set forth in SEQ ID NO: 18, CDR-L1 set forth in SEQ ID NO: 19, CDR-L2 set forth in SEQ ID NO: 20, and CDR-L3 set forth in SEQ ID NO: 21. The antibody product may comprise CDR-H1 set forth in SEQ ID NO: 22, CDR-H2 set forth in SEQ ID NO: 23, CDR-H3 set forth in SEQ ID NO: 24, CDR-L1 set forth in SEQ ID NO: 25, CDR-L2 set forth in SEQ ID NO: 26, and CDR-L3 set forth in SEQ ID NO: 21. The antibody product may comprise a heavy chain variable region comprising (a) an amino acid sequence at least 80% identical to SEQ ID NO: 1, 6, 7, 8, 9, or 10; or (b) an amino acid sequence set forth 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 at least 80% identical to SEQ ID NO: 2, 11, 12, 13, 14, or 15; or (b) an amino acid sequence set forth in SEQ ID NO: 2, 11, 12, 13, 14, or 15.The antibody products include (a) a heavy chain variable region comprising SEQ ID NO: 1; and a light chain variable region of SEQ ID NO: 2; (b) a heavy chain variable region of 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 comprising SEQ ID NO: 15; (g) a heavy chain variable region comprising SEQ ID NO: 7; and (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: (o) 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; 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 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.

[0022] 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 comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH), and a light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL). The antibody product may comprise (a) an amino acid sequence at least 80% identical to SEQ ID NO: 32; or (b) a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 32. The antibody product may comprise an amino acid sequence at least 80% identical to SEQ ID NO: 42; or (b) a light chain region comprising the amino acid sequence set forth in SEQ ID NO: 42. Exemplary antibody products comprise a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain region comprising the amino acid sequence set forth in SEQ ID NO: 42.

[0023] 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 comprising (a) an amino acid sequence 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) an IgG1 heavy chain amino acid sequence comprising the amino acid sequence set forth 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 (a) an amino acid sequence 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) an IgG4 amino acid sequence set forth 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.

[0024] The antibody products include (a) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO: 1; and a kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa light chain having an amino acid sequence comprising SEQ ID NO: 15.

[0025] The antibody product can specifically bind to human LILRB2 with a KD of, for example, 0.5 nM to 500 nM.

[0026] The antibody product can specifically bind to human LILRB2 expressed by cells, such as cancer myeloid cells or LILRB2-expressing cells. The antibody product can specifically bind to human immunosuppressive myeloid cells. The immunosuppressive myeloid cells can be present 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, for example, with a KD of 0.5 nM to 500 nM.

[0027] The antibody product may be a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibody product may be a monospecific, bispecific, trispecific, or multispecific antibody.

[0028] The antibody product may be bound by Fc receptors expressed on immunosuppressive macrophages or other myeloid cells, such as CD16 (FcγRIIIa), CD32 (FcγRII), or CD64 (FcγRI), which are expressed on immunosuppressive macrophages or other myeloid cells.

[0029] The present disclosure provides a method for providing cancer immunotherapy to a subject in need thereof, wherein the cancer is associated with the presence of immunosuppressive macrophages, comprising administering a therapeutically effective amount of an antibody product provided herein to the subject. In this method, the antibody product binds to macrophages, and the binding of the antibody product to the macrophages can have 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 the polarization of macrophages toward an immunosuppressive phenotype; and (d) enhancing the innate anti-tumor response. Activation of CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof can be measured as an increase in the levels of IFN-γ, TNF-α, or perforin, or any combination thereof. In this method, the binding of the antibody product to macrophages may or may not be cytotoxic to the macrophages. In the method, binding of the antibody product to macrophages can result in at least one of the following effects: (a) internalization of the antibody product by macrophages; (b) secretion of TNFα, IL-6, perforin, or any combination thereof; (c) reduced 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. The method can result in two or more of (a)-(f); three or more of (a)-(f); four or more of (a)-(f); five or more of (a)-(f); or all of (a)-(f). In the method, binding of the antibody product to macrophages can increase immunostimulatory activity in the tumor microenvironment. Binding of the antibody product to macrophages can reduce the immunosuppressive activity of macrophages. Binding of the antibody product to macrophages can reduce the tumor-promoting activity of macrophages.Binding of the antibody product can promote CD4+ T cell activation, CD4+ T cell proliferation, or both CD4+ T cell activation and proliferation. Binding to the antibody product can promote CD8+ T cell activation, CD8+ T cell proliferation, or both CD8+ T cell activation and proliferation. Binding of the antibody product can promote cytotoxic lymphocyte-mediated killing of cancer cells. Binding of the antibody product can promote NK cell-mediated tumor cell killing. Binding of the antibody product to macrophages can reduce the suppression of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment. The cancer can be, for example, a sarcoma, carcinoma, or blood-borne cancer. The cancer may include, for example, glioblastoma multiforme, head and neck cancer, kidney renal clear cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, skin 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. Cells of the cancer may express or overexpress LILRB2.

[0030] The methods provided herein may further include administering to the subject an effective amount of an anti-cancer therapeutic agent. The anti-cancer therapeutic agent may be an immune checkpoint inhibitor, including but not limited to, a PD-1 antagonist. The effective amount of the PD-1 antagonist may be an amount effective to reduce immunosuppression of T cells. The immunosuppression of T cells may be mediated through the interaction of T cells with myeloid cells expressing PD-L1.

[0031] 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 a composition provided herein and a container.

[0032] The present disclosure contemplates the use of an antibody product or composition provided herein for the manufacture of a medicament for the treatment of cancer in a subject in need thereof, wherein the cancer expresses LILRB2.

[0033] The present disclosure provides isolated nucleic acids comprising nucleotide sequences encoding part or all of the antibody products provided herein. The present disclosure provides expression vectors comprising the nucleic acids provided herein. The present disclosure provides host cells comprising the expression vectors provided herein.

[0034] The present disclosure provides methods for producing an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain, the method comprising: (a) growing a 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.

[0035] The present disclosure provides a pharmaceutical composition comprising an antibody product provided herein and a pharmaceutically acceptable excipient, which can be used to treat a subject with a cancer that expresses LILRB2.

[0036] The present disclosure provides methods for detecting LILRB2 in a sample, tissue, or cell using the antibody products provided herein, the method comprising contacting the sample, tissue, or cell with the antibody product and detecting the antibody product.

[0037] 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 an antibody product or pharmaceutical composition provided herein, wherein the antibody product can mediate depletion of at least one cancer cell that expresses LILRB2.

[0038] The present disclosure provides a method of promoting an immune response in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein.

[0039] The present disclosure provides a method of providing cancer immunotherapy to a subject in need thereof, wherein the subject's cancer cells express LILRB2, the method comprising administering a therapeutically effective amount of an antibody product provided herein to the subject. The method may comprise administering the antibody product in an amount effective to mediate cancer cell death via antibody-dependent cellular toxicity. The method may comprise administering the antibody product in an amount effective to reduce LILRB2-mediated suppression of T cells in the subject. The method may further comprise administering to the subject a PD-1 antagonist in an amount sufficient to reduce PD-1 / PD-L1 axis-mediated immunosuppression of T cells in the subject. The PD-1 antagonist may be a PD-1 antibody product.

[0040] The antibody products provided herein may be detectably labeled or may contain conjugated toxins, drugs, receptors, enzymes, receptor ligands, etc. The antibody products may contain therapeutic or cytotoxic agents.

[0041] Pharmaceutical compositions containing the antibody products provided herein can reduce or prevent binding of LILRB2 to its ligand and / or reduce or prevent LILRB2-mediated signaling and physiologically acceptable carriers or excipients. 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.

[0042] The present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition provided herein to the subject, wherein the subject has a cancer comprising cells that express or overexpress a ligand of LILRB2. In the method, the antibody product or antigen-binding fragment thereof enhances the immune response, slows or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates the tumor, depletes or blocks the activity of tumor-associated macrophages, alters their activity, reduces tumor-associated macrophage-mediated immunosuppression, reduces or reverses T-cell suppression, or a combination thereof. The cancer or tumor may contain macrophages that express LILRB2. The method may further comprise administering a second therapeutic agent to the subject. The second therapeutic agent may be an immune checkpoint inhibitor.

[0043] The following figures and detailed description (including examples) illustrate various non-limiting aspects of the subject matter contemplated herein.

[0044] Aspects of the present disclosure are illustrated by the following figures: [Brief explanation of the drawings]

[0045] [Figure 1A] Binding of B2A-IgG1 and B2A-IgG4 chimeras to human and cynomolgus LILRB2 Fc by ELISA and in LILRB2-expressing HEK293 cells. [Figure 1B] Binding of B2A-IgG1 and B2A-IgG4 chimeras to human and cynomolgus LILRB2 Fc by ELISA and in LILRB2-expressing HEK293 cells. [Figure 2] B2A-IgG4 blocks LILRB2 binding to human ANGPTL-2 and -5 by ELISA. [Figure 3A] Binding curves of human anti-LILRB2 chimeric antibodies to human monocytes and M0 and M2c macrophages. [Figure 3B]Binding curves of human anti-LILRB2 chimeric antibodies to human monocytes and M0 and M2c macrophages. [Figure 3C] Binding curves of human anti-LILRB2 chimeric antibodies to human monocytes and M0 and M2c macrophages. [Figure 4] Enhanced secretion of IFN-γ by LPS-stimulated PBMCs after treatment with anti-LILRB2 chimeric B2A-IgG1 and B2A-IgG4. [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. [Figure 6A] Rescue of CD8+ T cell proliferation by chimeric LILRB2 antibodies in M2c and CD8+ T cell co-culture assays, and rescue of IFN-γ and perforin responses by chimeric LILRB2 antibodies in M2c and CD8+ T cell co-culture assays. [Figure 6B] Rescue of CD8+ T cell proliferation by chimeric LILRB2 antibodies in M2c and CD8+ T cell co-culture assays, and rescue of IFN-γ and perforin responses by chimeric LILRB2 antibodies in M2c and CD8+ T cell co-culture assays. [Figure 7] Chimeric B2A-IgG1 LILRB2 antibody rescues IFN-γ release by exhausted T cells from M2c-mediated immune suppression. [Figure 8] Pharmacokinetic profiling of chimeric B2A-IgG4 and B2A-IgG1 in humanized FcRn mice. [Figure 9A] Antitumor efficacy of chimeric B2A-IgG4 in humanized NSG-SGM3 mice and in the subcutaneous SK-MEL-5 human melanoma tumor model. [Figure 9B] Antitumor efficacy of chimeric B2A-IgG4 in humanized NSG-SGM3 mice and in the subcutaneous SK-MEL-5 human melanoma tumor model. [Figure 10] Binding curves of humanized LILRB2 variants to monocytes, M0 and M2c macrophages. [Figure 11] The humanized LILRB2 antibody binds to a variety of human myeloid cells, including classical, intermediate, and non-classical monocytes, as well as myeloid dendritic cells. [Figure 12] Humanized LILRB2 variants bind to human neutrophils. [Figure 13] The humanized anti-LILRB2 variant B2H1-55 does not induce neutrophil activation in whole blood of healthy subjects. [Figure 14] Humanized LILRB2 antibodies induce the secretion of IFN-γ by LPS-stimulated PBMCs. [Figure 15] Rescue of CD8+ T cell proliferation and cytokine responses by humanized LILRB2 antibodies in M2c and CD8+ T cell proliferation assays. [Figure 16] Humanized LILRB2 mutants block the development of immunosuppressive macrophages. [Figure 17A] Alleviation of M2c-mediated immunosuppression in M2c / CD4 + T cell cocultures with selected humanized variants of B2H1-55. [Figure 17B] Alleviation of M2c-mediated immunosuppression in M2c / CD4 + T cell cocultures with selected humanized variants of B2H1-55. [Figure 18] Humanized LILRB2 antibody rescues IFN-γ release by exhausted T cells from M2c-mediated immune suppression. [Figure 19] The humanized anti-LILRB2 variant B2H1-55, in combination with a PD-1 antibody, enhances IFN-γ release by T cells exhausted from M2c-mediated immune suppression. [Figure 20] Humanized anti-LILRB2 variants induce a minimal cytokine response in whole blood of healthy subjects. [Figure 21A] The humanized anti-LILRB2 variants do not mediate ADCC of human monocytes but induce ADCC on HEK293 cells expressing human LILRB2. [Figure 21B] The humanized anti-LILRB2 variants do not mediate ADCC of human monocytes but induce ADCC on HEK293 cells expressing human LILRB2. [Figure 22]Pharmacokinetic profile of IgG1 humanized anti-LILRB2 variants in humanized FcRn mice. [Figure 23] Humanized anti-LILRB2 variants inhibit LPS-mediated IL-10 secretion by human PBMCs. DETAILED DESCRIPTION OF THE INVENTION

[0046] Unless otherwise defined herein, scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0047] antibody products The present disclosure provides antibody products that specifically bind to human LILRB2.

[0048] "PD-1 antagonist" refers to any chemical compound or biological molecule 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 on 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 of the therapeutic methods, medicaments, and uses disclosed herein, in which a human individual is 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 at NCBI Locus No. NP 005009. The human PD-L1 and PD-L2 amino acid sequences can be found at NCBI Locus No. NP 054862 and NP 079515, respectively.

[0049] "LILRB2 antagonist" means any chemical compound or biological molecule that blocks the binding of LILRB2 to HLA-G, HLA-A, HLA-B, HLA-F, or angiopoietin-like proteins (ANGPTLs, such as ANGPTL2 or ANGPTL5). Alternative names or synonyms for LILRB2 and its ligands include, but are not limited to, for ILT4, ILT4, ILT-4, MIR10, MIR-10, LIR2, LIR-2, CD85D; for HLA-G, MHC-G or major histocompatibility complex class I, G; for HLA-A, major histocompatibility complex class I, A; for HLA-B, AS, B-4901, major histocompatibility complex class I, B; for HLA-F, CDA12, HLA-CDA12, or major histocompatibility complex class I, F; for ANGPTL2, angiopoietin-3, ANG3, ANGPT3, ARP1, UNQ162, angiopoietin-like 2; for ANGPTL5, ARP4, HF ARP, PGAR, UNQ171, angiopoietin-like 54; and CDT6. In any of the therapeutic methods, medicaments, and disclosed uses in which a human individual is 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 amino acid sequence of human LILRB2 precursor can be found at NCBI Locus No.: AAB88119.1. The amino acid sequences of human HLA-G, HLA-A, HLA-B, HLA-C, and HLA-F precursors can be found at NCBI Locus No.: P17693.1, P04439.2, P01889.3, P10321-1, and P30511.3, respectively. The amino acid sequences of human ANGPTL2 and ANGPTL5 precursors can be found at NCBI Locus No.: Q9UKU9-1 and Q86XS5, respectively.

[0050] 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 found in a gene cluster on chromosome 19q13.4. The encoded protein belongs to the subfamily B class of LIR receptors, which 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, binds to MHC class I molecules on antigen-presenting cells, and transmits negative signals that inhibit stimulation of the immune response. It is thought to regulate inflammatory responses and cytotoxicity, promoting the concentration of the immune response and limiting autoreactivity. Multiple transcript variants encoding different isoforms have been found for this gene. The nucleotide sequence of the human cDNA encoding LILRB2 (NM_005874.5) is shown in SEQ ID NO: 53.

[0051] The terms "polypeptide" and "protein" are used interchangeably herein in the conventional manner to refer to molecules formed from amino acids. Polypeptides are not limited to a particular length. Peptides are included within polypeptides unless otherwise specified. These terms do not specify or exclude post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications, both naturally occurring and non-naturally occurring, known in the art. Polypeptides of interest in the context of the antibodies of the present disclosure include, but are not limited to, polypeptide fragments comprising a CDR capable of binding to LILRB2 protein expressed by myeloid cells or cancer cells.

[0052] 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. Fragments are approximately 5 to 500 amino acids in length. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Polypeptide fragments include immunologically functional fragments of antibodies containing the binding domain. In the case of LILRB2 antibodies, useful fragments include, but are not limited to, CDR regions, heavy or light chain variable domains, portions of the antibody chains, or only the variable regions containing two CDRs.

[0053] As used herein, the term "isolated protein" means that the protein of interest (1) is free from at least some other proteins with which it is normally found; (2) is essentially free from other proteins from the same source; (3) is expressed by cells from a different species; (4) is 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 noncovalent interactions) with polypeptides with which it is not naturally associated; or (6) is not naturally occurring. Genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof, can encode such an isolated protein. Preferably, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.

[0054] A "variant" of a polypeptide (e.g., an antibody) includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted in the amino acid sequence compared to another polypeptide sequence. Variants include fusion proteins.

[0055] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified in some way that differs from an insertion, deletion, or substitution mutant, for example, by conjugation to another chemical moiety.

[0056] The term "antibody" generally refers to an immunologically functional immunoglobulin protein comprising one or more polypeptide chains and specifically binding to an antigen. In humans, antibodies typically comprise four linked polypeptide chains, a "tetramer" comprising two identical "heavy" chains and two smaller, identical "light" chains. Each of the two heavy chains is linked to a light chain and is also linked in parallel to each other. This binding gives the antibody a roughly Y-shaped structure, with the binding portions of the heavy chains forming the legs of the "Y" and each light chain (together with the portion of the heavy chain to which it is bound) forming an arm of the "Y." Each arm of the antibody contains an antigen-binding site, so a typical antibody can bind to two of the antigens. In humans, there are five basic types or classes of antibodies: IgG, IgA, IgE, IgD, and IgM, distinguished by the structure of the heavy regions and their functional purpose. Several classes of intact antibodies in humans can differ from the typical tetrameric "Y" structural unit, e.g., circulating IgM antibodies, which contain five such units linked at their bases in a roughly circular array. Further details about antibody structure and function are provided elsewhere herein.

[0057] In a typical antibody, each pair or couplet of tetrameric units contains one full-length "light" chain (approximately 25 kDa) and one full-length "heavy" chain (approximately 50-70 kDa). Individual immunoglobulin chains are composed of several "immunoglobulin domains," each consisting of approximately 90-110 amino acids and expressing a characteristic folding pattern. These domains are the basic units from which antibody polypeptide chains are constructed. The amino-terminal portion of each chain typically contains a variable domain, which is responsible for antigen recognition. The carboxy-terminal portion is more evolutionarily conserved than the amino terminus of the chain and is called the "constant region" or "C region."

[0058] The term "heavy chain" includes full-length immunoglobulin heavy chains and fragments thereof containing sufficient variable domain sequence to confer binding specificity, either alone or together with a light chain variable domain. Heavy chains are typically classified as mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, which define the antibody's isotype 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 isotypes contain five heavy chains and five light chains. The heavy chain C region typically contains one or more domains capable of effector function. The number of heavy chain constant region domains depends on the isotype. For example, each full-length IgG heavy chain contains three C region domains known as CH1, CH2, and CH3, with CH3 being the closest to the carboxy terminus. The provided antibody product can have any of these isotypes and subtypes. For example, the LILRB2 antibody product can be an intact antibody of IgG1 or IgG4 subtype.

[0059] The term "light chain" encompasses full-length immunoglobulin light chains and fragments thereof containing sufficient variable domain sequence to confer binding specificity, either alone or together with a heavy chain variable domain. Human light chains are generally classified as kappa (κ) or lambda (λ) light chains. A full-length light chain includes an amino-terminal variable domain (VL) and a carboxy-terminal constant domain (CL).

[0060] In light and heavy chains, the variable and constant regions are naturally joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, ed.) 1989, New York: Raven Press.

[0061] The variable domains of immunoglobulin chains generally exhibit the same overall structure, including relatively conserved framework regions (FRs) connected by three hypervariable regions, more frequently referred to as "complementarity-determining regions" or CDRs. The CDRs from the two chains of each heavy / light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a specific epitope on a target protein (e.g., LILRB2). From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying each position in these domains.

[0062] Current technology utilizes various numbering schemes with different definitions of CDR length and position. For example, the Kabat numbering scheme is based on sequence alignment and uses the "variability parameter" of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most commonly occurring amino acid at that position) to predict CDRs [Kabat et al. in 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 that aligns antibody crystal structures 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 framework regions of different lengths, rather than conventional ones [Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," in: Kontermann and Dubel, 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 that includes 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 structures of variable regions and uses structurally conserved Cα positions to estimate the framework and CDR lengths [Honegger et al., J Mol Biol. 2001 309(3):657-70]. Those skilled in the art will understand that the definition of a CDR varies based on the method used.

[0063] Some of the antibody products provided typically have a structure related to naturally occurring antibodies. Thus, the term "antibody product" includes intact antibodies of any class or subclass or fragments thereof that can compete with intact antibodies for specific binding to a target antigen, including chimeric, humanized, fully human, and bispecific antibodies, as well as other forms. As noted above, intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some instances, they may contain fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains, and VNAR domains from sharks. Antibody products may be derived from only a single source, or may be "chimeric," i.e., different portions of the antibody may be derived from two different antibodies. For example, the complementarity-determining regions that confer the antibody's binding specificity may be derived from a rat or mouse source, while the framework regions of the variable regions are derived from a different species, such as a human. In other chimeric forms, the light and heavy variable domains (optionally with constant domains) may be derived from one species and one or more constant domains from another species. See, e.g., U.S. Patent No. 1,135,2444. The antibody products provided may be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody product" includes antibodies comprising two full-length heavy chains and two full-length light chains (such as IgG antibodies), as well as antibodies of other isotypes, derivatives, variants, and fragments thereof. The antibody products provided include, but are not limited to, monoclonal antibodies, human antibodies, chimeric antibodies, and humanized antibodies. The immunologically functional antibody fragments provided include, but are not limited to, scFv, Fab, Fab', F(ab')2, and domain antibody products.

[0064] As used herein, an "immunologically functional fragment" (or simply "fragment") of an immunoglobulin refers to a portion of an antibody that contains a light or heavy chain (or both) and is capable of specifically binding to an antigen, but where the light or heavy chain (or both) lacks at least some of the amino acids present in the full-length chain. Such fragments are biologically active in that they specifically bind to a target antigen and can compete with the intact antibody for specific binding to a given epitope. Such fragments retain at least one CDR present in a full-length light or heavy chain and can include a single heavy and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab'), 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. It is further contemplated that a functional portion of an antibody of the invention, e.g., one or more CDRs, can be covalently linked to a second protein or small molecule to create a therapeutic agent directed to a specific target in the body, with bifunctional therapeutic properties, or with a long serum half-life.

[0065] A "Fab fragment" comprises one light chain (VL+CL) and a portion of a heavy chain (VH+CH1) containing the variable and CH1 domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0066] The "Fc" region contains two heavy chain fragments, each containing the CH2 and CH3 domains of an antibody, and optionally the lower hinge region. The two heavy chain fragments are held together by two or more disulfide bonds (typically in the hinge region) and hydrophobic interactions of the CH3 domains.

[0067] A "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, and interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0068] An "F(ab')2 fragment" contains two light chains and two heavy chains, each containing a portion of the constant region between the CH1 and CH2 domains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0069] The "Fv region" comprises the variable domains from both the heavy and light chains, but lacks the constant domains.

[0070] A "single-chain antibody" is an Fv molecule in which the heavy and light chain variable domains are joined by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail, for example, in PCT Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

[0071] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable domain of the heavy chain or the variable domain of the light chain. In some instances, two or more VH domains are covalently linked with a peptide linker to create a bivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0072] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific (see below).

[0073] A "multispecific antibody" is one that targets more than one antigen or epitope.

[0074] A "bispecific (dual-specific)" or "bifunctional" antibody is a hybrid antibody having two different antigen-binding sites. Bispecific antibodies are a type of multispecific antibody and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking 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 bind to two different epitopes, which may be present on the same or different protein targets. A "trispecific" antibody has three different antigen-binding sites.

[0075] The term "neutralizing antibody" refers to an antibody that binds to a ligand, prevents the ligand from binding to its binding partner, or otherwise interrupts a biological response resulting from the binding of the ligand to its binding partner. When assessing the binding and specificity of an antibody or immunologically functional fragment thereof, an antibody or fragment substantially inhibits the binding of a ligand to its binding partner if excess antibody reduces the amount of binding partner bound to 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, a neutralizing antibody product reduces 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).

[0076] The term "competition" when used in the context of antibody products competing for the same epitope means that competition between the antibodies is determined by an assay in which the antibody product under test blocks or inhibits the specific binding of a reference antibody product to a common antigen (e.g., LILRB2 or a fragment thereof). Numerous types of competitive binding assays can be used, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays [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 label assays, solid-phase direct label sandwich assays [e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)]; solid-phase direct label RIA using I-125 labels [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]. Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either 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. The test antibody is usually present in excess. Antibodies identified by competitive assays (competing antibodies) include those that bind to the same epitope as the reference antibody and those that bind to an epitope sufficiently close to the epitope bound by the reference antibody so that steric hindrance occurs. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more by a selective binding agent, such as an antibody, which can be used in an animal to generate antibodies capable of binding to the antigen. An antigen can have one or more epitopes that can interact with different antibodies.

[0077] The term "epitope" includes any determinant capable of specific binding to an antibody or T-cell receptor. An epitope is the region of an antigen bound by an antibody that specifically targets that antigen, and, if the antigen is a protein, includes specific amino acids that directly contact the antibody. In most cases, epitopes reside on proteins, but in some instances, they can reside on other types of molecules, such as nucleic acids. Epitopes can include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural and / or charge characteristics. Generally, antibodies specific for a particular target antigen preferentially recognize epitopes on the target antigen in a complex mixture of proteins and / or macromolecules.

[0078] Dissociation constant (K d An antibody product "specifically binds" to its target antigen if its K d is "high affinity" if the K is less than 10 nM; d Antibody products specifically bind to antigens with "ultra-high affinity" when their K is less than 0.5 nM. Antibody products have K values ​​in the range of about 0.5 nM to about 500 nM. d The antibody product may have a K in the range of about 100 to about 500 nM. d Those skilled in the art will recognize that specific binding does not mean exclusive binding, but rather it allows for some degree of non-specific binding as is typical in biological reactions between groups that have affinity for one another.

[0079] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two drugs, the equilibrium dissociation constant, K D , the calculated ratio of the dissociation constant to the association constant between an antibody and its antigen (K off / K on ) The affinity can be expressed as K D The association constant K is the reciprocal of A The antibody products disclosed herein can also be expressed as K D The binding affinity, as measured by -4 M or less or 10 -16 M or less (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 The antibodies described herein are capable of binding to 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, 10 -7 M or less, or 10 -8 K below M D The human LILRB2 polypeptide or the human LILRB1 polypeptide can specifically bind to the human LILRB2 polypeptide or the human LILRB1 polypeptide at a specific affinity. Methods for determining the affinity between two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (SPR), biolayer interferometry (BLI), and the like.

[0080] As used herein, an antibody product is said to be "immunospecific" or "specific" or "specifically binds" if the antibody product forms a complex with an antigen that is relatively stable under physiological conditions. The terms "preferentially bind" or "specifically bind" mean that the antibody or fragment thereof binds to an epitope with higher affinity than it binds to an unrelated amino acid sequence, and is non-toxic at levels formulated for use in human administration, provided it is cross-reactive with other polypeptides containing the epitope. Such affinity may be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody product for an unrelated amino acid sequence. The term is also applicable, for example, when an antibody product is specific for a particular epitope carried by more than one antigen, in which case an antibody or antigen-binding fragment thereof carrying an antigen-binding domain can specifically bind to epitopes found on different antigens.

[0081] 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" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment, if any, must be addressed 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 Computational Molecular Biology, (Lesk, Ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds.), 1994, New Jersey: Humana Press; Sequence Analysis in Molecular Biology, (von Heinje), 1987, New York: Academic Press; Sequence Analysis Primer, (Gribskov and Devereux, eds.), 1991, New York: M. Stockton Press; Carillo et al., SIAM J Applied Math. 1988 48(5):1073-82.

[0082] When calculating percent identity, the sequences to be compared are aligned to maximize the sequence identity. An exemplary computer program used to determine percent identity is the GCG program package, including 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 whose percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acids or nucleotides (the "matched span" determined by the algorithm). A gap opening penalty (calculated as 3 times the average diagonal, where "average diagonal" is the average of the diagonal of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by a specific comparison matrix) and a gap extension penalty (usually 1 / 10 of the gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62 are used with the algorithm. Standard comparison matrices (e.g., for the PAM 250 comparison matrix, see 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.

[0083] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program 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 end gaps); gap length penalty: 4; similarity threshold: 0.

[0084] Certain alignment schemes for aligning two amino acid sequences may result in matching only short regions of the two sequences, and these small aligned regions may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) can be adjusted, if desired, to result in alignment over at least 50 consecutive amino acids of the target polypeptide.

[0085] Other exemplary programs for comparing and aligning pairs of sequences 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); 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(1 Pt 1):387-95).

[0086] "Amino acid" includes its ordinary meaning in the art. The 20 naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis, 2nd ed. (Golub and Gren, Eds.), Sinauer Associates: Sunderland, Mass. (1991). Stereoisomers of the 20 common amino acids (e.g., D-amino acids), unnatural amino acids such as α-, α-disubstituted amino acids, N-alkylamino acids, and other unusual amino acids may be suitable components. Examples of unusual amino acids include 4-hydroxyproline, gamma-carboxyglutamate, 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, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0087] Table 10 below shows the heavy and light chain variable domains of the LILRB2-specific antibody designated B2A, and the corresponding full-length heavy (hIgG1 and hIgG4) and light (κ) chains provided herein. The IMGT and Kabat CDRs are shown in Table 11. The CDRs of the two variable domains are shown in double underline for IMGT and in bold for Kabat.

[0088] [Table 10]

[0089] [Table 11]

[0090] Humanized variants of the B2A antibody were prepared as described in the Examples. Representative humanized heavy and light chain variable domains are shown in Table 12.

[0091] [Table 12]

[0092] Those skilled in the art will recognize that antibody products, such as full-length intact antibodies and LILRB2-binding antibody fragments, can be prepared based on the heavy and light chain variable domains shown in Tables 10 and 12, or based on the CDRs shown in Table 11. 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 shown in Table 12 (SEQ ID NOS: 6-10) were prepared in combination with kappa light chains containing the variable domains shown in Table 12 (SEQ ID NOS: 11-15). Full-length IgG1 heavy chain sequences are shown as SEQ ID NOS: 28-32. Full-length IgG4 heavy chain sequences are shown as SEQ ID NOS: 33-37. Full-length kappa light chain sequences are shown as SEQ ID NOS: 38-42. LILRB2-binding antibody products were prepared using all combinations of heavy and light chains, and testing of several of these in various non-clinical assays is described in the Examples.

[0093] The antibody product can comprise a light chain variable domain comprising a sequence of amino acids that differs by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from the sequences of the light chain variable domains described herein, where each such sequence difference is independently either a deletion, insertion, or substitution of a single amino acid. The light chain variable region in some antibodies comprises 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.

[0094] The antibody products can include a heavy chain variable domain that comprises a sequence of amino acids that differs from the sequence of the heavy chain variable domain provided herein by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, and each such sequence difference is independently either a deletion, insertion, or substitution of a single amino acid. The heavy chain variable region in some antibodies comprises 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 heavy chain variable region in Table 10 or Table 12. Still other antibody products are encompassed that include variant forms of the variant light chains and variant heavy chains just described.

[0095] The provided antibody products may include one, two, three, four, five, or all six CDRs. Some antibody products include both light chain and heavy chain CDR3s. Certain antibody products have variant forms of CDRs in which one or more (i.e., two, three, four, five, or six) of the CDRs have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CDR sequences, respectively. For example, the antibody product may include both light chain and heavy chain CDR3s that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the light chain and heavy chain CDR3 sequences, respectively. The CDR sequences of the provided antibody products may also differ from the CDR sequences of Table 10 or Table 12 such that the amino acid sequence of any given CDR differs by no more than 1, 2, 3, 4, or 5 amino acid residues from the sequences listed in Table 10 or Table 12. The differences from the listed sequences are conservative substitutions.

[0096] 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 identified by a particular residue (e.g., one or more specific segments of the LILRB2 protein). In binding to a LILRB2 epitope, the antibody does not necessarily contact every residue in the LILRB2 peptide, including that particular residue. Furthermore, every single amino acid substitution or deletion in a LILRB2 peptide does not necessarily significantly affect binding affinity. The precise epitope specificity of an antibody can be determined in various ways. One approach involves, for example, testing a collection of overlapping peptides of approximately 15 amino acids that span the LILRB2 sequence and differ in increments of a small number of amino acids (e.g., three amino acids). The peptides are immobilized in the wells of a microtiter dish. 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 is useful for identifying end-specific antibodies. Optionally, additional peptides terminating at a specific amino acid of interest can be included. This approach is useful for identifying end-specific antibodies to internal fragments of LILRB2. The antibody products are screened for specific binding to each of the various peptides. An epitope is defined as existing within a segment of amino acids common to all peptides to which the antibody shows specific binding.

[0097] Antibody products that compete with one of the exemplified antibodies for specific binding to LILRB2 are also provided. Such antibody products may also bind to the same epitope as one of the exemplified antibodies. Antibody products that compete with or bind to the same epitope as the exemplified antibodies or fragments are expected to exhibit similar functional properties. Exemplary antibody products include those having heavy and light chains, variable domains, and CDRs provided in Table 10, Table 11, or Table 12. Competitive antibody products may include those that bind to the epitopes described in the antibody and epitope sections above.

[0098] The antibody products provided include monoclonal antibodies that bind to LILRB2. Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from transgenic animals after the completion of the immunization schedule. The spleen cells can be immortalized using any technique known in the art, for example, by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in the hybridoma-producing fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that prevent them from growing in a specific selective medium that supports the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in mouse cell fusion 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 / 5XXO Bul. Examples of cell lines used in rat cell fusion include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion include U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0099] In some examples, hybridoma cell lines are produced by immunizing an animal (e.g., a transgenic animal having a human immunoglobulin sequence) with a LILRB2 immunogen; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells with a myeloma cell line to thereby generate hybridoma cells; and establishing hybridoma cell lines from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies that bind to LILRB2 polypeptides. Such hybridoma cell lines and the LILRB2 monoclonal antibodies produced thereby are provided herein.

[0100] The monoclonal antibodies secreted by the hybridoma cell lines can be purified using any useful technique known in the antibody art. The hybridomas or monoclonal antibodies can be further screened to identify monoclonal antibodies with particular properties. Examples of such screens are provided in the Examples below.

[0101] Chimeric and humanized antibodies based on the aforementioned sequences are also provided. Monoclonal antibodies for use as therapeutic agents can be modified in various ways before use. One example is a "chimeric" antibody, which is an antibody composed of protein segments from different antibodies covalently linked to produce a functional immunoglobulin light or heavy chain, or an immunologically functional portion thereof. Generally, a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies 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., Proc Natl Acad Sci USA. 1985 81:6851-5. CDR grafting is described, for example, in US Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089 and 5,530,101.

[0102] Generally, the goal in creating chimeric antibodies is to create chimeras in which the number of amino acids derived 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, with the remainder of the antibody chain(s) being identical or homologous to the corresponding sequences of an antibody from another species or belonging to another antibody class or subclass. For use in humans, V regions or selected CDRs from a rodent antibody are often grafted into a human antibody, replacing the naturally occurring V regions or CDRs of the human antibody.

[0103] "Humanized" antibody products are provided. Generally, humanized antibodies are produced from monoclonal antibodies initially generated in non-human animals. Certain amino acid residues in the monoclonal antibody, typically from non-antigen-recognizing portions of the antibody, are modified to become homologous to corresponding residues in human antibodies of the corresponding isotype. Humanization can be achieved using various methods, for example, by substituting at least a portion of a rodent variable region for the corresponding region of a human antibody (e.g., U.S. Pat. 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 human variable region(s) to produce hybrid antibodies.

[0104] Fully human antibodies are also provided. Methods are known for producing fully human antibodies specific to a given antigen without exposing humans to the antigen ("fully human antibodies"). One means for achieving the production of fully human antibodies is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means for producing fully human monoclonal antibodies (MAbs) in mice, animals that can be immunized with any desired antigen. The use of fully human antibodies can minimize the immunogenicity and allergic responses that can sometimes be caused by administering mouse or mouse-derivatized monoclonal antibodies to humans as therapeutic agents.

[0105] Fully human antibodies can be produced 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 consecutive amino acids and are optionally conjugated to a carrier such as a hapten. See, for example, Jakobovits et al., Proc Natl Acad Sci USA. 1993 90:2551-5; Jakobovits et al., Nature. 1993 362:255-8; and Bruggemann et al., Year Immunol. 1993 7:33-40. In one example of such a method, a transgenic animal is generated by disabling the endogenous mouse immunoglobulin loci encoding mouse heavy and light immunoglobulin chains therein and inserting large fragments of human genomic DNA containing loci encoding human heavy and light chain proteins into the mouse genome. The partially modified animals with less than a full complement of human immunoglobulin loci are then bred to obtain animals with all of the desired immune system modifications. When administered with an immunogen, these transgenic animals produce antibodies that are immunospecific to the immunogen but have human amino acid sequences rather than mouse amino acid sequences, including the variable regions. For further details of such methods, see, for example, International Publication No. 96 / 33735 and International Publication No. WO94 / 02602. Further methods relating to transgenic mice for producing human antibodies are described in U.S. Pat. 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; WO 91 / 10741; WO 90 / 04036; and European Patent Application Publication No. 546073B1.Transgenic mice, referred to herein as "HuMab" mice, contain human immunoglobulin gene minilocuses encoding unrearranged human heavy (gamma and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous gamma and kappa chain loci (Lonberg et al., Nature. 1994 368:856-9). Thus, these mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGκ monoclonal antibodies. An exemplary mouse carrying the entire human immunoglobulin locus in its germline is the XenoMouse (Abgenix). Another is the VelociImmune mouse (Regeneron Pharmaceuticals). Others include the RenMab and RenLite mice (Biocytogen), and more recently, the AlivaMab mouse (Ablexis).

[0106] Using hybridoma technology, antigen-specific human monoclonal antibodies with desired specificity can be produced and selected from the transgenic mice described above. Such antibodies can be cloned and expressed using appropriate vectors and host cells, or the antibodies can be recovered from cultured hybridoma cells.

[0107] Fully human antibodies can also be derived from phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 1992 227(2):381-8; Marks et al., J. Mol. Biol. 1991 222:581-97). Phage display technology mimics immune selection by displaying antibody repertoires on the surface of filamentous bacteriophage, followed by selection of phages by binding to selected antigens. One such technique is described in PCT Publication No. WO 99 / 10494, which describes the isolation of high-affinity and functional agonist antibodies against c-Mpl and MuSK receptors using such an approach.

[0108] Single-chain antibodies are provided. Single-chain antibodies are formed by linking a heavy chain variable domain (Fv region) fragment and a light chain variable domain (Fv region) fragment (such as those shown in Table 10 or Table 12) via an amino acid bridge (short peptide linker) to yield a single polypeptide chain. Such single-chain Fvs (scFvs) can be prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). The resulting polypeptide can fold back on itself to form an antigen-binding monomer or can form a multimer (e.g., a dimer, trimer, or tetramer), depending on the length of the flexible linker between the two variable domains. Techniques developed for the production of single-chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird et al., Science. 1988 242:423-6; Huston et al., Proc Natl Acad Sci USA. 1988 85:5879-83; Ward et al., Nature. 1989 334:544-6; and de Graaf et al., Methods Mol Biol. 2002 178:379-87. "Diabodies" are dimers of scFv.

[0109] Antibodies provided herein of one subclass can be converted to antibodies from a different subclass using subclass switching techniques. For example, the variable domains shown in Table 10 or Table 12 can be combined with constant domains of any desired Ig subtype. Such techniques allow for the preparation of new antibodies that have the antigen-binding properties of a given antibody (parent antibody) but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA technology can be used. Cloned DNA encoding specific antibody polypeptides, such as DNA encoding the constant domain of an antibody of a desired isotype, can be used in such procedures. See, for example, Lantto et al., Methods Mol Biol. 2002 178:303-16. Thus, the provided antibodies encompass the desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE, and IgD).

[0110] The antibody products provided can comprise one or more CDRs of any of the heavy chain variable domains exemplified herein, where such CDRs are determined according to IMGT, Kabat, or other methods: (i) a CDR-H1 having at least 80% sequence identity with CDR-H1 of SEQ ID NO: 1, 6, 7, 8, 9, or 10; (ii) a CDR-H2 having at least 80% sequence identity with CDR-H2 of SEQ ID NO: 1, 6, 7, 8, 9, or 10; and (iii) a CDR-H3 having at least 80% sequence identity with CDR-H3 of SEQ ID NO: 1, 6, 7, 8, 9, or 10. The antibody products provided may include one or more CDRs of any of the light chain variable domains exemplified herein, where such CDRs are determined according to IMGT, Kabat, or other methods: (i) a CDR-L1 having at least 80% sequence identity with CDR-L1 of SEQ ID NO: 2, 11, 12, 13, 14, or 15; (ii) a CDR-L2 having at least 80% sequence identity with CDR-L2 of SEQ ID NO: 2, 11, 12, 13, 14, or 15; and (iii) a CDR-L3 having at least 80% sequence identity with CDR-L3 of SEQ ID NO: 2, 11, 12, 13, 14, or 15. In some embodiments, the CDRs have at least 85%, at least 90%, at least 95%, or at least 99% identity to the determined CDR sequences. The antibody product may include one, two, three, four, five or all six of the aforementioned CDRs, so long as it specifically binds to hLILRB2.

[0111] The provided antibody products may include one or more of the following exemplary heavy chain IMGT CDRs: (i) CDR-H1 having at least 80% sequence identity to SEQ ID NO: 16; (ii) CDR-H2 having at least 80% sequence identity to SEQ ID NO: 17; and (iii) CDR-H3 having at least 80% sequence identity to SEQ ID NO: 18. The provided antibody products may include one or more of the following light chain CDRs: (i) CDR-L1 having at least 80% sequence identity to SEQ ID NO: 19; (ii) CDR-L2 having at least 80% sequence identity to SEQ ID NO: 20; and (iii) CDR-L3 having at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the CDRs have at least 85%, at least 90%, at least 95%, or at least 99% identity to the designated CDR sequences. The antibody product may include one, two, three, four, five or all six of the aforementioned CDRs, so long as it specifically binds to hLILRB2.

[0112] The provided antibody products may comprise one or more of the following exemplary heavy chain Kabat CDRs: (i) CDR-H1 having at least 80% sequence identity to SEQ ID NO: 22; (ii) CDR-H2 having at least 80% sequence identity to SEQ ID NO: 23; and (iii) CDR-H3 having at least 80% sequence identity to SEQ ID NO: 24. The provided antibody products may include one or more of the following light chain CDRs: (i) CDR-L1 having at least 80% sequence identity to SEQ ID NO: 25; (ii) CDR-L2 having at least 80% sequence identity to SEQ ID NO: 26; and (iii) CDR-L3 having at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the CDRs have at least 85%, at least 90%, at least 95%, or at least 99% identity to the designated CDR sequences. The antibody product may include one, two, three, four, five or all six of the aforementioned CDRs, so long as it specifically binds to hLILRB2.

[0113] The antibody products provided may include: (a) a heavy chain variable region having 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more 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 more 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).

[0114] Other antibody products are provided that compete with antibodies such as those described above for specific binding to LILRB2 polypeptides. For example, antibody products are provided that compete with antibodies consisting of two identical heavy chains and two identical light chains, wherein the heavy chains comprise SEQ ID NO: 1, 6, 7, 8, 9, or 10, and the light chains comprise SEQ ID NO: 2, 11, 12, 13, 14, or 15.

[0115] LILRB2 antibody products are provided that have a half-life of at least 1 day in vitro or in vivo (e.g., when administered to a human subject). The antibody product may have a half-life of at least 3 days. The antibody product may have a half-life of 4 days or more. The antibody product may have a half-life of 8 days or more.

[0116] Mutants Provided herein are variants of the LILRB2 antibody products (e.g., variant forms of the 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 chain variable regions or light chain variable regions or CDRs listed in Tables 10 and 12.

[0117] Naturally occurring amino acids can be divided into classes based on common side chain properties: 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, and; 5) residues that affect chain orientation: Gly, Pro; 6) aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions can involve replacing a member of one of these classes with another member of the same class. Conservative amino acid substitutions can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other inverted or reversed forms of amino acid moieties.

[0118] Non-conservative substitutions may involve exchanging a member of one of the above classes for a member of another class. Such substituted residues can be introduced into regions of the antibody product that are homologous to human antibodies or into non-homologous regions of the molecule.

[0119] When making such changes, the hydropathic index of the amino acids can be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical "hydrophilicity index" to each amino acid and then iteratively averaging these values ​​along the peptide chain. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are as follows: 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); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0120] The importance of hydrophobicity profiles in conferring interactive biological function to proteins is understood in the art [e.g., Kyte and Doolittle, J Mol Biol. 1982 157:105-31]. It is known that certain amino acids can be substituted for other amino acids with similar hydropathic indices or scores and still retain similar biological activity. When making changes based on hydropathic index, substitutions of amino acids with hydropathic indices within ±0.2 can be made. Substitutions of amino acids with hydropathic indices within ±0.1 can also be made. Substitutions of amino acids with hydropathic indices within ±0.5 can also be made.

[0121] It is also understood that substitution of like residues in an amino acid sequence can be made effectively based on the relative hydrophilicity or hydrophobicity of the residues, particularly when the biologically functional protein or peptide thereby produced is intended for use in an immunological molecule, as in the present case. The greatest local average hydrophobicity characteristic of a protein, as governed by the hydrophilicity of its adjacent amino acids, can correlate with its immunogenicity and antigen binding or immunogenicity, i.e., a biological property of the protein.

[0122] Various methods are known for estimating the hydrophilicity or hydrophobicity of amino acid residues in proteins. A comparative survey of such methods is described in Biswas et al., J Chromatogr A. 1000(1-2):637-55. Hopp and Woods (Mol Immunol. 1983 20(4):483-9) assigned hydrophilicity values ​​to the following amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 0.1); glutamate (+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 a positive value, and less hydrophilic residues are assigned a negative value. When making changes based on similar hydrophilicity values, substitutions of amino acids with hydrophilicity values ​​within ±0.2 are included; otherwise, substitutions of amino acids with hydrophilicity values ​​within ±0.1 are included, or substitutions of amino acids with hydrophilicity values ​​within ±0.5 are included. In some cases, epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also called "epitope core regions."

[0123] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein.Those skilled in the art can identify suitable regions of molecules that can be changed without destroying activity by targeting regions that are not considered important for activity.Those skilled in the art can also identify the residues and parts of molecules that are conserved between similar polypeptides.Even regions that may be important for biological activity or structure can undergo conservative amino acid substitution without destroying biological activity or adversely affecting polypeptide structure.

[0124] Furthermore, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for the activity or structure of similar proteins. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0125] Those skilled in the art can also analyze the three-dimensional structure of similar polypeptides and the amino acid sequence associated with that structure. Taking this information into account, those skilled in the art can predict the alignment of the amino acid residues of an antibody relative to its three-dimensional structure. Those skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can create test variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using an assay for LILRB2 activity (see Examples below), thus providing information on which amino acids can be changed and which should not be changed. In other words, based on the information gathered from such routine experiments, those skilled in the art can easily determine the amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations.

[0126] Substantial alterations in the functional and / or biochemical characteristics of the antibody products described herein can be achieved by creating substitutions in the amino acid sequences of the heavy and light chains that differ significantly in their effect on (a) the structure of the molecular backbone in the region of the substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk. "Conservative amino acid substitutions" can involve replacing a native amino acid residue with a standard residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Additionally, any native residue in a polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis.

[0127] Amino acid substitutions (whether conservative or non-conservative) of the subject antibody can be made by those skilled in the art by applying routine techniques. Amino acid substitutions include, but are not limited to, substitutions that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in portions of antibodies outside the domain(s) that form intermolecular contacts. Conservative amino acid substitutions that do not substantially change the structural characteristics of the parent sequence (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes the parent antibody or the native antibody) can be used. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed.), 1984, New York: W.H. Freeman and Company; Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton et al., Nature. 1991 354(6349):105-6, each of which is incorporated herein by reference.

[0128] Glycosylation variants of antibody products are provided in which the number and / or type of glycosylation site(s) is / are altered compared to the amino acid sequence of the parent polypeptide. The antibody product variants may contain more or fewer 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 except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence prevent the addition of an N-linked carbohydrate chain present in the native polypeptide. For example, glycosylation can be reduced by deleting Asn or substituting Asn with a different amino acid. For example, one or more new N-linked sites can be created. Antibodies typically have N-linked glycosylation sites in the Fc region.

[0129] Further antibody product variants include cysteine ​​variants in which one or more cysteine ​​residues in the parent or native amino acid sequence are deleted or substituted with another amino acid (e.g., serine). Cysteine ​​variants are particularly useful when antibodies must be refolded into a biologically active conformation. Cysteine ​​variants can have fewer cysteine ​​residues than native antibodies, typically an even number, to minimize interactions caused by unpaired cysteines.

[0130] Effector function Antibody structure influences the role that antibodies play in the immune system and the effects they can induce or affect. See, for example, Vidarsson et al., Front Immunol. 2014 5(Art. 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. Typically, Fc-mediated functions involve binding of the Fc portion of an antibody to a specialized receptor molecule, "Fc receptor," or "FcR," expressed by the cell whose function is affected.

[0131] IgG is considered the most versatile immunoglobulin because, in some embodiments, it performs all of the functions of immunoglobulin molecules. IgG is the predominant 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. As a result of binding to Fc receptors on PMNs, monocytes, and macrophages, the cells are now better able to internalize antigens in some cases. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other cell types results in the activation of other functions.

[0132] In certain embodiments, the FcR is a native-sequence human FcR. Furthermore, preferred FcRs are those that bind IgG antibodies (gamma ("γ") receptors), including receptors of the FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) subclasses, including allelic variants; alternatively spliced ​​forms of these receptors, FcγRII receptors, include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain.

[0133] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are required for such killing. Primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. To assess the ADCC activity of a molecule of interest, in some embodiments, an in vitro ADCC assay is performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0134] Alternatively, or additionally, in some embodiments, ADCC activity of the molecule of interest is assessed in vivo, e.g., in an animal model.

[0135] In some embodiments, antibodies of the present disclosure bind to surface membrane proteins of M2-like macrophages and are internalized by them. This internalization process is thought to be responsible for the observed change in the functional immunosuppressive properties of these cells, i.e., differentiating the cells from an M2 state to a slightly activated state without killing the cells or inhibiting their proliferation. In some embodiments, upon internalization, the antibodies decrease the expression of immunosuppressive soluble factors and increase the expression of soluble factors that stimulate or promote the activity or proliferation of T cells, including CD4+ helper T cells and cytotoxic lymphocytes.

[0136] In certain therapeutic applications, the internalization process is used to kill target cells expressing LILRB2 protein or reduce the activity or proliferation of target cells. The number of internalized antibody molecules will be sufficient or appropriate to kill the cells or inhibit their growth. Depending on the potency of the antibody or antibody conjugate, in some cases, 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 are so potent in killing that the internalization of one molecule of the toxin conjugated to an antibody is sufficient to kill the target cell.

[0137] In some embodiments, the LILRB2 antibody or antigen-binding fragment provided herein is 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 a compound and a label includes any means 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 antigen-binding fragment thereof is conjugated to an affinity tag (e.g., a purification tag) or recombinantly engineered with an affinity tag. For example, affinity tags such as polyhistidine (e.g., His6) tags are conventional in the art.

[0138] In some embodiments, the LILRB2 antibody or antigen-binding fragment further comprises a detectable moiety. Detection is accomplished, for example, in vitro, in vivo, or ex vivo. For example, in vitro assays for detecting and / or determining (quantitating, qualifying, etc.) hLILRB2 protein expressed by macrophages using an antibody or its antigen-binding fragment include, but are not limited to, ELISA, RIA, and Western blot. In some embodiments, in vitro detection, diagnosis, or monitoring of the antibody's antigen is performed by obtaining a sample (e.g., a blood sample) from a subject and testing the sample, for example, in a standard ELISA assay.

[0139] derivative Derivatives of the LILRB2 antibody products described herein are also provided. Derivatized antibody products can include any molecule or substance that confers desirable properties to the antibody product, such as extended half-life for a particular use. Derivatized antibody products can include, for example, a detectable (or labeled) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, a detectable bead (e.g., a magnetic bead or a high-density (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 pharmaceutically active moiety), or a molecule that enhances 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 use). Examples of molecules that can be used to derivatize antibody products include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-conjugated and PEGylated derivatives of antibody products can be prepared using techniques well known in the art. The antibody may be conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant may be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol.

[0140] Other derivatives include covalent or aggregative conjugates of LILRB2 antibody products with other proteins or polypeptides, such as by expressing recombinant fusion proteins containing heterologous polypeptides fused to the N- 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 alpha-factor leader, or a peptide such as an epitope tag. LILRB2 antibody product-containing fusion proteins can include a peptide added to facilitate purification or identification of the LILRB2 antibody product (e.g., poly-His). LILRB2 antibody products can also be conjugated to a FLAG peptide, as described in Hopp et al., Bio / Technology 1988 6:1204-10, and U.S. Pat. No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by specific monoclonal antibodies (mAbs), allowing for rapid assay and easy purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0141] 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 bonded dimers, trimers, or higher. It is contemplated to use oligomers containing two or more LILRB2 antibody products, and one example is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0142] An oligomer can comprise multiple LILRB2 antibody products linked via covalent or non-covalent interactions between peptide moieties fused to the LILRB2 antibody polypeptide. Such peptides can be peptide linkers (spacers) or peptides with oligomerization-promoting properties. Certain polypeptides derived from leucine zippers and antibodies are among the peptides that can promote oligomerization of LILRB2 antibody products linked thereto, as described in more detail below.

[0143] The oligomer can contain 2 to 4 LILRB2 antibody products. The LILRB2 product portion of the oligomer can be in any of the forms described above, such as a variant or fragment. The oligomer contains a LILRB2 antibody product that has LILRB2-binding activity.

[0144] The preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, in Ashkenazi et al., Proc Natl Acad Sci USA. 1991 88(23):10535-9; Byrn et al., Nature 1990 344(6267):677-670; and Hollenbaugh and Aruffo, Curr Protoc Immunol. 2002 48(1):10.19A.1-10.19A.11.

[0145] A dimer is provided, comprising two fusion proteins prepared by fusing a LILRB2-binding fragment of a LILRB2 antibody to the Fc region of an antibody. The dimer can be prepared, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and assembling the expressed fusion proteins into closely related antibody molecules, in which an interchain disulfide bond is formed between the Fc portions to form a dimer.

[0146] As used herein, the term "Fc polypeptide" encompasses native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also encompassed. Fusion proteins containing an Fc portion (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns.

[0147] One exemplary Fc polypeptide, described in WO 93 / 10151 and U.S. Pat. Nos. 5,426,048 and 5,262,522 (each incorporated herein by reference), is a single-chain polypeptide extending from the N-terminal hinge region of a human IgG1 antibody to the native C-terminus of the Fc region. Another exemplary Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and Baum et al., EMBO J. 1994 13:3992-4001 (1994). The amino acid sequence of this mutein is identical to the native Fc sequence set forth in PCT Publication No. WO 93 / 10151, except that the 19th amino acid is changed from Leu to Ala, the 20th amino acid is changed from Leu to Glu, and the 22nd amino acid is changed from Gly to Ala. The mutein exhibits reduced affinity for the Fc receptor.

[0148] Alternatively, the oligomer is a fusion protein comprising multiple LILRB2 antibody polypeptides, with or without a peptide linker (spacer peptide). Suitable peptide linkers include those described in U.S. Patent Nos. 4,751,180 and 4,935,233.

[0149] Another method for preparing oligomeric LILRB2 antibody product derivatives involves the use of leucine zippers. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT Publication No. WO 94 / 10308, and the leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., FEBS Lett. 1994 344:191-5. The use of a modified leucine zipper that allows 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 containing an LILRB2 antibody fragment fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric LILRB2 antibody product is recovered from the culture supernatant.

[0150] The LILRB2 antibody products described herein can also be derivatized or modified so that the product has a longer half-life compared to underivatized or unmodified antibodies. For example, the antibody product can contain point mutations that increase serum half-life, as described in PCT Publication No. WO 00 / 09560.

[0151] Nucleic Acids and Cells Also provided are nucleic acids encoding one or more chains of the antibody products herein, polynucleotides sufficient for use as hybridization probes, PCR or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding the polypeptides, antisense nucleic acids for inhibiting expression of the polynucleotides, and complementary sequences of the foregoing.

[0152] The nucleic acids provided encode the antibody products disclosed herein, e.g., a light chain variable region shown in Table 10 or Table 12, and / or a heavy chain variable region 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 than those listed in Table 10 or Table 12. The disclosure provides each degenerate nucleotide sequence encoding each antibody product.

[0153] The term "polynucleotide" or "nucleic acid" refers to a single- or double-stranded polymer. The nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate. The term encompasses both single- and double-stranded forms.

[0154] By "isolated nucleic acid molecule" is meant DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, where the isolated polynucleotide is not related to all or a portion of a polynucleotide found in nature or is linked to a polynucleotide with which it is not linked in nature. For purposes of this disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include coding sequences for up to 10, or even up to 20, other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0155] Unless otherwise specified, the left end of any single-stranded polynucleotide sequence discussed herein is the 5' end. The leftward direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction. The sequence region on the DNA strand that is located at the 5' end to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence." The sequence region on the DNA strand that is located at the 3' end to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0156] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend on the host organism. For example, eukaryotic control sequences can include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. "Control sequences" can also include leader sequences and / or fusion partner sequences.

[0157] The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0158] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in conjunction with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, affect RNA splicing of the coding region operably linked thereto.

[0159] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under appropriate conditions. For example, a control sequence in a vector "operably linked" to a protein-coding sequence is ligated such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0160] The term "host cell" means a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0161] DNA encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full-length) can be isolated from B cells of mice immunized with LILRB2 or an immunogenic fragment thereof. DNA can be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another example of a known technique by which nucleotide sequences encoding antibody polypeptides can be selected.

[0162] 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 pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6xSSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing about 50% formamide and a hybridization temperature of 42°C), and wash conditions in 0.5xSSC, 0.1% SDS at 60°C. Stringent hybridization conditions include hybridization in 6xSSC at 45°C, followed by one or more washes in 0.1xSSC, 0.2% SDS at 68°C. Moreover, one skilled in the art can manipulate the hybridization and / or wash 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 will typically remain hybridized to each other.

[0163] Basic parameters influencing the selection of hybridization conditions and guidelines for devising appropriate conditions are provided, for example, by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4) and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0164] Changes can be introduced by mutation into a nucleic acid, thereby resulting in a change in the amino acid sequence of the polypeptide (e.g., an antibody or antibody derivative) it encodes. Mutations can be introduced using any technique known in the art. One or more specific amino acid residues can be substituted, for example, using site-directed mutagenesis protocols. One or more randomly selected residues can be substituted, for example, using random mutagenesis protocols. Once generated, however, the mutant polypeptides can be expressed and screened for desired properties.

[0165] Polypeptides that are components of the desired antibody product are expressed in any suitable recombinant expression system.

[0166] An expression vector is provided that contains a nucleic acid encoding a LILRB2 antibody product. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0167] Typically, expression vectors used in any of the host cells contain sequences for plasmid or viral maintenance, and sequences for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences," typically 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 including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element.

[0168] Optionally, the vector may contain a sequence encoding a "tag," i.e., an oligonucleotide molecule located at the 5' or 3' end of the coding sequence, an oligonucleotide sequence encoding polyHis (e.g., hexaHis), or another "tag" for which a commercially available antibody exists, such as FLAG, HA (hemagglutinin from influenza virus), or myc. The tag is typically fused to the antibody protein upon expression and can serve as a means for affinity purification of the antibody from host cells. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified antibody polypeptide by various means, such as using specific peptidases for cleavage.

[0169] Flanking sequences in an 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 two or more sources), synthetic, or natural. Thus, the source of the flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate, or any plant, so long as the flanking sequences are functional in and can be activated by the host cell machinery.

[0170] Expression and cloning vectors typically contain a promoter recognized by the host organism and operably linked to the nucleic acid encoding the LILRB2 antibody product. Promoters are traditionally classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased transcription levels from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, initiate continuous gene product production; i.e., there is little or no experimental control over gene expression. Numerous promoters recognized by a variety of potential host cells are known. A suitable promoter is operably linked to the DNA encoding the LILRB2 antibody product by removing the promoter from the source DNA by restriction enzyme digestion or by amplifying the promoter by polymerase chain reaction and inserting the desired promoter sequence into the vector.

[0171] Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus, and most preferably, simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0172] Exemplary promoters useful in recombinant expression vectors include 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 sarcoma virus (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 sequence of the metallothionine gene (Brinster et al., Nature. 1982 296:39-42); prokaryotic expression vectors such as the beta-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 1978 75:3727-31). USA.1983 80:21-5). The following animal transcriptional control regions are also available for use, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, which 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, which is active in pancreatic beta cells (Hanahan, Nature. 1985 315:115-22); the mouse mammary tumor virus control region, which is active in testis, breast, lymphocytes, and mast cells (Leder et al., Cell. 1986 45:485-95); and the albumin gene control region, which is active in the liver (Pinkert et al., Genes Dev 1987 1:268-76); the liver-active alpha-fetoprotein gene regulatory region (Krumlauf et al., Mol Cell Biol. 1985 5:1639-48; Hammer et al., Science.1987 235:53-8); the alpha 1-antitrypsin gene control region, which is active in the liver (Kelsey et al., Genes Dev. 1987 1:161-71); the beta globin gene control region, which is active in bone marrow cells (Mogram et al., Nature. 1985 315:338-40; Kollias et al., Cell. 1986 46:89-94); the myelin basic protein gene control region, which is active in oligodendrocytes in the brain (Readhead et al., Cell. 1987 48:703-12); the myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, Nature. 1985 314:283-6); the gonadotropin-releasing hormone gene control region, which is active in the hypothalamus (Mason et al., Science. 1986 234:1372-8); and most specifically, immunoglobulin gene control regions that are active in lymphoid cells (Grosschedl et al., Cell. 1984 38:647-58; Adams et al., Nature. 1985 318:533-8; Alexander et al., Mol Cell Biol. 1987 7:1436-44).

[0173] An enhancer sequence can be inserted into the vector to increase transcription of the nucleic acid encoding the LILRB2 antibody product described herein in higher eukaryotes. Various enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, and insulin). Enhancer sequences derived from viruses can also be used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancing elements for activating eukaryotic promoters. The enhancer can be spliced ​​into the vector at a 5' or 3' position relative to the nucleic acid molecule, but is typically located at a 5' position relative to the promoter.

[0174] In expression vectors, transcription termination sequence is typically located 3' of the end of polypeptide coding region, and serves to terminate transcription.The transcription termination sequence used for expression in prokaryotic cells is typically a GC-rich fragment, followed by a poly-T sequence.Sequence can be easily cloned from library or even purchased commercially as part of vector, but can also be easily synthesized using the method for nucleic acid synthesis as described herein.

[0175] A selection gene can be used to amplify the gene to be expressed. Amplification is the process by which a gene that cannot be expressed in a single copy is expressed at a high enough level to allow the cell to survive and grow under specific selective conditions, and is tandemly repeated in the chromosome of successive generations of recombinant cells. Examples of suitable amplifiable selection markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase. In using these markers, mammalian cell transformants are placed under selection pressure in which only transformants are uniquely adapted to survive due to the selection gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby allowing only cells in which the selection gene has been amplified to survive. Under these conditions, DNA adjacent to the selection gene, such as DNA encoding an antibody, is co-amplified with the selection gene. As a result, greater amounts of LILRB2 polypeptide are synthesized from the amplified DNA.

[0176] A ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

[0177] In some cases, for example, when glycosylation is desired in a eukaryotic host cell expression system, various presequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered or a prosequence can be added, which can also affect glycosylation. The final protein product can have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that are not completely removed upon expression. For example, the final protein product can have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites can result in a slightly truncated but active form of the desired polypeptide, if the enzyme cleaves at such a region within the mature polypeptide.

[0178] If a commercially available expression vector lacks some of the desired flanking sequences described above, the vector can be modified by individually ligating these sequences into the vector. After a vector has been selected and modified as desired, a nucleic acid molecule encoding a LILRB2 antibody product is inserted into the appropriate site of the vector.

[0179] The completed vector containing the sequence encoding the antibody product is inserted into an appropriate host cell for amplification and / or polypeptide expression. Transformation of the LILRB2 antibody product expression vector into the selected host cell can be achieved by well-known methods, including transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE-dextran, or other known techniques. The method selected will depend, in part, on the type of host cell used. These and other suitable methods are well known to those skilled in the art.

[0180] The antibody can be expressed in a hybridoma cell line or a cell line other than a hybridoma. An expression construct encoding the antibody 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 packaging the polynucleotide into a virus or bacteriophage and transducing the construct into the host cell using transfection procedures known in the art, as exemplified, for example, by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The optimal transformation procedure used depends on the type of host cell being 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 polynucleotide(s) into liposomes, mixing nucleic acids with positively charged lipids, and direct microinjection of DNA into the nucleus.

[0181] When cultured under appropriate conditions, the transformed host cells will synthesize a LILRB2 antibody product, which can then be recovered from the culture medium (if the host cells secrete it into the medium) or directly from the host cells that produce the LILRB2 antibody product (if they are not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, polypeptide modifications desirable or necessary for activity (e.g., glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0182] Mammalian cell lines available as expression hosts 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 (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney (HEK) cells (e.g., HEK293), and several other cell lines. The best cell line for expressing a particular DNA construct can be selected by testing various cell lines to determine which cell line has the highest expression level and produces an antibody product with the desired LILRB2-binding properties.

[0183] Nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences are also provided. The nucleic acid molecules may contain only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer, or a fragment that encodes an active portion of the polypeptide (e.g., a LILRB2-binding portion).

[0184] composition Also provided is a composition comprising the LILRB2 antibody product. Pharmaceutical compositions typically include one or more of buffers, pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers and preservatives. Also provided is the use of the antibody product in preparing pharmaceutical compositions or medicaments.

[0185] Acceptable formulation components of pharmaceutical formulations are nontoxic to recipients at the dosages and concentrations employed. In addition to the antibody products provided herein, compositions may contain components to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. Suitable materials for formulating pharmaceutical compositions include 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, glycine, etc.); 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); colorants, flavorings, and diluents; emulsifiers; 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 glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapar); stability enhancing agents (such as sucrose or sorbitol); isotonicity agents (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.(such as glycine, glutamine, asparagine, arginine or lysine) (see Remington's Pharmaceutical Sciences, 23rd ed., (Adejare, ed.), 2020, Elsevier Academic Press).

[0186] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. Suitable vehicles or carriers for such compositions include water for injection, saline, or artificial cerebrospinal fluid, and may be supplemented with other ingredients common to compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Compositions containing LILRB2 antibody products can be prepared for storage by mixing a selected composition having the desired purity with optional formulation agents in the form of a lyophilized cake or aqueous solution. Furthermore, LILRB2 antibody products can be formulated as lyophilizates using appropriate excipients, such as sucrose.

[0187] The formulation components are present in concentrations acceptable to the site of administration. A buffer is advantageously used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 4.0 to about 8.5, or alternatively, about 5.0 to 8.0. The pharmaceutical composition may comprise a TRIS buffer of about pH 6.5 to 8.5, or an acetate buffer of about pH 4.0 to 5.5, which may further contain sorbitol or a suitable substitute thereof.

[0188] Other pharmaceutical compositions are in the form of sustained or controlled delivery formulations.Can use various other sustained or controlled delivery means formulation techniques, such as liposome carriers, bioerodible microparticles or porous beads and depot injections (see, for example, PCT Publication No. WO 93 / 15722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions). Sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules, polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919 and European Patent No. 058,481), copolymers of L-glutamic acid and gamma-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., supra), or poly-D(-)-3-hydroxybutyric acid (European Patent No. 133,988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc Natl Acad Sci USA. 1985 82:3688-92; EPO Publication No. EP 036676; EP 088046 and EP 143949.

[0189] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0190] The components used to formulate pharmaceutical compositions are preferably highly pure and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

[0191] The kits can be provided in multiple dose units or single dose units. For example, each kit can include both a first container with a dried protein and a second container with an aqueous diluent, including, for example, single- and multi-chamber pre-filled syringes (e.g., liquid syringes, frozen syringes, or needleless syringes).

[0192] Pharmaceutical compositions can be delivered parenterally, typically by injection. Injections can be intraocular, intraperitoneal, intraportal, intramuscular, intravenous, intrathecal, intracerebral (intraparenchymal), intraventricular, intraarterial, intralesional, perilesional, or subcutaneous. Eye drops can be used for intraocular administration. In some instances, injections can be localized near a specific bone or bones targeted for treatment. For parenteral administration, antibodies can be administered in a pyrogen-free, parenterally acceptable aqueous solution containing 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 has been formulated as a sterile, isotonic solution, properly preserved.

[0193] Pharmaceutical compositions containing the subject LILRB2 antibody products can be administered by bolus injection, infusion, implantation device, sustained-release system, or other means for achieving sustained release. Pharmaceutical compositions can also be administered locally via implantation of a membrane, sponge, or another suitable material into which the desired molecule has been absorbed or encapsulated. When an implantation device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, sustained bolus, or continuous release. Preparations can be formulated with drugs, e.g., injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid; polyglycolic acid; or copoly(lactic / glycolic) acid (PLGA)), beads, or liposomes, which can provide controlled or sustained release of the product and can then be delivered via depot injection. Formulation with hyaluronic acid has the effect of promoting sustained release in the circulation.

[0194] The subject compositions containing LILRB2 antibody products can also be used ex vivo. In such cases, cells, tissues, or organs removed from a patient are exposed to or cultured with LILRB2 antibody products. The cultured cells can then be reimplanted into the patient or another patient, or used for other purposes.

[0195] LILRB2 antibody products can be delivered by implanting specific cells that have been genetically engineered to express and secrete the polypeptide, using methods such as those described herein. Such cells can be animal or human, and can be autologous, heterologous, or xenogeneic, or can be immortalized. To reduce the possibility of an immunological response, cells can be encapsulated to avoid infiltration of surrounding tissues. The encapsulating material is typically a biocompatible, semipermeable polymer enclosure or membrane that allows release of the protein product(s) but prevents destruction of the cells by the patient's immune system or other harmful factors from surrounding tissues.

[0196] As used herein, "substantially pure" means that the described molecular species is the predominant species present, i.e., more abundant on a molar basis 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 contain at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. The target species can also be purified to essential homogeneity, such that contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

[0197] Dosage The provided pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatments.

[0198] As used herein, the terms "therapy / treatment," "treating," and the like refer to administering a drug or performing a procedure with the intent of achieving an effect. The effect is preventative, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it results in a partial or complete cure of the disease and / or its symptoms. As used herein, "therapy / treatment" includes the treatment of a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes (a) preventing a disease or disease symptoms from occurring in a subject who is predisposed to the disease but has not yet been diagnosed with it (e.g., including diseases associated with or caused by a primary disease); (b) inhibiting the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing regression of the disease. Treatment refers to any clinical manifestation of successful treatment, amelioration, or prevention, e.g., any objective or subjective parameter, such as relief, remission, relief of symptoms, or improved patient tolerance of the disease state, slowing the rate of decline or decline, or reducing decline in the final stages of decline. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term "treating" includes administration of a compound or agent of the present disclosure to prevent or delay, alleviate, or arrest or inhibit the onset of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptoms, or disease side effects in a subject. For example, a subject is "treated" for a disease or disorder if, after receiving a therapeutic amount of a combination of LILRB2 antibody products provided herein, the patient exhibits one or more observable and / or measurable changes in the endpoints or symptoms of the disease state.

[0199] According to the present disclosure, an "effective response" is achieved when a subject experiences partial or complete relief or alleviation of signs or symptoms of the disease, and in the case of cancer treatment, specifically includes, but is not limited to, symptomatic improvement, delayed progression, cure, remission, prolonged survival, or other objective response. Expected progression-free survival can be measured in months to years, depending on prognostic factors, including the number of recurrences, stage of disease, and other factors. Prolonged survival includes, but is not limited to, periods of at least 1 month (month), about 2 months or more, about 3 months or more, about 4 months or more, about 6 months or more, about 1 year or more, about 2 years or more, about 3 years or more, etc. Overall survival is also measured, for example, in months to years. Alternatively, an effective response can be when the subject's symptoms remain static.

[0200] In a prophylactic method, a therapeutic agent is administered before the symptoms of an undesirable disease or disorder appear, thereby preventing or slowing the progression of the disease or disorder. Thus, when used in conjunction with a prophylactic method, the term "therapeutically effective" means that (on average) fewer subjects will develop an undesirable disease or disorder or progress in the severity of the symptoms after treatment.

[0201] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. A "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sport, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. The mammal may be a human.

[0202] Generally, the toxicity and therapeutic efficacy of antibody products can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, e.g., by LD 50 (lethal dose for 50% of the population) and ED 50This involves determining the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices are preferred.

[0203] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosage for human use. The dosage of the active ingredient is typically administered at or above the ED 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0204] The effective amount of a pharmaceutical composition containing a LILRB2 antibody product used therapeutically or prophylactically will depend, for example, on the context and purpose of the treatment. Therefore, those skilled in the art will understand that appropriate dosage levels for treatment will vary depending, in part, on the molecule being delivered, the indication for which the LILRB2 antibody is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and / or condition (age and general health). Clinicians can titrate the dosage and modify the route of administration to achieve optimal therapeutic efficacy. Typical dosages range from about 1 mg / kg up to about 1600 mg / kg or more, depending on the factors described above. Dosages can range from 1 mg / kg to about 200 mg / kg; or 1 mg / kg to about 1200 mg / kg; or 1 μg / kg to about 1600 mg / kg.

[0205] The frequency of administration depends on the pharmacokinetic parameters of the LILRB2 antibody product in the formulation. For example, a clinician administers the composition until a dosage that achieves the desired effect is reached. Thus, the composition can be administered as a single dose, as two or more doses (containing equal amounts of the desired molecule) over time, or as a continuous infusion via an implanted device or catheter. Treatment can be continuous or intermittent over time. Further refinement of the appropriate dosage is routine and within the scope of those skilled in the art. Appropriate dosages can be confirmed through the use of appropriate dose-response data. An exemplary administration schedule is every 2-3 weeks.

[0206] To treat a disease condition by targeting LILRB2, a composition containing a subject LILRB2 antibody product is administered to a patient in an amount and for a time sufficient to induce a sustained improvement in at least one indicator reflecting the severity of the disorder. An improvement is considered "sustained" if the patient shows improvement at least twice, separated by at least 1-7 days, and optionally 1-6 weeks. The appropriate interval depends in part on the disease condition being treated. Determining an appropriate interval for determining whether improvement is sustained is within the knowledge of one of ordinary skill in the art. The degree of improvement can be determined based on signs or symptoms, and can also be assessed using questionnaires administered to the patient, such as quality of life questionnaires.

[0207] To determine whether the amount and duration of treatment are sufficient, various indicators that reflect the extent of the patient's illness can be evaluated.The baseline value of one or more selected indicators is established by examining the patient before administering the first dose of antibody.Preferably, the baseline examination is performed within about 60 days after administering the first dose.When the antibody is administered to treat acute symptoms, such as to treat broken bone, the first dose is administered as soon as practically possible after the injury occurs.

[0208] Improvement is induced by administering a subject LILRB2 antibody product until the patient shows improvement over baseline in one or more selected indicators. In the treatment of chronic conditions, this degree of improvement can be achieved by repeatedly administering the medicament for at least one month or more, for example, one, two, or three months or more, or indefinitely. A period of one to six weeks, or even a single administration, is often sufficient to treat acute conditions. In the case of injuries or acute conditions, a single administration may be sufficient.

[0209] Although a patient's condition after treatment may appear to improve according to one or more indicators, treatment can be continued indefinitely at the same level or at a reduced dose or frequency. Once treatment is reduced or discontinued, it can be subsequently resumed at the original level if symptoms reappear.

[0210] How to use The LILRB2 antibody products disclosed herein have various utilities. Some of the antibodies and fragments are useful, for example, in specific binding assays, affinity purification of LILRB2 or its ligands, and screening assays to identify other antagonists of LILRB2 activity. The antibody products can be used to treat various diseases associated with LILRB2 activity.

[0211] LILRB2 antibody products can be used to detect LILRB2 in biological samples. Such use allows for the identification of cells or tissues that produce the protein or serve as a diagnostic for detecting pathological conditions in which LILRB2 is overproduced or underproduced. Strong expression of LILRB2 by macrophages, osteoclasts, and other bone marrow cells can be a marker of the activity of those cells, and detection of LILRB2 expression on bone marrow cells can be used as a marker for diseases or disorders characterized by the cell type in question. Similarly, detection of LILRB2 expression by cancer cells can be used to identify subjects whose cancer may be suitable for treatment with the therapeutic LILRB2 antibody product methods disclosed herein.

[0212] Thus, provided is a method for detecting cellular activity in a biological sample, such as an in vitro culture medium sample or a tissue sample from a subject, or in vivo in a subject, comprising contacting a cell expressing LILRB2 with a LILRB2 antibody product provided herein. The LILRB2 antibody product can be conjugated to a detectable moiety, and the method can include directly detecting the moiety. The method can also include indirectly detecting binding of the LILRB2 antibody product to the cell via a detectable moiety that binds to the antibody. For example, an IgG antibody conjugated to a detectable moiety can be used to bind to a LILRB2 antibody presented as an IgG isotype. The cell can be a tumor cell. The cell can be a bone marrow cell (e.g., a monocyte, dendritic cell, macrophage, myeloid-derived suppressor cell, tumor-associated macrophage, immunosuppressive macrophage, or M2-like macrophage) or an osteoclast.

[0213] 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, a LILRB2 molecule or 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 binding between the antibody product and LILRB2 indicates that the candidate molecule binds to LILRB2. Binding of the antibody product can be detected by various methods, for example, ELISA. Detection of binding between the LILRB2 antibody product and LILRB2 can be simplified by detectably labeling the antibody. In some methods, molecules that show binding in the initial screen are further analyzed to determine whether they inhibit or modulate LILRB2 activity.

[0214] The LILRB2 antibody products provided herein are useful for the treatment of human diseases, including cancer.

[0215] The LILRB2 antibody products described herein can be used alone or in combination with another anti-cancer therapeutic agent to treat cancer. The cancers to be treated are those whose cancer cells are known to express LILRB2 or have previously been observed to express LILRB2. Certain cancers that are EGFR mutant have been found to express LILRB2 at higher levels and are therefore contemplated for treatment with the antibodies disclosed herein. In contrast, LILRB2 expression in cancer cells has also been found to be inversely correlated with PD-L1 expression. Therefore, the anti-LILRB2 treatments described herein are also contemplated when PD-L1 expression by cancer cells is not observed. Such treatments are indicated when therapeutic interventions in the PD-1 / PD-L1 axis are ineffective or expected to be ineffective.

[0216] Provided herein are methods of treating patients with cancer in which LILRB2 antibody products mediate the killing of cancer cells.

[0217] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer encompasses skin, tissue, organs, bone, cartilage, blood vessels, and diseases of the blood vessels. The term "cancer" further encompasses both primary and metastatic cancers.

[0218] Examples of cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cancer of the bladder, blood, bone, bone marrow, brain, breast, cervix, colon, esophagus, gastrointestinal tract, rectum, head and neck, kidney, larynx, liver, lung, oronasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testicle, thyroid, tongue, and uterus.

[0219] Cancers to be treated include, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, Hodgkin's disease; Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's 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 carcinoma, 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, kidney renal clear cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, skin cutaneous melanoma, gastric adenocarcinoma, testicular germ cell carcinoma, gastric carcinoma, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid carcinoma, cutaneous squamous cell carcinoma, or ovarian cancer.

[0220] Furthermore, the cancer may specifically be of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrimatous carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; Papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ear 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, breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian Stromal tumors, malignant; meningiomas, malignant; granulosa cell tumors, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumors, malignant; lipid cell tumors, malignant; paragangliomas, malignant; extramammary paragangliomas, malignant; pheochromocytoma; breast angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant cellular blue nevus; sarcomas; fibrosarcomas; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumors, malignant; mixed Müllerian tumor; nephroblastoma; hepatoblastoma ;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian stroma, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Paracortical osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;Paragranuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; mycosis fungoides or cutaneous T-cell lymphoma; other specified non-Hodgkin's lymphoma; malignant histiocytic proliferation; 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; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0221] As an example, a method for treating a subject suffering from melanoma is provided. As used herein, "melanoma" refers to a condition characterized by the growth of tumors originating from the melanocyte system in the skin and other organs. Most melanocytes are found in the skin, but they can also be found in the meninges, gastrointestinal tract, lymph nodes, and eyes. When melanoma occurs in the skin, it is called cutaneous melanoma. Melanoma can also occur in the eye, and is called ocular melanoma or intraocular melanoma. Melanoma rarely occurs in the meninges, gastrointestinal tract, lymph nodes, or other areas where melanocytes are found.

[0222] Cells of a cancer treated with the methods provided herein can express LILRB2. Cells of a cancer can overexpress LILRB2.

[0223] One way that LILRB2 antibody products can mediate cancer cell death is through antibody-dependent cellular cytotoxicity ("ADCC"), a process in which antibodies coat target cells (e.g., cancer or bacterial cells) and recruit effector cells via a non-phagocytic mechanism to induce target cell death.

[0224] As described above, in therapeutic methods, the LILRB2 antibody products provided herein can be used as monotherapy or combination therapy. "Combination" therapy refers to the administration of one therapeutic agent before, during, or after the administration of another therapeutic agent to a subject.

[0225] In combination cancer treatments, LILRB2 antibody products are used in combination with one or more other anti-cancer therapies to treat a subject's cancer. The anti-cancer therapies can be chemotherapy or biological molecules. The anti-cancer therapies can be immunotherapeutic molecules. The immunotherapeutic agents can be checkpoint inhibitors. The checkpoint inhibitors can be PD-1 antagonists. The checkpoint inhibitors can be PD-L1 antagonists.

[0226] Provided herein are methods of treating a patient with cancer, comprising administering to the patient a LILRB2 antibody product described herein in combination with immunotherapy.

[0227] As an example of a combination with immunotherapy, provided is a method of treating a patient with cancer comprising administering to the patient a therapeutically effective amount of a LILRB2 antibody product and one or more immune checkpoint inhibitors.

[0228] The term "immune checkpoint inhibitor" or "checkpoint inhibitor" generally refers to an agent that modulates immune checkpoint proteins ("checkpoint proteins"). Checkpoint inhibitors can achieve total or partial reduction, inhibition, or interference with the activity of checkpoint proteins, or can cause other changes to the structure of the checkpoint protein to alter binding of the checkpoint protein to its ligand and / or affect pathways associated with checkpoint protein activity, for example, by acting as antagonists to the checkpoint protein or its ligand. Immune checkpoint inhibitors can be compounds such as antibodies or other proteins that bind to and antagonize 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.

[0229] As disclosed herein, checkpoint proteins can interfere with T cell-mediated killing of cancer cells in certain circumstances and conditions. Although checkpoint inhibitors can reverse the interference of checkpoint proteins, interference with checkpoint proteins is not sufficient in certain types of cancer (e.g., certain solid tumors). The present disclosure contemplates that by combining the antibody products provided herein with another checkpoint inhibitor, macrophage-mediated T cell exhaustion can be alleviated and T cell effector function can be stimulated.

[0230] Checkpoint inhibitors can inhibit one or more checkpoint proteins, including, but not limited to, 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, SIRP alpha, DC-SIGN, CD200R, DR3, CDCHK1, CHK2, A2aR, or B-7 family proteins.

[0231] Checkpoint inhibitors can interact with ligands of checkpoint proteins. For example, non-limiting examples of checkpoint protein ligands 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.).

[0232] A checkpoint inhibitor can be an antagonist. For example, a checkpoint inhibitor can antagonize a checkpoint protein. A checkpoint inhibitor can be an antagonist for a ligand of a checkpoint protein. An antagonist can be a biological molecule such as a biological therapeutic agent. A checkpoint inhibitor can be an antibody or an antigen-binding portion thereof, such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. A checkpoint inhibitor can be a small molecule. A checkpoint inhibitor can be a rationally designed peptide. A checkpoint inhibitor can be a cell or a cell preparation (e.g., a cell expressing a checkpoint inhibitor).

[0233] Checkpoint inhibitors can inhibit PD-1. Programmed cell death 1 (PD-1) is a key checkpoint receptor expressed by activated T cells and activated B cells and mediates immunosuppression. Specifically, PD-1 limits T cell activity in peripheral tissues during the inflammatory response to infection. Furthermore, as a checkpoint protein, PD-1 blockade can enhance T cell proliferation and cytokine production in response to antigen stimulation by specific antigen targets or allogeneic cells in a mixed lymphocyte reaction.

[0234] Without intending to be bound by theory, it is believed that PD-1 blockade, in combination with the LILRB2 antibody products disclosed herein, reduces macrophage-mediated T cell suppression / exhaustion, increases T cell proliferation and cytokine production, and improves immune cell effector function. PD-1 blockade can be achieved by various 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 a checkpoint inhibitor that is a PD-1 antagonist. For example, the PD-1 antagonist can be a PD-1 antibody (e.g., nivolumab, pembrolizumab, etc.). The PD-1 antagonist can be a small molecule (e.g., INCB-086550 (Incyte) or a small molecule disclosed below, e.g., 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). The PD-1 antagonist can be or include a rationally designed peptide (e.g., APi2568). The PD-1 antagonist can be or include a cell or cell preparation (e.g., a cell expressing a PD-1 binding agent, e.g., a PD-1 antibody, e.g., HerinCAR-PD1).

[0235] Exemplary PD-1 antibodies suitable for use in the present methods include nivolumab (ONO-4538, BMS-936558, MDX1106, Opdivo®; Bristol-Myers Squibb), pembrolizumab (MK-3475, Keytruda®; Merck), cemipilimab (e.g., cemiplimab-rwlc (Libtayo™; Regeneron)), dostarlimab (e.g., dostarlimab-gxly (Jemperli™; GlaxoSmithKline)), pimivalimab (IgG4) (JTX-4014; Jounce Therapeutics), spartalizumab (IgG4) (PDR001; Novartis), canrelizumab (SHR1210; Jiangsu Engineering Pharmaceuticals), sintilimab (IBI308; Innovent and Eli Lilly), tislelizumab (BGBA317; BeiGene), toripalimab (JS 001; Shanghai Junzhi 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 the PD-1 binding domain of any thereof.

[0236] Another exemplary PD-1 antagonist is a rationally designed peptide, such as APi2568, that contains a B cell epitope (amino acids 92-110 from PD-1) linked via a four amino acid linker to a promiscuous T cell epitope (amino acid residues 288-302 from the measles virus fusion protein) and is combined with water for injection (WFI) to form formulation IMU-201, which becomes PD1-Vaxx when emulsified with the excipient Montanide ISA 720 VG.

[0237] Another exemplary PD-1 antagonist is a cell that expresses a PD-1 antibody, for example, a PD-1 antibody-expressing CAR-T cell (e.g., HerinCAR-PD1 cell).

[0238] In some embodiments, the checkpoint inhibitor is a PD-L1 antagonist, e.g., a PD-L1 antibody. In some embodiments, the PD-L1 antibody is selected from the group consisting of avelumab, durvalumab, atezolizumab, embafolimab, cosibelimab, LY3300054 CA-170, BMS-936559, and a PD-L1-binding fragment or a combination thereof. In some embodiments, the PD-L1 antagonist comprises a PD-L1-binding domain comprising the CDRs of an antibody selected from the group consisting of AUNP-12, BMS-986189, avelumab, durvalumab, atezolizumab, embafolimab, cosibelimab (CK-301), LY3300054, CA-170, and BMS-936559, and an active fragment thereof, or a combination thereof.

[0239] 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 ligand CD80 / CD86, which is expressed on antigen-presenting cells. Therefore, CTLA-4 inhibitors that block the interaction of CTLA-4 with its ligand can block the negative downregulation of immune responses induced by the interaction of these molecules. Therefore, 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. Further, exemplary CTLA-4 antibodies include ipilimumab (10D1, MDX-D010, Yervoy™; Bristol-Myers Squibb), tremelimumab (ticilimumab, CP-675,206; MedImmune), and quabonlimab (MK-1308; Merck). CTLA-4 antagonists can include the CTLA-4 binding domain of any CTLA-4 antagonist, or a fragment thereof. CTLA-4 antagonists can include small molecules (see, e.g., Wang et al., Biochim Biophys Acta Rev Cancer. 2019 1871(2):199-224).

[0240] Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a CD4-associated transmembrane protein that competitively binds to MHC II and acts 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 / Prima Biomed), MK-4280 (Merck Sharp & Dohme), REGN3767 (Regeneron Pharmaceuticals), leratolimab (BMS-986016, Bristol-Myers Squibb), and BI 754111 (Boehringer Ingelheim).

[0241] 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 widely expressed on lymphocytes, liver, small intestine, thymus, kidney, spleen, lung, muscle, reticulocyte, and brain tissues. Binding of Gal-9 by the TIM-3 receptor triggers downstream signaling to negatively regulate T cell survival and function. Checkpoint inhibitors can be agents that inhibit TIM-3. Checkpoint inhibitors can be TIM-3 antagonists, such as TIM-3 antibodies or antibodies against TIM-3 ligands. TIM-3 antagonists can include 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, e.g., U.S. Patent Nos. 9,103,832, 8,552,156, 8,647,623, and 8,841,418; U.S. Patent Application Publication Nos. 2016 / 0200815, 2015 / 0284468, 2014 / 0134639, and 2016 / 0200815. and WO 2016 / 068802, WO 2016 / 068803, WO 2016 / 071448, WO 2011 / 155607 and WO 2013 / 006490.

[0242] 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 the function of T cells and natural killer (NK) cells. The checkpoint inhibitor can be a TIGIT antagonist. The TIGIT antagonist can bind to TIGIT or a TIGIT ligand. The TIGIT antagonist can be a TIGIT antibody or an antibody against a TIGIT ligand. The TIGIT antagonist includes the TIGIT-binding domain of any TIGIT antagonist or a fragment thereof. 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).

[0243] Exemplary anti-CD27 agonists include MK-5890 (Merck).

[0244] Exemplary ICOS antibodies include vopratelimab (JTX-2011; Jounce).

[0245] 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 with a co-formulation of a PD-1 antagonist, such as a PD-1 antibody, and a CTLA-4 antagonist, such as a CTLA-4 antibody, and the dosage of each component is controlled to provide a safe and effective treatment to the subject. In some embodiments, the LILRB2 antibody is administered with a co-formulation of pembrolizumab / quavonlimab (MK-1308A; Merck).

[0246] Also provided are methods for treating cancer, including administering therapeutically effective amounts of a LILRB2 antibody product provided herein and a colony-stimulating factor 1 (CSF1) antagonist. Colony-stimulating factor 1 receptor (CSF1R) inhibitors have been developed for cancer treatment. 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 cabilalizumab.

[0247] Also provided are methods of treating cancer comprising administering therapeutically effective amounts of a LILRB2 antibody product and an agonistic CD40 antibody provided herein.

[0248] Also provided are methods of treating cancer comprising administering therapeutically effective amounts of a LILRB2 antibody product and an inhibitory CD47 antibody provided herein.

[0249] Also provided are methods of treating cancer comprising administering a therapeutically effective amount of a LILRB2 antibody product provided herein and an effective amount of a class IIa histone deacetylase (HDAC) inhibitor, such as TMP195.

[0250] Also provided are methods for treating cancer, including administering a therapeutically effective amount of a LILRB2 antibody product 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 for either or both of these receptors can promote the functional orientation of tumor-associated macrophages toward an M1-like phenotype. Resiquimod-loaded β-cyclodextrin nanoparticles have been reported to have antitumor effects that can be enhanced in the presence of a PD-1 antagonist. Rodell et al., Nat Biomed Eng. 2018 2:578-88. Examples of such TLR7 / TLR8 agonists include resiquimod (B848), motolimod (VTX-2337), and imiquimod.

[0251] Also provided is a method for treating a disease state in a subject, wherein the disease state is characterized by or mediated through LILRB2 expression by bone marrow cells. The bone marrow cells can be macrophages. The bone marrow cells can be osteoclasts or osteoclast precursors. The method includes administering a therapeutically effective amount of a LILRB2 antibody product disclosed herein to the subject.

[0252] Also provided is a method for treating or preventing a bone metabolic disorder in a subject.The bone metabolic disorder can be osteoporosis, bone destruction associated with rheumatoid arthritis, cancer-related hypercalcemia, bone destruction associated with multiple myeloma or cancer metastasis to bone, giant cell tumor, osteopenia, tooth loss due to periodontitis, periprosthetic osteolysis, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, or osteogenesis imperfecta.The bone metabolic disorder can be osteoporosis.The osteoporosis can be postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of therapeutic agents such as steroids or immunosuppressants, or osteoporosis associated with rheumatoid arthritis.

[0253] Other terms As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies.

[0254] As used herein, all numerical values ​​or ranges include whole integers within or encompassing such ranges, and fractions of values ​​or integers within such ranges or encompassing ranges, unless the context clearly dictates otherwise. Thus, for example, a reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, reference to a range of 1 to 5,000 times encompasses 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x or 20x, etc., as well as 1.1x, 1.2x, 1.3x, 1.4x or 1.5x, etc., 2.1x, 2.2x, 2.3x, 2.4x or 2.5x, etc.

[0255] As used herein, the term "about" a number refers to a range that is inclusive of the number and extends from 10% below to 10% above the number. The range "about" refers to 10% below the lower limit of the range and 10% above the upper limit of the range.

[0256] As used herein, "can," "can be," "may," and "may be" all refer to what is contemplated by the inventors as being functional and available as part of the provided subject matter. [Example]

[0257] The following examples illustrate specific embodiments, but variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on this invention.

[0258] To generate anti-human LILRB2 antibodies from rabbit B cells, rabbits were immunized with human LILRB2 protein. B cells from immunized rabbits were cultured to clonal density, and IgG antibodies in the supernatants were evaluated for (a) binding to human and cynomolgus LILRB2 by enzyme-linked immunosorbent assay (ELISA), (b) blocking of LILRB2 binding to HLA-G, (c) binding to cells expressing LILRB2, and (d) lack of binding to other LIL(A / B) family members. The variable regions from positive hits were sequenced, cloned, and expressed as recombinant rabbit / human IgG4 and IgG1 Fc chimeras.

[0259] Top clones were selected based on activity in a panel of functional and phenotypic assays using primary human macrophages and T cells, as well as antitumor activity in xenograft models. Selected clones were humanized in silico, and the humanized antibodies were screened in vitro for their ability to rescue T cell functional activity (activation and proliferation) from M2c macrophage-mediated immune suppression.

[0260] Example 1: Immunization, cloning and initial screening The antibody designated B2A was among those 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. The rabbits received booster immunizations on days 21, 42, and 73 after the primary immunization, respectively. Pre-immunization and test bleeds were assessed for specific antibody titers by indirect ELISA. On day 83 after the primary immunization, heparinized whole blood was collected for rabbit monoclonal antibody development. Peripheral blood B cells from both rabbits were collected after the final boost, then isolated, purified, and cultured to clonal density using a proprietary method. 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 ELISAs were performed, probing with an anti-rabbit IgG secondary antibody (ImmunoPrecise). To recover antibodies specific to the target antigen, negative screening of positive candidates was performed against an irrelevant human-Fc fusion protein (ImmunoPrecise). B cell supernatants were also evaluated for their ability to block LILRB2-Fc (R&D Systems, No. 2078-T4) binding to HLA-G. The top 96 responding wells were stored in standard RNA lysis buffer (ImmunoPrecise) for antibody RNA isolation and generation of recombinant plasmid DNA.

[0261] The heavy and light chain variable regions (kappa) of the rabbit antibodies were cloned into separate mammalian expression vectors containing the heavy and kappa constant regions of human IgG from the top positive clones. Recombinant monoclonal antibodies were produced using transfected HEK-293 cells, purified using standard methods, and evaluated in several biochemical and cell-based functional assays.

[0262] Example 2: Binding of chimeric antibodies to LILRB2 (human and cynomolgus monkey) by ELISA To confirm binding to human and cynomolgus monkey LILRB2, antibodies in the supernatant were assessed for binding 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™) and incubated overnight at 4°C. The plate was washed three times with wash buffer (0.05% Tween® 20 / PBS) using a microplate washer and then blocked with 90 μL / well of ELISA Blocking Buffer A (1% BSA / PBS) at room temperature (RT) for 1 hour. After blocking, 25 μL / well of LILRB2 antibody or isotype control was added to the plate and incubated at RT for 1 hour. After primary antibody binding, the plate was 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 / PBS) and added at 25 μL / well to the plate and incubated at RT for 1 hour protected from light. After incubation, the plate was washed four times with Wash buffer using a microplate washer. After removing the final wash, 25 μL / well of 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher, No. 34028) was added, and the plate was incubated at RT for 5–10 minutes protected from light. After development, the reaction was stopped by adding 25 μL / well of 0.3 M HCl, and the plate was read at 450 nm using an EnVision (Perkin Elmer) microplate reader. EC 50Values ​​were calculated based on the log concentration of the primary antibody at optical density at 450 nm. Binding of anti-LILRB2 B2A-IgG1 to human and cynomolgus monkey LILRB2 is shown in Figure 1a, demonstrating binding to human LILRB2 and EC 50 The binding to cynomolgus monkey LILRB2 was 3.81 ng / mL, but no binding to cynomolgus monkey LILRB2 was demonstrated.

[0263] Example 3: Preparation of cells expressing human LILRB2 Cells stably expressing human LILRB2 were generated. Human embryonic kidney cells (HEK293T / 17; ATCC, CRL-11268) were cultured in DMEM medium (Gibco, No. 11965-084) supplemented with 10% fetal bovine serum (FBS; HyClone, No. SH30396.03; heat-inactivated before use) according to ATCC guidelines. Prepackaged lentiviral particles containing a puromycin-selectable lentivector backbone were purchased from G&P Biosciences: without the gene of interest (negative control; No. LTV0001) or with human LILRB2 (SEQ ID NO: 52) (No. LTV2992). HEK293T cells (5 × 10 4(100 cells) were transduced with human LILRB2 lentiviral particles at a multiplicity of infection (MOI) of 10 in the presence of 8 μg / mL Polybrene Infection / Transfection Reagent (Millipore, No. TR-1003-G) at 37°C and 5% CO2 for 24 hours. The virus-containing medium was then removed, and the cells were allowed to recover in fresh medium for 2 days before selection in medium containing 0.75 μg / mL puromycin (Thermo Fisher, No. A1113803). A population of LILRB2-expressing cells was isolated and cultured by selecting for stable LILRB2 expression using puromycin as a selectable marker. LILRB2 expression in the puromycin-resistant cell population was confirmed by flow cytometry using a BD FACSymphony™ cytometer and a LILRB2-specific antibody. In addition to LILRB2 transduction, cells were also transduced with negative control lentiviral particles using the same protocol to generate a stable puromycin-resistant control cell line (containing the lentivector backbone without the gene insert) for use in the assay.

[0264] Once target expression was confirmed, cells were expanded. A polyclonal pool of selected cells was banked at 4-5 million cells / mL per vial to create a master stock. Cell banking involves pelleting cells from culture by centrifugation, removing the medium, and resuspending the cells first in 4°C DMEM + 10% FBS, then adding an equal volume of 2x ATCC-recommended freezing medium (DMEM + 10% FBS + 10% DMSO) so that the cells reach a density of 4-5 million cells / mL in 1x freezing medium (DMEM + 10% FBS + 5% DMSO). Aliquots of the cell suspension were placed in a 4°C Mr. Frosty™ alcohol-based slow-freezing system and immediately stored at -80°C. These frozen cell aliquots were stored at -80°C for 1 day and then transferred to a liquid nitrogen vapor tank.

[0265] For use in the assay, the master stock vial was thawed in a 37°C water bath for 1 minute and centrifuged to pellet the cells. The cells were suspended in DMEM + 10% FBS and target protein expression was confirmed by flow cytometry. These cells were expanded in culture to create 30 working stocks, and target protein expression was confirmed by flow cytometry. These cells were expanded in culture to create 30 working stocks.

[0266] Example 4: LILRB2 antibodies bind to cells expressing human LILRB2 To confirm the binding of the LILRB2 antibody, a flow cytometry binding assay was performed using HEK293T cells stably expressing human LILRB2 (Example 3). The modified HEK293T cells were incubated at 4°C for 30 minutes in Blocking Buffer B (PBS, 1% FBS + 0.05% NaN3 (Ricca Chemical, No. 7144.8-16, diluted), 2 mM EDTA) containing 10% FBS to block nonspecific binding. LILRB2 antibody conjugated to AF647 (Thermo Fisher Scientific, No. 20106) and an isotype control antibody (OncoResponse) were prepared at different concentrations and added directly to the HEK293T cells in Blocking Buffer B. The cells were then incubated at 4°C for 1 hour. Cells were washed twice with FACS buffer and then incubated with AF647-goat anti-human IgG Fc (Jackson ImmunoResearch, No. 109-605-098) for 30 minutes. Cells were washed twice with FACS buffer and then stained with Zombie Violet fixable viability dye (BioLegend, No. 423114) for 10 minutes at room temperature, washed with FACS buffer, resuspended in 100 μL of FACS buffer, and acquired on a BD FACSymphony™ or BD FACSCanto™ II flow cytometer (BD Biosciences). The mean fluorescence intensity (MFI) binding of the LILRB2 antibody was calculated using EC 50FlowJO software (10.5.3, FlowJO, LLC) and GraphPad Prism were used to gate on live cells for binding calculations. B2A-IgG1 and B2A-IgG4 chimeras bound to LILRB2-expressing HEK293T cells and expressed EC 50 The values ​​were comparable, 0.21 nM and 0.30 nM, respectively. A representative example is shown in Figure 1B.

[0267] Example 5: LILRB2 antibodies do not bind to other LILRB or LILRA family members To determine whether the anti-LILRB2 antibody specifically binds to LILRB2 and not to other LILRB and LILRA family members, we performed a cross-reactivity assay. Cross-reactivity of the LILRB2 antibody to other LILRB proteins was assessed by antibody binding to transiently transfected HEK293-6E cells and measured by flow cytometry. Binding was assessed to HEK293-6E cells transiently transfected with human LILRB-1, -2, -3, -4, and -5 plasmids (Origene, numbers RC219949, RC217935, RC211228, RC220932, and RC206516) or human LILRA-1, -2, -4, -5, and -6 plasmids (Origene, numbers RC210808, RC205626, RC220452, RC212310, and RC212965). AF647 mouse anti-human IgG-Fc secondary antibody (Jackson ImmunoResearch, No. 109-605-098) was used to detect bound human anti-LILRB2 antibody on the cells. Mouse primary antibodies against LILRA and LILRB targets and corresponding isotype controls were used to confirm specific binding of all targets tested (R&D Systems), and AF647 F(ab')2 fragment goat anti-mouse antibody (Jackson ImmunoResearch, No. 115-606-062) was used to detect bound positive control antibodies. The binding of B2A-IgG1 and B2A-IgG4 to other LILRA and LILRB family members is shown in Tables 1A and 1B. No binding of B2A-IgG4 or B2A-IgG1 to LILRB-1, -3, -4, or -5 was observed, and no binding of any of the antibodies to LILRA-1, -2, -4, -5, or -6 was observed, confirming the specificity of these antibodies.

[0268] [Table 1A]

[0269] [Table 1B]

[0270] Example 6: LILRB2 antibodies block LILRB2-Fc binding to ANGPTL-2 and -5 Angiopoietin and angiopoietin-like (ANGPTL) proteins are secreted glycoproteins that play a role in angiogenesis, lipid metabolism, hematopoietic stem cell proliferation, and inflammation. While angiopoietins signal through the tyrosine kinase receptors Tie1 or Tie2, 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. Proteins were purchased from R&D Systems (No. 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 for overnight incubation at 4°C. After incubation, the plate was rinsed with Wash buffer and then blocked with Blocking Buffer C (diluted 3% BSA supplemented with 0.05% Tween® 20 / PBS) for 2 hours at 37°C. The anti-LILRB2 antibody was incubated with the LILRB2-Fc protein for 1 hour at RT. Blocking Buffer C was removed from the plate, and the anti-LILRB2 antibody titration / LILRB2-Fc mixture was then incubated in the wells overnight at 4°C. The plate was washed with Wash buffer, and then a 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 RT and then rinsed 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 RT in the dark. After rinsing the plate with wash buffer, 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 absorbance was measured at 450 nm. As shown in Figure 2, B2A-IgG4 dose-dependently blocked the binding of LILRB2-Fc to ANGPTL-2 and -5.

[0271] Example 7A: LILRB2 antibody B2A-IgG4 blocks LILRB2-Fc binding to HLA-G Tumor-associated HLA-G is an HLA class I ligand for LILRB2. Binding of HLA-G to LILRB2 induces immunosuppressive signals in LILRB2-expressing myeloid cells. To determine whether anti-LILRB2 antibodies block HLA-G-LILRB2 interactions and prevent the induction of inhibitory signals, a dose-titration blocking ELISA assay was performed. A 384-well plate was coated with 5 μg / mL of monomeric HLA-G (Fred Hutchinson Cancer Research Center, No. bHLA-G), diluted with PBS, and then incubated at room temperature for 1 hour. 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. The LILRB2-Fc protein was then incubated with the antibody titrations at room temperature for 30 minutes to allow binding before addition to the plate. After rinsing the plate three times with Wash buffer, 25 μL of the protein / antibody mixture was added per well and incubated at RT for 1 hour. After antibody incubation, the plate was washed three times with Wash buffer. Anti-human IgG Fc-HRP secondary antibody was diluted and added at 25 μL / well. The plate was incubated in the dark at RT for 1 hour. After secondary antibody incubation, the plate was washed four times with Wash buffer. 25 μL / well of ultra-neat TMB was added to each well for development and incubated at RT for 5 minutes. The reaction was stopped with 0.3 M HCl (25 μL / well). Absorbance was read at 450 nM using Envision. IC of HLA-G blocking by clone B2A-IgG4 50 was 0.066 μg / mL.

[0272] Example 7B: LILRB2 antibody B2A-IgG4 blocks LILRB2-Fc binding to HLA-G expressed on tumor cells 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 crucial for the efficacy of anti-LILRB2 antibodies. LILRB2 antibodies were evaluated for blocking the binding of recombinant LILRB2-Fc protein to HLA-G expressed on 721,221 B lymphoma cells by flow cytometry.

[0273] Recombinant human LILRB2-Fc-Avitag protein (AcroBiosystems, No. LI2-H82F5) was diluted to 30 μg / mL in FACS buffer (PBS containing 2 mM EDTA, 1% FBS, and 0.05% NaN) and combined with an equal volume of anti-LILRB2 antibody at concentrations of 40, 20, 10, or 5 μg / mL, followed by incubation for 1 hour at 4°C. B cells (721.221) were washed with PBS, counted, and then aliquoted at 1.3 × 10 in Fc Block (10% NGS / PBS (Sigma, No. G6767), 2.5% FBS, 1% anti-CD32 (BD Biosciences, No. LI2-H82F5), 2 mM EDTA, and 0.05% NaN) in FACS buffer. 6 / mL (50 × 10 3 The cells were resuspended at a cell density of 1000 cells / well and then incubated at RT for 30 minutes. Cells (50 μL) were added to wells containing the LILRB2-Fc-Avitag protein / antibody preincubation solution to a final concentration of 7.5 μg / mL LILRB2-Fc-Avitag protein and 20, 10, 5, or 2.5 μg / mL antibody and then incubated at RT for 1 hour. The cells were then washed with FACS buffer (100 μL / well) and pelleted by centrifugation at 450 × g for 5 minutes. The supernatant was removed and a second wash with 250 μL / well of FACS buffer was performed, followed by a subsequent centrifugation at 450 × g for 5 minutes to pellet the cells.

[0274] A 1:250 dilution of streptavidin-PE (BioLegend, No. 405204) (75 μL / well) was added, and the cell / antibody mixture was incubated at RT for 30 minutes protected from light. The cells were then washed twice in PBS containing 1 mM EDTA at a volume of 200 μL / well, followed by centrifugation at 450 × g for 5 minutes to pellet the cells. The cells were resuspended in 50 μL of cell viability dye (Zombie Violet, BioLegend, No. 423114) at a 1:2000 dilution and incubated at RT for 10 minutes protected from light. A final wash of 200 μL / well of FACS buffer was performed, and the cells were then pelleted by centrifugation at 450 × g for 5 minutes. The supernatant was removed, and the cells were resuspended in 100 μL of FACS buffer for flow cytometry analysis, as described in Example 4. The B2A-IgG4 clone blocked LILRB2-Fc binding to HLA-G-expressing 721.221 cells in a dose-dependent manner, whereas the IgG4 isotype control had no effect on LILRB2-Fc binding to 721.221 cells. Complete inhibition of binding by the B2A-IgG4 clone was observed at concentrations of 20 μg / mL and 10 μg / mL, and partial blocking (normalized) of 82% and 32% was observed at concentrations of 5 μg / mL and 2.5 μg / mL, respectively.

[0275] Example 8: Binning of LILRB2 antibodies Anti-LILRB2 antibodies were evaluated in blocking flow cytometry experiments using HEK293T-LILRB2 cells stably expressing LILRB2 (Example 3) and biotin-conjugated benchmark LILRB2 antibodies prepared based on the literature: B2Comp1 (PCT Publication WO 2021 / 138079 A1, designated MK-4830) and B2Comp2 (US Patent Application Publication No. 2019 / 0194327, designated J-19.h1). The variable domains of these antibodies were cloned into IgG4 and lambda (B2Comp1) or kappa (B2Comp2) constant regions. LILRB2 flow cytometry binning separates antibodies into different bins according to their ability to block benchmark antibody binding 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 antibody in FACS buffer was added to HEK293T-LILRB2 cells at a final concentration of 0.1, 1, or 10 μg / mL and then incubated at RT for 1 hour. Then, "hot" biotinylated benchmark antibody was added at a final assay concentration of 5 ng / mL. After 45 minutes of incubation at RT, cells were washed with FACS buffer and incubated with streptavidin-APC (Invitrogen, No. S32357), and binding of benchmark to HEK293T-LILRB2 cells was quantified at RT for 45 minutes. Cells were then washed with FACS buffer and stained with cell viability dye eFluor™ (e780; Thermo Fisher Scientific, No. 65-0865-14) 780 for 15 minutes at RT. Cells were washed with FACS buffer and resuspended in 100 μL of FACS buffer for acquisition by flow cytometry as described in Example 4.

[0276] Chimeric anti-LILRB2 antibodies were binned into four separate categories:

[0277] Bin 1: Antibodies that block the binding of B2Comp1 and B2Comp2 to HEK293T-LILRB2 cells and share an epitope with both B2Comp1 and B2Comp2

[0278] Bin 2: Antibodies that block the binding of B2Comp1 but not B2Comp2 to HEK293T-LILRB2 cells and share an epitope with B2Comp1

[0279] Bin 3: Antibodies that do not block the binding of B2Comp1 and B2Comp2 to HEK293T-LILRB2 cells

[0280] Bin 4 antibody blocks the binding of B2Comp2, but not B2Comp1, to HEK293T-LILRB2 cells and shares an epitope with B2Comp2

[0281] The binning data are shown in Table 2. B2A-IgG4 did not block binding of any of the comparable LILRB2 antibodies to HEK293T-LILRB2 cells at 0.1, 1, and 10 μg / mL, 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 a novel epitope not shared with certain other published anti-LILRB2 antibodies.

[0282] [Table 2]

[0283] Example 9: Isolation and differentiation of human primary cells Evaluation of anti-LILRB2 antibodies in immunological assays requires the isolation of human T cells and monocytes and the differentiation of monocytes into immunosuppressive macrophages. Various techniques are known in the art, including those described below for the cells used in these examples. Apheresis products are collected from subjects, and autologous monocytes and T cells are isolated using the techniques described or other techniques 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, Washington). Peripheral blood mononuclear cells (PBMCs) are purified from LeukoPak by standard density gradient centrifugation (FicollPaque® Premium 1.073 or 1.077, GE Healthcare, No. 17-5449-52, or Cytiva No. 17144003). Discard the supernatant and resuspend the pellet in 20 mL of EasySep™ buffer (STEMCELL Technologies, No. 20144) for PBMC enumeration and further isolation of monocytes and T cells. Monocytes are isolated using the EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies, No. 19359) according to the manufacturer's instructions. Total CD3+, CD4+, and CD8+ T cells are isolated using the EasySep™ Human CD3+, CD4+, and CD8+ T Cell Isolation Kit (STEMCELL Technologies, Nos. 19051, 17952, and 17953, respectively) according to the manufacturer's instructions. These negative selection kits use antibodies to label undesired cell types for removal, allowing the isolation of desired target cells from untreated samples.

[0284] Example 10: Macrophage generation Macrophages can be generated from PBMC-derived monocytes using commonly used techniques such as those described below.

[0285] Generation of M0 macrophages: On day 0, monocytes from individual subjects (isolated as described in Example 9) were cultured at 25-50 x 10 cells / mL in M0 culture medium (90% X-VIVO™ 15 + 10% FBS + 100 ng / mL human M-CSF (PeproTech, No. 300-25)). 3 Cells were seeded into a 96-well culture plate (Thermo Fisher (Costar), No. 09-761-175) at 100 μL per well. The cells were incubated at 37°C and 5% CO for 5–6 days to generate M0 macrophages.

[0286] 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 culture medium (M0 medium + 20 ng / mL human IL-10 (PeproTech, No. 200-10)). The cells were incubated at 37 °C and 5% CO for 2 days. On days 7–8 of culture, M2c macrophages were ready for co-culture assay setup. M2c macrophages were detached from the plate by incubation with Macrophage Detachment Solution DXF (PromoCell) and washed with PBS before downstream assays.

[0287] Example 11: Generation of exhausted T cells Exhausted T cells are an indicator of an immunosuppressive tumor microenvironment and contribute to cancer immune evasion. To mimic exhausted T cells in the TME, exhausted T cells were generated by repeated stimulation. Exhausted T cells with blast-like morphology were generated from human PBMCs by repeated (3X) phytohemagglutinin (PHA) stimulation. Cells were counted and cultured at 1 x 10 in T cell blast culture medium (90% IMDM (Thermo Fisher (Gibco) No. 12440053) + 10% human serum + 2 μg / mL PHA-L (Sigma-Aldrich (Roche), No. 11249738001) + 4 ng / mL recombinant human IL-2 (R&D Systems, No. 202-IL)). 6Cells were incubated at 1:200 cells / mL. Cells were split 1:2 or 1:3 every 3–4 days and cultured for a total of 10 days (2 splits over 10 days; 3 total PHA stimulations). Fresh T cell blast culture medium was added to the cells at each split. Cells were harvested on day 10 and either set up for coculture assays or frozen for future use. The exhausted T cell phenotype was confirmed by expression of PD-1, TIM-3, and TIGIT, as well as the transcription factor Eomes (data not shown).

[0288] Example 12: LILRB2 antibodies bind to human monocytes and M0 and M2c macrophages Anti-LILRB2 antibodies were evaluated for their ability to bind to LILRB2-expressing myeloid cell subsets, including monocytes, M0 macrophages, and M2c macrophages. M2c macrophages are used as a surrogate for tumor-associated macrophages, which are suppressive macrophages commonly found in the tumor microenvironment. Frozen monocytes were removed from storage in vapor-phase liquid nitrogen, thawed by gentle swirling in a 37°C water bath, and then resuspended in X-VIVO™ 15 medium containing 10% FBS. PBMCs were placed in a 15 mL conical tube and centrifuged at 300 × g for 5 minutes, and the supernatant was then removed. Cells were diluted to 2.5 × 10 in assay medium (X-VIVO™ 15 + 10% FBS). 5 The cells were resuspended at a cell density of 1000 cells / mL. Cells were plated at 100 μL / well (25K cells / well) in a 96-well flat-bottom plate. M0 and M2c macrophages were generated as described in Example 10 and harvested from flasks by incubation in Macrophage Detachment Solution DXF for 15 minutes at RT, then removed from the flasks into X-VIVO™ 15 medium. After centrifugation, the cells were resuspended in FACSBlocking Buffer D (FACS buffer + 10% FBS + 0.5 mg / mL human IgG1) and then incubated at 4°C for 30 minutes. 2.5 x 10 cells in 25 μL of FACSBlocking Buffer D were harvested. 4Cells / 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× the final assay concentration. Cells were incubated with the antibody for 1 hour at 4°C. Cells were washed twice with FACS buffer, stained with Zombie UV live / dead stain viability dye (BioLegend, No. 423107) (1:500 dilution) for 15 minutes at RT in the dark, 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 EC 50 Used in binding calculations. B2A-IgG1 and B2A-IgG4 bind to human monocytes and M0 and M2c macrophages (Figure 3).

[0289] Example 13: LILRB2 antibodies enhanced IFN-γ responses in LPS-stimulated PBMCs To determine whether blocking LILRB2 enhances innate immune responses, which in turn enhances IFN-γ secretion and attenuates the immunosuppressive TME, the effect of B2A-IgG1 and IgG4 chimeras on IFN-γ secretion by PBMCs in response to lipopolysaccharide (LPS) was evaluated. Frozen PBMCs were removed from storage in vapor-phase liquid nitrogen, thawed by gentle swirling in a 37°C water bath, and then resuspended in assay medium. PBMCs were placed in a 15 mL conical tube and centrifuged at 300 × g for 5 minutes, and the supernatant was then removed. 3 × 10 cells were cultured. 6 Cells were resuspended in assay medium at 100 μL / mL. Cells were plated at 100 μL / well (300K cells / well) in a 96-well flat-bottom plate. This cell density was optimized for the average level of IFN-γ response per subject. If a subject was known to produce a higher cytokine response, fewer cells were plated per well. Cells were incubated at 37°C, 5% CO2 for 1 hour.

[0290] The LILRB2 antibody was diluted to a 4x concentration in assay medium (final concentration 1 μg / mL) and 50 μL of diluted antibody was added per well. For wells that did not receive antibody, 50 μL of assay medium was added per well. Cells containing the antibody dilutions were incubated for 2 hours at 37°C, 5% CO2 before LPS stimulation. LPS was diluted to a 4x concentration in assay medium (final concentration in the assay: 1 μg / mL), and 50 μL per well was added to the cell / antibody mixture, except for the control samples with known higher IFN-γ responses, where the final LPS concentration was 0.1 μg / mL. For control wells (no LPS), 50 μL of assay medium was added per well. Cells were incubated for 24 hours at 37°C, 5% CO2 in a final volume of 200 μL per well. Supernatants (150 μL) were collected after 24 h of incubation and either frozen at -80°C or directly tested for IFN-γ secretion by ELISA (R&D Systems). Anti-LILRB2 antibodies contributed to the pro-inflammatory phenotype by demonstrating enhanced IFN-γ secretion from LPS-stimulated PBMCs. Both B2A-IgG1 and B2A-IgG4 induced similar IFN-γ secretion profiles, shown for three representative subjects in Figure 4.

[0291] Example 14: LILRB2 antibody enhanced TNF-α secretion in CD40-activated macrophages CD40L expressed on T cells is the primary molecule responsible for activating macrophages in the TME through cell-cell contact and binding to CD40 expressed on macrophages. 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 inflammatory cytokine production by macrophages treated with LILRB2 antibody but without the presence of T cells. A HEK293 cell line modified to express CD40L (CrownBio, No. C2041) was used to mimic binding and subsequent stimulation by activated T cells. M0 macrophages were cultured using monocytes (25 × 10 3The CD40L-expressing HEK293 cells were generated from 5 × 10 cells / well. The medium was removed from the wells, and 100 μL / well of fresh assay medium was added. 5x concentrated LILRB2 antibodies (B2A-IgG1 and B2A-IgG4) were added at 50 μL / well and incubated on the macrophages for 2 hours at 37°C and 5% CO2. During the 2-hour incubation, CD40L-expressing HEK293 cells were harvested from the flask and irradiated at 40 Gy. After pre-incubation with the LILRB2 antibody, CD40L-expressing HEK293 cells (5 × 10 cells / well) were incubated for 2 hours at 37°C and 5% CO2. 3 (B2A-IgG1 and B2A-IgG4) were added at a volume of 100 μL / well to a final well volume of 250 μL / well in a 96-well plate and incubated overnight at 37°C, 5% CO2. After incubation, 200 μL of supernatant was collected from the wells and either frozen at -80°C or immediately assayed for TNF-α secretion by HTRF according to the manufacturer's instructions (CisBio, No. 62HTNFAPET). Both B2A-IgG1 and B2A-IgG4 show comparable enhancement of TNF-α secretion by CD40L-activated macrophages in four different subjects (Figure 5).

[0292] Example 15: LILRB2 antibodies attenuate M2c-mediated immunosuppression in M2c / CD8+ or M2c / CD4+ T cell co-cultures Crosstalk between immunosuppressive myeloid cells expressing LILRB2 and T cells contributes to T cell exhaustion and the lack of antitumor immune responses in the TME. This crosstalk can be modeled by in vitro coculture of autologous monocyte-derived M2c macrophages with anti-CD3-stimulated CD8+ or CD4+ T cells. Reduced immunosuppression can be assessed by T cell proliferation and quantification of IFN-γ and perforin secretion as surrogates of T cell activation and antitumor activity. After polarization of M0 macrophages into M2c macrophages as described in Example 10, supernatant was removed from the macrophages in the 96-well culture plate and replaced with 100 μL of assay medium containing OKT3 (BioLegend #317326) at a final concentration of 0.25 or 0.63 μg / mL. LILRB2 antibody (dose titration from 20 μg / mL to 0.0015 μg / mL) was added in a volume of 50 μL / well, and the plate was then incubated for 1-2 hours at 37°C, 5% CO2. While incubating the M2c macrophages with the antibody, autologous CD4+ or CD8+ T cells were isolated from PBMCs as described in Example 9. The isolated CD8+ or CD4+ T cells were stained with CellTrace™ Violet (Thermo Biosciences, Inc.) as described in Example 7B. Excess CellTrace™ was washed away with pre-warmed assay medium, and labeled CD8+ or CD4+ T cells were resuspended in assay medium and then diluted at 5 x 10 in a volume of 100 μL at a 1:1 ratio of M2c:CD4+ or M2c:CD8+ T cells. 5 cells / mL were added to the M2c / antibody preparation. The cells were then incubated at 37°C, 5% CO for 72 hours.

[0293] The T cell-containing supernatant was transferred to a V-bottom 96-well plate and centrifuged to pellet the T cells. The culture supernatant was collected and frozen at -80°C for quantification of human IFN-γ and perforin levels by MSD-ELISA (Meso Scale Discovery, custom U-plex assay). The T cell pellet was stained with e780 viability dye for 10 minutes at room temperature in the dark, washed with 150 μL of FACS buffer, and resuspended in 100 μL of FACS buffer for acquisition on a BD FACSymphony™ or FACSCanto™ flow cytometer (BD Biosciences). The percentage and total number of proliferating CD8+ or CD4+ T cells were analyzed using FlowJO software and reported as percent CellTrace+ dividing cells or total number of CellTrace+ dividing cells.

[0294] The chimeric antibody clones attenuated M2c macrophage-mediated immunosuppression in these M2c / T cell coculture assays, as measured by restoring CD8+ T cell proliferation and IFN-γ secretion and perforin release (Figures 6A and 6B, respectively).

[0295] Example 16: LILRB2 antibodies rescue IFN-γ responses of exhausted T cells in co-culture with M2c macrophages A hallmark of ineffective anti-cancer immune responses 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 activity is a promising strategy for treating cancer. The ability of LILRB2 antibodies to rescue the functional activity of exhausted T cells from LILRB2-mediated immunosuppression was evaluated by an assay utilizing exhausted T cells and M2c cells in coculture to measure macrophage-mediated immunosuppression.

[0296] T cell blast and M2c co-culture assay To measure the ability of LILRB2 antibody to rescue the functional activity of T cell blasts from M2c-mediated immunosuppression, the culture medium from M2c macrophages was removed and replaced with assay medium containing LILRB2 or an isotype control antibody and incubated at 37°C, 5% CO for 2 hours. OKT3 antibody (final concentration: 0.25 μg / mL) was added to the wells and incubated at 37°C, 5% CO for 30 minutes. T cell blasts (Example 11) were finally added to the M2c / LILRB2 antibody mixture + OKT3 at a 1:1 ratio and incubated at 37°C, 5% CO for 72 hours. IFN-γ levels were quantified by ELISA from supernatants collected 72 hours after OKT3 stimulation (R&D or Meso Scale). B2A-IgG1 rescued the IFN-γ response of exhausted T cells from M2c macrophage-mediated immunosuppression (Figure 7). B2A-IgG4 has no activity in this assay (data not shown).

[0297] Example 17: Pharmacokinetic profiling of chimeric LILRB2 antibodies in humanized FcRn mice Therapeutic monoclonal antibodies for cancer treatment are typically of the immunoglobulin G (IgG) subclass. Because human IgG binds to the mouse neonatal Fc receptor (FcRn) with higher affinity than human FcRn, the half-life of monoclonal antibodies tested in mice does not correlate with that observed in humans. 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 the lysosome, preventing its degradation, and FcRn-bound IgG antibodies are recycled to the extracellular surface, where they dissociate from FcRn and return to the circulation, which in turn allows for an extended half-life of these IgG monoclonal antibodies.

[0298] Different FcRn transgenic mice were generated to knock out mouse FcRn and express human FcRn. Several groups have demonstrated that the PK of human therapeutic antibodies in these humanized FcRn mice correlates with human PK and exhibits 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 possess all mouse immune cells; in these models, only the mouse FcRn is deleted and human FcRn is expressed.

[0299] Female homozygous Tg32 FcRn mice were purchased at 6–8 weeks of age (Jackson Laboratory, No. 014565) and housed in microisolator cages under specific pathogen-free conditions in the Bloodworks Northwest vivarium. All procedures were performed under the institutional guidelines of Bloodworks Northwest's Institutional Animal Care and Use Committee Protocol #5390-01. Mice were identified using ear tags. All mice were acclimated for a minimum of 5 days before the start of antibody dosing. On the day of dosing, the initial body weight of each mouse was recorded. Mice were assigned to two LILRB2 antibody treatment groups: B2A-IgG4 and B2A-IgG1, with 12 mice per group. The 12 mice from each treatment group were divided into three bleed groups with four mice per group and administered a single intraperitoneal (IP) dose of 20 mg / kg B2A-IgG4 or B2A-IgG1 antibody. Blood was collected from each group at three alternating time points as follows: Group 1: 0.25, 4, 96 hours; Group 2: 1, 24, 168 hours; Group 3: 2, 48, 240 hours.

[0300] Blood collection and serum preparation Blood was collected via retroorbital (0.25, 1, and 2 hours), submandibular (4, 24, and 48 hours), and terminal cardiac puncture (96, 168, and 240 hours) bleeding. Whole blood was allowed to clot for a minimum of 30 minutes at room temperature. Clots were removed by centrifugation (2000 × g) at 4°C for 10 minutes. Serum was aliquoted into four new microfuge tubes and frozen at -80°C until analysis.

[0301] ELISA assay for measuring anti-LILRB2 antibody concentrations in mouse serum A human LILRB2 capture ELISA was performed to determine the antibody concentration of each group. Recombinant human LILRB2-HIS tagged protein was diluted to 2 μg / mL in PBS and added to a 384-well high-binding ELISA plate (Greiner Bio-One Microlon™) at 25 μL / well and incubated overnight at 4°C. The plate was washed four times with wash buffer using a microplate washer and then blocked with 90 μL / well of Blocking Buffer E (3% BSA / PBS) for 1 hour at RT. B2A-IgG1 and B2A-IgG4 antibody stocks were diluted in Assay buffer (0.05% BSA / TBS-T (0.05% Tween® 20) / Tris-buffered saline (pH 7.4)) to prepare a top standard concentration of 40 ng / mL. Fifteen 2-fold serial dilutions of the standard were diluted with 0.001 to 0.002% mouse serum. Serum samples from anti-LILRB2-treated mice were also diluted in Assay Buffer. Different dilutions were tested depending on the time point of serum collection and the antibody administered. Each standard dilution and mouse serum dilution was assayed in duplicate wells. After blocking, 25 μL / well of diluted standard and diluted mouse serum was added to the assay plate and incubated at RT for 1 hour. After primary antibody binding, the plate was washed five times with Wash Buffer using a microplate washer. The secondary detection antibody (HRP-goat anti-human IgG Fc specific) was diluted 1:5000 in Assay Diluent (1% BSA / PBS) and added at 25 μL / well to the plate and incubated at RT in the dark for 1 hour. The plate was washed five times with Wash Buffer using a microplate washer. After removing the final wash, 25 μL / well of 1-Step™ Ultra TMB-ELISA Substrate Solution was added and the plate was incubated at RT for 5–10 minutes, protected from light. After development, the reaction was stopped 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.

[0302] PK analysis Anti-LILRB2 antibody serum levels were determined from separate blood draws taken 0.25 and 240 hours after anti-LILRB2 antibody administration. Mean B2A-IgG4 and B2A-IgG1 antibody serum levels were generated from four mice per time point to create composite PK profiles. Non-compartmental PK parameters after IP injection were analyzed using the Microsoft Excel PK solver 2.0 add-in on the mean serum concentration profiles over time, and PK parameters were calculated using the PK solver software (Table 4). Antibody serum exposure was graphed using GraphPad Prism for Windows (GraphPad Software). B2A-IgG1 and B2A-IgG4 antibodies exhibited half-lives of 7.0 and 9.9 days in humanized FcRn mice (Table 3 and Figure 8).

[0303] [Table 3]

[0304] Example 18: LILRB2 antibodies inhibit tumor growth in humanized NSG-SGM3 mice bearing subcutaneous human SK-MEL-5 melanoma The antitumor effect of B2A-IgG4 was tested in an in vivo humanized tumor model. Female humanized NSG-SGM3 mice were purchased from Jackson Laboratory (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 intravenously injecting human UBC CD34+ hematopoietic stem cells into irradiated 3-week-old NSG-SGM3 mice. Engraftment of human CD45+ cells to assess humanization in peripheral blood was monitored weekly by Jackson Laboratory. Only animals with a minimum engraftment of 25% human CD45+ cells were received from Jackson Laboratory and enrolled in the study.

[0305] In vivo tumor xenograft model and LILRB2 antibody administration:

[0306] Female humanized NSG-SGM3 mice were housed in microisolator cages under specific pathogen-free conditions in the Bloodworks Northwest vivarium. All procedures were performed under the institutional guidelines of Bloodworks Northwest's IACUC Protocol #5390-02. Mice were identified using ear tags. All mice were allowed to acclimate for a minimum of 5 days before the start of the study. Mice were implanted in the right flank with 2 x 10 guinea pigs in 100 μL of PBS containing 20% ​​Matrigel (R&D Systems, No. 3632-005-02). 6 SK-MEL-5 human melanoma cells (HLA class A*02:01; ATCC) were inoculated subcutaneously (SC).

[0307] Tumor size was measured twice a week using a digital caliper. Tumor volume was calculated: tumor volume (mm 3 )=(L×W 2 / 2) (where L is the maximum dimension and W is the minimum dimension). Approximately 50 mm 3 When the mean tumor size reached 100 μg / mL, mice were randomized into groups based on both tumor size and human CD45+ cell engraftment, with each group containing 8–9 mice. Mice were intraperitoneally (IP) administered 20 mg / kg of B2A-IgG4 or IgG4 isotype control on the day of randomization (day 9) and again 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).

[0308] The mean tumor growth inhibition (TGI) was calculated using the following formula for all data collection days up to day 27 (Table 5).

[0309] JPEG2025535387000009.jpg1993

[0310] Statistical differences in tumor volume were determined using a parametric RM (repeated measures) two-way ANOVA with Geisser-Greenhouse correction using GraphPad Prism software. The standard error of the mean was calculated for tumor volume for each day. P values ​​were considered significant as follows: *P<0.05; **P<0.01 and ***P<0.001.

[0311] Mice treated with anti-LILRB2 B2A-IgG4 achieved 79% tumor growth inhibition (TGI) and 33% tumor regression by day 41 compared to the IgG4 control-treated group, suggesting the ability of B2A-IgG4 to slow tumor growth (Table 5 and Figure 9a).

[0312] B2A-IgG4 achieved anti-tumor activity in this humanized NSG-SGM3 human SK-MEL-5 melanoma mouse model (Figures 9A and 9B and Table 4), whereas an IgG4 control antibody showed a lack of anti-tumor activity in the same tumor model (Figures 9A and 9B).

[0313] [Table 4]

[0314] Example 19: Humanization of antibodies The identified rabbit / human chimeric B2A-IgG4 mAb was selected for humanization. Humanization was performed in silico using a proprietary methodology (Fusion Antibody, Belfast, Northern Ireland). This system generates a model of the parent variable domain, allowing for structure-guided humanization. The sequence is aligned to a panel of human germline sequences selected for preferential manufacturability properties, and non-human amino acids are grafted into the human sequence using the proprietary CDRx™ humanization platform. The first round of humanization performed on the clone was EC 50 Since the criteria were met, only the first round of humanization was performed.

[0315] Five heavy and five light chains of B2A-IgG4 were generated. The humanized amino acid sequences were submitted to GenScript, which reverse-translated and codon-optimized each variable region sequence for mammalian cell expression using a proprietary methodology. Genes encoding the signal sequences and variable regions were synthesized by GenScript and cloned in frame with the human IgG4 constant region for the heavy chain and the human kappa constant region for the light chain into the pTT5 vector. 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 Canada (NRC)), and conditioned medium was harvested after 7 days. The recombinant antibodies were purified by Protein A affinity chromatography. These affinity-purified antibodies were then used to generate binding data.

[0316] Success was defined as achieving a binding constant within 2-fold of the parent mAb, in this case EC 50 was determined by ELISA using rabbit variable regions fused to human IgG4 constant regions as the parent chimeric mAb.

[0317] Twenty-five IgG4 variants were expressed in mammalian cell culture as a combinatorial library of five light chains and five heavy chains. Additionally, B2A-IgG4 was included as a transfection control. Conditioned media from these transient transfections were assayed for human IgG concentration. A plate-based ELISA (see Example 2) was performed to immobilize LILRB2-His and detect binding of the humanized mAb variants via an anti-human IgG4 HRP-labeled secondary antibody. Transfection of the parent rabbit / human chimeric clones yielded benchmark EC 50 Served as a positive control. Antibody titers and EC 50 The values ​​are shown in Table 5.

[0318] [Table 5]

[0319] Example 20: Humanized variant antibodies bind to LILRB2 by ELISA and to cells expressing LILRB2 Selected humanized variant antibodies were purified and tested for their ability to bind to human LILRB2 by ELISA using the method described in Example 2, and to HEK293T cells expressing human LILRB2 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 parent chimeric antibody.

[0320] [Table 6]

[0321] Example 21: Humanized LILRB2 antibodies do not bind to other LILRB or LILRA family members Selected humanized variant antibodies were tested for their ability to bind to other LILRB or LILRA family members using the methods of Example 5 and a commercially available positive control antibody. The humanized LILRB2 antibody variant did not bind to any members of the LILRA family or to any additional LILRB family members. Representative data are shown in Tables 7A and 7B.

[0322] [Table 7A]

[0323] [Table 7B]

[0324] Example 22: Humanized LILRB2 mutant antibodies block LILRB2-Fc binding to HLA-G Selected humanized IgG4 mutant antibodies were tested for their ability to block the binding of human LILRB2-Fc to monomeric HLA-G by ELISA using the method described in Example 7. The humanized mutants blocked the binding of LILRB2-Fc to HLA-G. Representative data are shown in Table 9.

[0325] [Table 8]

[0326] Example 23: Humanized variant B2H1-55 antibodies bind to monocytes and M0 and M2c macrophages Humanized variant B2H1-55 was tested for binding to monocytes, M0 and M2c macrophages according to the methods described in Example 12. Data compiled from 15 subjects (monocytes), 7 subjects (M0 macrophages), and 10 subjects (M2c macrophages) in which dose escalation of AF647-conjugated B2H1-55 was tested is shown in Figure 10. Humanized variant B2H1-55 showed dose-dependent binding to monocytes, M0 and M2c macrophages, with a mean EC 50 are 35.6, 74.8 and 24.3 ng / mL, respectively.

[0327] Example 24: Humanized LILRB2 antibody B2H1-55 binds to a panel of myeloid cells but not lymphocytes LILRB2 is predominantly expressed in whole blood myeloid cells. Since we determined that the LILRB2 antibody does not bind to whole blood lymphocytes, we performed a whole blood immunophenotyping assay to test the binding of the humanized antibody B2H1-55 to whole blood myeloid cell populations. Whole blood from healthy subjects was purchased (Bloodworks Northwest). The blood-containing tube was gently inverted to evenly distribute the plasma and cells. The blood was then thoroughly mixed with Blocking Mix (10% FBS + 500 μg / mL human IgG1 myeloma plasma (Athens Research) + 0.05% NaN3), pipetted into a 96-well deep plate, and then incubated at 4°C for 1 hour. Cells were resuspended at 30 minutes during incubation with the Blocking Mix. Antibody titration was 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 at 4°C for 45 minutes, protected from light, and then at room temperature for 15 minutes, for a total incubation time of 1 hour. Red blood cells (RBCs) were lysed using 1x RBC Lysis buffer (BD Pharm Lyse™; BD Biosciences, No. 555899) in three consecutive RBC lysis steps by adding the RBC Lysis buffer to the blood mixture, pipetting up and down to mix thoroughly, and then incubating at room temperature for 10 minutes, protected from light. After incubation, the cell plate was sealed and centrifuged at 200 x g for 5 minutes, and the supernatant was then aspirated from the wells. PBS (1x) was added to stop RBC lysis, followed by centrifugation at 200 x g for 5 minutes and removal of the supernatant. The cells were then transferred to a 96-well V-bottom plate. Viability staining was performed by adding a 1:500 dilution of viability dye (Zombie Violet, BioLegend, No. 423114) and incubating for 20 minutes at room temperature protected from light. Cells were resuspended in FACS buffer, centrifuged to remove the viability dye, and the supernatant was removed. Cells were then resuspended in FACSBlocking Buffer D and incubated for 15 minutes at room temperature.Flow cytometry antibody cocktail (fluorophore-conjugated BV421 anti-human CD3 (BioLegend, No. 300434), BV711 anti-human CD4 (BioLegend, No. 300558), APC / Cy7 anti-human CD8 (BioLegend, No. 344714), PE anti-human CD11c (BioLegend, No. 337206), BUV496 anti-human CD14 (BD Biosciences, No. 741200), BUV805 anti-human CD15 (BD Biosciences, No. 742057), BV786 anti-human CD16 (BD Biosciences, No. 742057)) Anti-human CD19 (BioLegend, No. 563690), BV605 anti-human CD19 (BioLegend, No. 302244), FITC anti-human CD56 (BioLegend, No. 318304), and PE / Cy7 anti-human HLA-DR (BioLegend, No. 307616) were added directly to the wells on top of the blocking buffer and then incubated for 30 minutes at RT protected from light. After incubation, FACS buffer was added to the wells for washing, and the plate was then centrifuged at 350 x g for 5 minutes and 350 μL of FACS buffer was added for acquisition on a FACSymphony™ cytometer. The antibodies were resuspended in IgG1 buffer. Data from three subjects are shown in Figure 11, where B2H1-55 was assayed for binding at two concentrations compared to an hIgG1 isotype control. The humanized LILRB2 antibody B2H1-55 binds exclusively to myeloid cells, including classical, non-classical, and intermediate monocytes, myeloid dendritic cells, and neutrophils. B2H1-55 did not interact with human T cells, B cells, or NK cells.

[0328] Example 25: Humanized variant B2H1-55 binds to neutrophils Human neutrophils have been reported to express LILRB2. To determine whether the humanized anti-LILRB2 variants bind to and activate human neutrophils, we evaluated the binding of the humanized antibody to LILRB2 expressed on neutrophils in whole blood from healthy donors (Bloodworks NW, Seattle, WA). Neutrophils were isolated directly from whole blood by immunomagnetic negative selection according to the manufacturer's instructions (StemCell, No. 19666). After neutrophil isolation, 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 × 10 cells / well in a 96-well plate. 5 Cells were seeded at 1000 cells / mL. Cells were incubated in Blocking Buffer G at 4°C for 30 minutes. The titer of B2H1-55 conjugated to AF647 in-house (Alexa Fluor™ 647 NHS Ester, Thermo Fisher Scientific, No. A20106) was measured in FACS buffer at final concentrations of 10, 1, and 0.1 μg / mL. The diluted antibody was added to neutrophils at a 1:1 dilution and then incubated at 4°C for 120 minutes protected from light. After antibody incubation, cells were rinsed with FACS buffer and then centrifuged at 450 × g for 5 minutes to pellet the cells. Cells were resuspended in viability dye (dead cell labeling reagent) and incubated at RT for 10 minutes in the dark. After staining with the viability dye, cells were rinsed with FACS buffer and then centrifuged at 450 × g for 5 minutes. The cells were resuspended in FACS buffer and analyzed by flow cytometry using a BD FACSymphony™ cytometer. Representative data are shown in Figure 12, in which dose-dependent binding of B2H1-55 to neutrophils was observed. Data shown are for six subjects from two independent experiments.

[0329] Example 26: Humanized LILRB2 antibodies do not induce neutrophil activation Humanized variants of B2H1-55 were tested for their potential to activate human neutrophils in whole blood from healthy subjects. Titrations of soluble B2H1-55 humanized variants, an IgG1 isotype control, and a commercially available positive control (BioLegend, anti-CD16 clone 3G8) were added to whole blood and incubated in a 96-well plate at 37°C for 2 hours. The final antibody concentrations in whole blood were 100, 50, 10, and 0.1 μg / ml. After antibody incubation, cells were rinsed with FACS buffer and then centrifuged at 300 × g for 5 minutes to pellet the cells, and the supernatant was discarded. Whole blood cells were then stained with fluorochrome conjugates BV421 anti-human CD11b (BioLegend, No. 301324), AF647 anti-human CD62L (BioLegend, No. 304818), FITC anti-human CD66b (BioLegend, No. 305104), and BUV395 anti-human CD15 (BD Biosciences, No. 740318) for 20 minutes at 4°C. Cells were rinsed with FACS buffer and then centrifuged at 300 × g for 5 minutes to pellet the cells, and the supernatant was discarded. RBC lysis was performed with 200 μL of 1× lysis solution (BD Biosciences, No. 740318). Biosciences, No. 349202, 1:10 dilution) was added to all wells, mixed gently by pipetting up and down, and incubated 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. Cells were washed with FACS buffer at 300 x g for 5 minutes, resuspended in FACS buffer, and evaluated by flow cytometry using a BD FACSymphony™ cytometer. Neutrophil frequency (CD66b+CD15+) (PMN in Figure 13) and changes in surface density of activation markers (increase in CD11b and decrease in CD62L) were analyzed using FlowJo software.

[0330] Representative data from two healthy subjects are shown in Figure 13, comparing the humanized variant B2H1-55 with an anti-CD16 positive control antibody (clone 3G8) and an isotype control antibody. Upon binding to neutrophils, B2H1-55 did not induce cell activation, as indicated by unchanged CD11b expression and retention of surface CD62L. The effect associated with the positive control antibody (Figure 13) is consistent with the reported activity for anti-CD16 antibodies.

[0331] Example 27: Humanized LILRB2 antibodies enhance IFN-γ responses in LPS-stimulated PBMCs To determine whether the humanized variants enhanced the innate immune response of human monocytes by blocking the interaction of LILRB2 with its HLA ligand, the effects of the variants on IFN-γ production and IL-10 secretion by LPS-stimulated human PBMCs were evaluated. The humanized variants were tested for their ability to enhance IFN-γ and IL-10 secretion by LPS-stimulated PBMCs according to the method described in Example 13. IFN-γ and IL-10 were quantified in supernatants collected 24 hours after LPS stimulation (R&D Systems). The humanized variants induced a proinflammatory innate Th1-like phenotype, and PBMCs showed enhanced IFN-γ secretion and reduced IL-10 production when stimulated with LPS. Representative IFN-γ data are shown in Figure 14, where the three humanized variants are compared with the B2A-IgG1 parent, the benchmark antibody B2Comp1, and the hIgG1 isotype control antibody. Data shown are for two representative subjects at two antibody concentrations. Humanized LILRB2 variants enhanced IFN-γ secretion from LPS-stimulated PBMCs. As shown in Figures 23A-C for representative humanized variants, humanized anti-LILRB2 variants dose-dependently inhibit LPS-mediated IL-10 release by human PBMCs.

[0332] Humanized anti-LILRB2 variants and Toll-like receptor 2 (TLR) ligands, such as heat-killed Listeria monocytogenes (HKLM, InvivoGen, No: Tlrl-hklm, 2.5 × 10 7 The humanized anti-LILRB2 variants were also tested in combination with HKLM (2.5 × 10 cells / mL) and Pam3CSK4 (InvivoGen, No:Tlrl-pms, 100 ng / mL). 7 cells / mL) or Pam3CSK4 (100 ng / mL)-treated PBMCs enhanced IFN-γ secretion and reduced IL-10 release (data not shown).

[0333] Example 28: Humanized LILRB2 antibodies alleviate M2c-mediated immunosuppression in an M2c / T cell co-culture assay The humanized variants were tested for their ability to restore proliferation, IFN-γ secretion, and perforin release by CD8+ T cells cocultured with immunosuppressive M2c macrophages according to the methods described in Example 15. Representative data are shown in Figure 15. Similar to the LILRB2 parent chimeric B2A-IgG1, the humanized LILRB2 variants attenuate M2c-mediated immunosuppression by dose-dependently enhancing CD8+ T cell proliferation, IFN-γ secretion, and perforin release.

[0334] Example 29: Humanized LILRB2 antibodies block the development of immunosuppressive macrophages To determine whether anti-LILRB2 antibodies can disrupt the generation of tumor-associated macrophages, we performed an M2c / CD8+ T cell coculture assay, as described in Example 15, with an additional "pre-regimen" step during M0-to-M2c polarization. For the pre-regimen step, M0 macrophages were polarized to M2c macrophages for 2 days in the presence of anti-LILRB2 antibodies or an isotype control ("during polarization"). After 2 days of M0-to-M2c macrophage polarization, the antibodies were washed out before 72 hours of co-culture with CD8+ T cells, and no treatment was given to M2c macrophages after the "post-polarization" step. The humanized variant B2H1-55 restored CD8+ T cell proliferation when added as a pre-regimen treatment, suggesting that the M2c-mediated immunosuppressive effect of these cells was alleviated. Representative data are shown in Figure 16.

[0335] Example 30: LILRB2 antibody attenuates M2c macrophage-mediated immunosuppression in M2c / CD4+ T cell cocultures Humanized variant B2H1-55 was tested for its ability to restore proliferation and IFN-γ and perforin secretion by CD4+ T cells cocultured with immunosuppressive M2c macrophages according to the methods described in Example 15. B2H1-55 and an IgG1 isotype control were tested at 5 or 10 μg / mL. Representative data are shown in Figures 17A and 17B. Humanized variant B2H1-55 alleviated M2c-mediated suppression, as indicated by rescue of CD4+ T cell proliferation (Figure 17A) and IFN-γ and perforin release (Figure 17B).

[0336] Example 31: Humanized variants rescue IFN-γ responses by exhausted T cells from M2c-mediated immune suppression Selected humanized variant antibodies were tested for their ability to rescue IFN-γ responses by exhausted T cells using the methods described in Example 16. Representative data from the IFN-γ assay are shown in Figure 18. Similar to the results shown with B2A-IgG1, treatment with the humanized variants attenuated M2c macrophage-mediated immunosuppression, as indicated by the secretion of IFN-γ by exhausted T cells.

[0337] Example 32: Humanized LILRB2 antibody in combination treatment with PD-1 antibody reduces M2c-mediated immune suppression of exhausted T cells To determine whether blocking LILRB2 enhances the efficacy of anti-PD-1 antibodies, the humanized variants were evaluated in combination with PD-1 antibodies in the M2c / exhausted T cell co-culture assay described above. The experiment followed the method of Example 16, with the addition of treatment of exhausted T cells with anti-PD-1 or isotype control.

[0338] The medium was removed from the wells of M2c macrophages and replaced with assay medium containing LILRB2 or isotype control antibody and incubated for 2 hours at 37°C, 5% CO2. The final concentrations of anti-LILRB2 antibody or IgG1 isotype were between 12 and 333 ng / mL. After the initial 2-hour pretreatment, 50 μL / well of anti-human CD3 clone OKT3 in assay medium (final assay concentration 0.250 μg / mL) was added, and the plates were incubated at 37°C for 30 minutes before the addition of exhausted T cells.

[0339] Exhausted T cells were preincubated with PD-1 (Pem-hIgG4 S228P) antibody (InvivoGen, No. hpd1pe-mab14) or an IgG4 isotype control for 2 hours, as shown in Figure 19. The final concentration of anti-PD-1 antibody was 1 μg / mL. 72 hours after T cell addition, supernatants were collected, and IFN-γ levels were determined by ELISA (R&D or Meso Scale). The results are shown in Figure 19. Blockade of LILRB2 enhanced the efficacy of anti-PD-1 antibody and rescued exhausted T cells from M2c-mediated immune suppression, as indicated by enhanced IFN-γ secretion.

[0340] Example 33: Humanized LILRB2 antibodies induce mild cytokine secretion in whole blood Immunomodulatory therapeutic antibodies carry the risk of cytokine release syndrome, a rapid systemic inflammatory response characterized by the secretion of proinflammatory cytokines by immune cells. Antibody-target binding can induce cytokines by directly activating lymphocytes and myeloid cells or by interacting with Fcγ receptors on myeloid and NK cells. A whole blood in vitro cytokine release assay is a standard assay used to assess the risk of therapeutic antibody-mediated cytokine release syndrome. We evaluated whether treatment with humanized LILRB2 variants induces the release of proinflammatory cytokines in whole blood from healthy study subjects.

[0341] The LILRB2 antibody and control antibody were diluted to a final concentration of 10x with 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 (Bloodworks NW, Seattle, Washington). To avoid cell lysis, whole blood (225 μL / well) was added to the LILRB2 antibody without mixing. The blood / antibody mixture was incubated at 37°C and 5% CO2 for 24–48 hours. After incubation, the plate was centrifuged at 350 × g for 5 minutes to pellet the cells. Plasma was collected from the surface of each well (65 μL) and immediately evaluated for IL-6, TNF-α, IFN-γ, and IL-1β cytokine secretion by MSD-ELISA according to the manufacturer's instructions (Meso Scale Discovery). The release of IL-1β, IL-6, IFN-γ, and TNF-α in response to treatment with the humanized variants was compared with cytokine induction by the corresponding human IgG1 isotype, an untreated control, and an anti-CD52 positive control antibody (alemtuzumab, CAS 216503-57-0, BOC Sciences No. B 0084-305393). Representative data from three healthy subjects are shown in Figure 20. In all subjects tested, treatment with the LILRB2 humanized variants did not induce 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 (Subject Y in Figure 20). In two subjects, minimal IL-6 secretion was observed after treatment with the humanized LILRB2 variants at the highest dose of 150 μg / mL, suggesting that these antibodies do not induce cytokine release syndrome in whole blood. Data for one representative subject (Subject X) is shown in Figure 20, demonstrating the occasional IL-6 response. Treatment with the humanized LILRB2 variants did not induce the release of IL-6, TNF-α, IFN-γ, or IL-1β cytokine secretion in whole blood in six of eight healthy subjects.The cytokine levels observed were similar to those induced by the comparative LILRB2 antibody B2Comp1 (data not shown) (IgG4 heavy chain (SEQ ID NO: 43), lambda light chain (SEQ ID NO: 44)) and lower than those induced by the CD52 antibody positive control.

[0342] Example 34: Humanized antibody variants do not induce ADCC in human monocytes or HEK293 cells expressing LILRB2 The humanized variants were tested for their ability to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against either human monocytes or HEK293T cells expressing human LILRB2. Cytotoxic activity was quantified by a widely used method of quantifying ADCC by flow cytometry (Yamashita M, 2016 Scientific Reports, 6:19772, DOI:10.1038 / srep19772). Monocytes from frozen PBMCs of healthy subjects were isolated as described in Example 9. NK cells were isolated from frozen PBMCs of healthy subjects using EasySep™ Human NK Cell Isolation Kit (STEMCELL Technologies, No. 17955) according to the manufacturer's instructions.

[0343] For NK cell and human monocyte ADCC assays, isolated monocytes (target cells) were added to 25 µL / well (10 4 Monocytes (1000kJ / well) were seeded into 96-well low-attachment plates and combined with 25 μL of 4X anti-LILRB2 antibody or isotype control diluted in medium at a final assay concentration of 10 μg / mL. Target cells and antibody were incubated for 1 hour at 37°C. Autologous NK cells (effector cells) were then added to the target cells (50 μL / well) at a final effector:target ratio of 8:1 in a final volume of 100 μL / well of effector + target + antibody. Cells were incubated for 4 hours at 37°C and 5% CO2.

[0344] For NK cell and HEK293T-LILRB2 ADCC assays, HEK293T cells expressing hLILRB2 (target cells) or mock-transfected cells (negative control target cells) were labeled with CellTrace™ Violet dye as described in Example 14. The K562 human CML line, which lacks HLA class I and does not express human LILRB2, was included as a positive control target for NK cell killing. K562 tumors were labeled with CellTrace™ Violet (CTV) in the same manner as HEK293T cells. Excess CTV was washed away with prewarmed assay medium, and the labeled HEK293T-LILRB2 or K562 were resuspended in assay medium and then dispensed into a 96-well low-attachment plate at 25 μL / well (10 3 Target cells were seeded with 100 μL of 4X anti-LILRB2 antibody or isotype control diluted in culture medium to a final assay concentration of 10 μg / mL (50 μL / well). Target cells and antibody were incubated for 1 hour at 37°C. Autologous NK cells (effector cells) were then added to the target cells (50 μL / well) at a final effector:target ratio of 8:1 in a final volume of 100 μL / well of effector + target + antibody. Cells were incubated for 4 hours at 37°C and 5% CO2.

[0345] Detection of NK cell killing

[0346] The cells were gently pipetted to detach them from the low-adhesion plate. They were transferred to a 96-well v-bottom plate and centrifuged at 300 × 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 each of human TruStain FcX™ (BioLegend, No. 422302), fluorine-labeled anti-human CD16 (BioLegend, No. 302046), and anti-human CD14 (BD Biosciences, No. 563561) antibodies (used to gate on monocytes) and incubated at 4°C for 20 minutes. The cells were rinsed with FACS buffer, centrifuged at 300 × g for 5 minutes, and the supernatant was discarded. The cells were resuspended in 50 μL / well of e780 viability dye (diluted 1:1000 in PBS) and incubated at RT for 15 minutes. Cells were rinsed, resuspended in FACS buffer, and then analyzed by flow cytometry using a BD FACSymphony™ cytometer and FlowJO software. NK killing was analyzed by gating and reporting the frequency of CD14+e780 positive cells.

[0347] Similar to the NK / monocyte assay, HEK293T or K562 cells were gently pipetted into the assay plate and transferred to a 96-well v-bottom plate for flow cytometry staining. Cells were resuspended in 50 μL / well of e780 viability dye (diluted 1:1000 in PBS) and incubated at RT for 15 minutes. Cells were rinsed with 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.

[0348] Combined data from 2 to 10 healthy subjects for the NK / monocyte ADCC assay are shown in Figure 21A, in which the 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 2 healthy subjects. Three representative subjects are shown in Figure 21B for the NK / HEK293T-LILRB2 assay. Compared to the IgG1 isotype control, the parental B2A-IgG1 and humanized variants do not induce ADCC of human monocytes but induce killing of HEK293T-LILRB2 cells (Figure 21).

[0349] Example 35: Pharmacokinetic profiling of humanized antibody variants in humanized FcRn mice The humanized variants were evaluated for their pharmacokinetic profiles in humanized FcRn mice according to the methods described in Example 17. Mice were assigned to three humanized LILRB2 antibody treatment groups: B2H1-55, B2H1-35, and B2H1-52, with 12 mice per group. The 12 mice from each treatment group were divided into three blood collection groups with four mice per group and administered a single intraperitoneal (IP) dose of 10 mg / kg of antibody. Blood was collected from each group at three alternating time points as follows: Group 1: 0.25, 4, and 96 hours; Group 2: 1, 24, and 168 hours; and Group 3: 2, 48, and 240 hours. Serum collection and PK analysis were described in Example 17. The PK profiles of variants B2H1-55, B2H1-35, and B2H1-52 are shown in Table 9 and Figure 22. The humanized anti-LILRB2 variants exhibited half-lives in the range of 5 to 10 days in humanized FcRn mice, typical of IgG1 antibodies.

[0350] [Table 9]

[0351] All documents mentioned in this application are incorporated herein by reference in their entirety.

Claims

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

21.

2. 2. The antibody product of claim 1, comprising CDR-H1 set forth in SEQ ID NO: 16, CDR-H2 set forth in SEQ ID NO: 17, CDR-H3 set forth in SEQ ID NO: 18, CDR-L1 set forth in SEQ ID NO: 19, CDR-L2 set forth in SEQ ID NO: 20, and CDR-L3 set forth in SEQ ID NO:

21.

3. 2. The antibody product of claim 1, comprising CDR-H1 set forth in SEQ ID NO:22, CDR-H2 set forth in SEQ ID NO:23, CDR-H3 set forth in SEQ ID NO:24, CDR-L1 set forth in SEQ ID NO:25, CDR-L2 set forth in SEQ ID NO:26, and CDR-L3 set forth in SEQ ID NO:

21.

4. (a) an amino acid sequence at least 80% identical to SEQ ID NO: 1, 6, 7, 8, 9, or 10; or (b) an amino acid sequence represented by SEQ ID NO: 1, 6, 7, 8, 9, or 10 The antibody product of any one of claims 1 to 3, comprising a heavy chain variable region comprising:

5. (a) an amino acid sequence at least 80% identical to SEQ ID NO: 2, 11, 12, 13, 14, or 15; or (b) an amino acid sequence represented by SEQ ID NO: 2, 11, 12, 13, 14, or 15 The antibody product of any one of claims 1 to 5, comprising a light chain variable region comprising:

6. (a) a heavy chain variable region comprising SEQ ID NO: 1; and a light chain variable region of SEQ ID NO: 2; (b) a heavy chain variable region of 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 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; 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; 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. The antibody product of any one of claims 1 to 5, comprising:

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

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

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

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

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

13. (a) an amino acid sequence 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) a heavy chain amino acid sequence comprising the amino acid sequence set forth 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 of any one of claims 1 to 12, comprising:

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

15. (a) an amino acid sequence 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) an amino acid sequence represented by 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 15. The antibody product of any one of claims 1 to 11 and 14, comprising a heavy chain amino acid sequence comprising:

16. An antibody product that binds to human LILRB2, (a) an IgG1 heavy chain having an amino acid sequence comprising SEQ ID NO:1; and a kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa 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 kappa light chain having an amino acid sequence comprising SEQ ID NO:

15. An antibody product comprising:

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

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

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

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

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

22. 21. The antibody product of any one of claims 1 to 20, which binds to human immunosuppressive myeloid cells.

23. 22. The antibody product of claim 21, wherein the immunosuppressive myeloid cells are in a tumor microenvironment.

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

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

26. K between 0.5 nM and 500 nM Ｄ 25. The antibody product of any one of claims 1 to 24, which specifically binds to human M2c macrophages at 25°C.

27. 26. The antibody product of any one of claims 1 to 25, which is a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single-chain antibody.

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

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

30. 29. The antibody product of claim 28, which is bound by CD16 (FcγRIIIa), CD32 (FcγRII) or CD64 (FcγRI) expressed on immunosuppressive macrophages or other myeloid cells.

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

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

33. 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 of any one of claims 1 to 32.

34. The antibody product binds to macrophages, and the binding of the antibody product to macrophages is (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 macrophage polarization into immunosuppressive macrophages; and (d) enhancing the innate anti-tumor response.

35. 34. The method of claim 33, wherein the activation of CD4+ T cells, CD8+ T cells, NK cells, or any combination thereof is measured as enhanced levels of IFN-γ, TNF-α, or perforin, or any combination thereof, or decreased levels of IL-10 release.

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

37. Binding of the antibody product to macrophages is (a) internalization of the antibody product by the macrophage; (b) secretion of TNFα, IL-6, perforin, or any combination thereof; (c) reduced 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) promoting tumor cell killing in the tumor microenvironment.

38. 38. The method of claim 37, wherein the coupling results in two or more of (a)-(f); three or more of (a)-(f); four or more of (a)-(f); five or more of (a)-(f); or all of (a)-(f).

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

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

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

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

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

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

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

46. 39. The method of any one of claims 33 to 38, wherein binding of the antibody product to macrophages reduces inhibition of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment.

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

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

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

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

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

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

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

54. 54. The method of claim 53, wherein said immunosuppression of T cells comprises immunosuppression mediated through interaction of said T cells with myeloid cells that express PD-L1.

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

56. 56. An article of manufacture comprising the composition of 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 manufacture of a medicament for the treatment of 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. 59. An expression vector comprising the nucleic acid of claim 58.

61. 60. An expression vector comprising the nucleic acid of claim 59.

62. 60. An expression vector comprising the nucleic acid of claims 58 and 59.

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

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

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

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

67. An antibody product according to any one of claims 1 to 32 or a pharmaceutical composition according to claim 55 for use in treating a subject with a cancer that expresses LILRB2.

68. 33. Use of the antibody product of any one of claims 1 to 32 in the manufacture of a medicament for treating a cancer that expresses LILRB2.

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

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

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

72. 71. The method of claim 70, wherein the subject has a tumor having a tumor microenvironment, and wherein an anti-tumor immune response within the tumor microenvironment is increased.

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

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

75. 75. The method of 74, wherein said administering said antibody product comprises administering said antibody product in an amount effective to mediate killing of cells of said cancer via antibody-dependent cellular cytotoxicity.

76. 75. The method of claim 74, wherein said administering said antibody product comprises administering said antibody product in an amount effective to reduce LILRB2-mediated suppression of T cells in said subject.

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

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

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

80. 80. The antibody product of any one of claims 79, comprising a therapeutic or cytotoxic agent.

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

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

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

85. 85. The method of claim 84, wherein the subject has a cancer comprising cells that express or overexpress a ligand of LILRB2.

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

87. 87. The method of any one of claims 84-86, wherein the cancer or tumor comprises macrophages that express LILRB2.

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

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

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

21.

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

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