LILRB2 / 1 antibody product and method

Abstract

This disclosure relates to a LILRB2 / 1 antibody product and its use. The antibody product specifically binds to LILRB1 and LILRB2 on cells such as bone marrow cells, lymphocytes, or cancer cells. The antibody product can be used in methods of treating diseases, such as cancer immunotherapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 459,096 filed on 13 April 2023 and U.S. Provisional Application No. 63 / 527,282 filed on 17 July 2023, both of which are incorporated herein by reference in their entirety.

[0002] Inclusion by referencing the sequence list This application includes, as another part of the disclosure, a computer-readable sequence listing (filename: 59003_SeqListing.xml, 54,121 bytes - an XML file created on 8 April 2024) which is incorporated herein in its entirety by reference.

[0003] This disclosure relates to LILRB2 / 1 antibody products and related methods. The products disclosed herein bind to certain LILRBs expressed in human 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] Checkpoint inhibitor (CPI) therapy targets immune checkpoints to manipulate and / or modulate the immune system, including both innate and adaptive immune responses. Recent developments of checkpoint inhibitor antibodies have revolutionized the way malignant solid tumors are treated. Despite the success of immunotherapy, CPIs have failed to improve outcomes in tumors that are not infiltrated by immune cells, so-called "cold" tumors. One reason for the failure of checkpoint inhibitors in cold tumors is the immunosuppressive nature of the tumor microenvironment (TME) [Heinhuis et al., JAMA Oncol. 2020 6(1):100-7]. Immunosuppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), are important components of the tumor mesenteric vascular effusion (TME) and contribute to immune evasion by many solid tumors [Fleming et al., Front Immunol. 2018 9:398 (doi:10.3389 / fimmu.2018.00398), Garvin et al., J Cancer Res Clin Oncol. 2018 144(7):1253-63]. High levels of tumor invasion by MDSCs and TAMs generally predict an unfavorable prognosis in solid tumors [Ai et al., BMC Cancer. 2018 18(1):1220; Chen et al., J Biomed Sci. 2019 26(1):78].

[0005] The LILR family comprises two broad subfamilies, LILRA and LILRB, each containing six stimulative and four inhibitory members. LILRB subfamily members include two or four extracellular immunoglobulin domains, transmembrane domains, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRs can exert inhibitory or stimulative immunomodulatory effects on a wide range of immune cells. See, for example, Lewis Marffy, Front Immunol. 2020 11:857 and De Louche and Roghanian, JCI Insight. 2022 7(2):e151553, doi:10.1172 / jci.insight.151553.

[0006] Leukocyte immunoglobulin-like receptor B1 (LILRB1, also known as ILT2, LIR1, PIRB, MIR7, and CD85j) and leukocyte immunoglobulin-like receptor B2 (LILRB2, also known as ILT4, LIR2, MIR10, and CD85d) are ITIM-containing immunosuppressive cell surface proteins expressed on immune cells and are known to inhibit the immune response. This inhibitory function is thought to regulate inflammatory responses and cytotoxicity, and may contribute to limiting the autoreactivity of the immune system. LILRB2 is mainly expressed on myeloid cells (monocytes, macrophages, dendritic cells, and granulocytes), while LILRB1 is more broadly expressed, not only on lymphocytes including B cells and subsets of CD8 T cells and NK cells, but also in the bone marrow. In TMEs, LILRB1 and LILRB2 are found in myeloid suppressor cells (MDSCs) and tumor-supporting tumor-associated macrophages (TAMs) [Chen et al., J Clin Invest. 2018 128(12):5647-62]. The presence of LILRB1 in additional immune cell types may confer greater functional differences between the two receptors, as LILRB1 expression has been shown to be increased in NK cells of cancer patients and correlate with resistance to NK cell cytotoxicity [Zhang, Mol Med Rep. 2012 5(4):910-6, Roberti et al., Eur J Immunol. 2015 45:1560-9, Chen et al. J Immunother Cancer. 2020 8(2):e000515]. NK cells play a crucial role in the cancer immune response via ADCC-mediated killing of cancer cells. Blocking LILRB1 may help to relieve tumor-induced immunosuppression of NK cell activity and enhance the antitumor response.

[0007] Ligands for LILRB2 include, in particular, HLA-A, -B, -C, -F, and -G, HLA-B27, CD1d, and CD1c, as well as angiopoietin-like proteins 2 and 5. Ligands for LILRB1 include, in particular, HLA-AA, B, C, F, and G, as well as HLA-B27 [Burshtyn and Morcos, J Immunol. 2016 196(3):947-55]. Trans or cis interactions between LILRB1 or LILRB2 and their ligands mediate immunosuppression by myeloid cells and promote tumor immune evasion in TMEs. The role of LILRB1 and LILRB2 interactions with non-classical HLA-Gs has been extensively investigated because altered HLA-G expression has been reported in tumor contexts and may be a marker of poor prognosis in cancer [Carosella et al., Trends Cancer. 2021 7(5):389-92, Chen et al., Int Immunopharmacol. 2022 109:108798, Lin and Yan, Mol Med. 2015 21(1):782-91]. See also PCT Publication No. 2009 / 100135A2.

[0008] LILRB1 is a type I transmembrane glycoprotein with four extracellular immunoglobulin-like (Ig-like) domains and four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasm for signal transduction [Cosman et al., Immunity. 1997 7:273-82]. LILRB1 is polymorphic and has various transcriptional variants expressed in the human population [Liu et al., Immunogenetics. 2022 74(6):513-25]. Two distal membrane Ig domains (D1-D2) are involved in interacting with HLA class I; the D1 domain engages with the α3 region of the HLA class I heavy chain, and the D2 domain interacts with beta-2 microglobulin (β2m) [Willcox et al., Nat Immunol. 2003 4(9):913-9]. Because the α3 and β2m regions of HLA class I antigens are relatively conserved, LILRB1 binds to a wide range of HLA class I antigens [Willcox et al., 2003, ibid.; Jones et al., J Immunol. 2011 186(5):2990-7; Liu et al., 2022, ibid.]. LILRB1 allotypes with amino acid changes in the D1-D2 region can be classified into nine variants, with variants 1, 2, and 3 representing the most common variants, and the nine variants showing the frequency distribution within the population [Liu et al., 2022, ibid.]. The effects of amino acid changes in the D1-D2 domains of LILRB1 on binding to selected HLA class I antigens have been investigated [Yu et al., J Clin Invest. 2018 128(4):1523-37, Kuroki et al., Hum Mol Genet. 2005 14(16):2469-80]. The four amino acid changes most tested in this region are L68P, A93T, I142T, and S155I, where the numbers correspond to the full-length LILRB1 protein sequence. LILRB1 D1-D2 variants bind to HLA class I antigens with different avidities [Liu et al., 2022, ibid.].These differences in binding avidity may be related to the diversity of immune responses between individuals, but how these polymorphisms affect LILRB1 avidity and functionality is not yet fully understood [Liu 2022, ibid., Wang et al., Cell Mol Immunol. 2020 17:966-75 (2020)].

[0009] Combinations of anti-LILRB2 antibodies with anti-PD-1 treatment (TGI) in humanized tumor models, as well as combinations of LILRB1 blocking antibodies with anti-PD-1 or anti-EGFR antibodies, have been investigated (Chen 2018, Mandel 2022). The effects of double-blocking LILRB1 and LILRB2 have also been investigated in vitro in tumor cells and in [Hu et al., Cancer Res. 2022 82(12_Suppl):3433]. Antibodies targeting and antagonizing LILRB2, such as MK-4830 (IgG4) (Agenus and Merck) and JTX-8064 (IgG4) (Jounce Therapeutics), are currently being evaluated in clinical trials for cancer treatment. See also Papadopoulos et al. ASCO abstract 2021 J Clin Oncol 39(15). Immune-Onc PCT Publication No. 2022 / 087188A1, Five Prime Publication No. 2020 / 014132A2, Mt.SINAI Publication No. 2020_061059A1, JOUNCE Publication No. 2019 / 126514A2, and JOUNCE Publication No. 2016 / 111947A2 disclose LILRB2 antibodies.

[0010] Merck PCT Publication 2021 / 138079A1 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. CTX-585 (Celldex Therapeutics), a LILRB2 / PD-1 bispecific antibody, appears to be in preclinical development in the context of solid tumors. See also PCT Publication 2022 / 217019A1.

[0011] Antibodies that target and antagonize both LILRB2 and LILRB1 are also under development, such as NGM707 (NGM Biopharmaceuticals). See PCT Publication No. 2021 / 222544A1. PCT Publications No. 2022 / 034524A2 (Biond), No. 2022 / 026360A2 (Univ.Texas), and LG Chemical Publication No. 2022 / 025585A1 disclose LILRB1 antibodies.

[0012] LILRB antibody products and methods for their therapeutic use are still needed in this field. [Overview of the project]

[0013] This disclosure aims to demonstrate that different patterns of expression and function of LILRB1 and LILRB2 enable complementary targeted approaches for cancer immunotherapy. Dual blockade of LILRB1 and LILRB2 receptors with a single antibody restoring both innate and adaptive immune responses is intended herein to enhance the efficacy of checkpoint inhibitors. This disclosure provides LILRB2 / 1 antibody products (sometimes referred herein as “anti-LILRB2 / 1 antibody products”) and compositions comprising such antibody products, as well as methods for their manufacture and use in the treatment of diseases, including cancer. Therapeutic modalities that block LILRB1 and LILRB2 binding to HLA ligands in the TME are provided to enhance the efficacy of T-cell checkpoint inhibitors.

[0014] The LILRB2 / 1 antibody products provided herein may be intact antibodies or may contain immunologically functional antibody fragments. Therefore, LILRB2 / 1 antibody products include antibodies having naturally occurring or recombinant structures, as well as other polypeptides such as antibody fragments having antigen-binding domains. Nucleic acids, molecules, vectors, and host cells useful for the production of antibody products are also provided. Antibody products and their compositions can be used to prevent or treat a variety of different disease conditions, including but not limited to the prevention or treatment of disease conditions such as cancer. The LILRB2 / 1 antibodies provided herein have useful half-lives and exhibit antitumor activity in humanized mouse models. The humanized LILRB2 / 1 antibodies provided herein restore effector function of 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 / 1 antibody products provided herein are expected to bind to LILRB1 and LILRB2 at epitopes different from those currently conjugated by other antibodies, either in development or clinically. In preclinical studies, the antibody products enhanced LPS-induced IFN-γ production by peripheral blood mononuclear cells (PBMCs), reduced IL-10 release, mitigated immunosuppression of tumor-promoting macrophages, and induced T cell activation (indicated by enhanced proliferation, IFN-gamma secretion, and perforin release). Representative antibody products have shown the ability to restore the ability of exhausted T cells to secrete IFN-γ in the presence of tumor-promoting macrophages, and have also significantly enhanced pembrolizumab activity in combination studies. Importantly, chimeric antibody products showed superior antitumor activity compared to comparators in humanized mouse tumor models, accompanied by significant tumor growth inhibition and tumor regression.

[0016] LILRB2 / 1 antibody products did not cross-react with non-human primate LILRB2, showed minimal binding to cynomolgus monkey LILRB1, and did not cross-react with other inhibitory or activating LILR family members. Treatment with representative antibody products did not trigger inflammatory cytokine release in human whole blood.

[0017] While we do not wish to be bound by any particular theory, it is intended herein that the immunostimulatory activity of the provided antibody appears to arise from two distinct but coordinated mechanisms: the association of the variable regions of LILRB1 / LILRB2, accompanied by the heavy chain association of the Fc receptor.

[0018] On the other hand, LILRB1 and LILRB2 expressed in bone marrow cells are thought to negatively regulate antitumor immunity through binding to HLA-G on tumor cells and HLA class 1 on bone marrow cells. The binding of LILRB1 and LILRB2 to these ligands involves Ca2 crossing the cell membrane of bone marrow cells. + By inhibiting signal transduction, recruiting Src homologous domain 2 containing the protein tyrosine phosphatase-1 (SHP1) and SHP2 phosphatases within the bone marrow cell membrane, and producing myeloid cytokines that can prevent checkpoint inhibitor (CPI) therapy from enhancing the antitumor T cell response, these mechanisms promote immunosuppressive myeloid cells in the tumor microenvironment. Individually or collectively, these phenomena can lead to a reduction in cancer cell death. LILRB1 and LILRB2 are also thought to further inhibit tumor cell killing by competing with cytotoxic T lymphocytes for binding to HLA class I.

[0019] On the other hand, activating Fc receptors (FcγRIA, FcγRIIA, and FcγRIIIA) on bone marrow cells is 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 immune receptor tyrosine-based activation motifs through SRC family kinases and spleen tyrosine kinases, resulting in transcriptional activation of several pro-inflammatory cytokines and chemokines that drive cell recruitment, migration, differentiation, and survival.

[0020] The antibodies disclosed herein appear to enable the coupling of LILRB2 / 1 antagonism and Fc receptor-mediated activation. The antibodies bind to a specific epitope on LILRB2, inhibiting not only myeloid-myelocyte interaction with tumor cell HLA-G but also cis-interaction with HLA class I, thereby inactivating both inhibitory signals on myeloid cells. This action promotes polarization of tumor-infiltrating myeloid cells into an inflammatory phenotype, while simultaneously releasing MHC class I on these cells to engage receptors necessary for optimal activation of cytotoxic T lymphocytes. Furthermore, the antibodies can enhance antitumor immunity by providing an immunostimulatory signal through engagement of FcγRIIIA to activation. This dual or co-engagement mechanism provides these antibodies with novel attributes for targeting myeloid cells to reverse CPI resistance, enhance tumor cell killing, and improve patient outcomes.

[0021] This disclosure provides an antibody product comprising a variable region that specifically binds to human LILRB2 and LILRB1, the variable region comprising, when specified using the IMGT CDR definition, a first domain comprising CDR-H1 shown in SEQ ID NO: 16, CDR-H2 shown in SEQ ID NO: 17, and CDR-H3 shown in SEQ ID NO: 18, and a second domain comprising CDR-L1 shown in SEQ ID NO: 19, CDR-L2 shown in SEQ ID NO: 20, and CDR-L3 shown in SEQ ID NO: 21, or, when specified using the Kabat CDR definition, a first domain comprising CDR-H1 shown in SEQ ID NO: 22, CDR-H2 shown in SEQ ID NO: 23, and CDR-H3 shown in SEQ ID NO: 24, and a second domain comprising CDR-L1 shown in SEQ ID NO: 25, CDR-L2 shown in SEQ ID NO: 26, and CDR-L3 shown in SEQ ID NO: 27.

[0022] The variable region may include a heavy chain variable domain that has an amino acid sequence at least 80% identical to that of SEQ ID NOs: 1, 6, 7, 8, 9, or 10, or that includes the amino acid sequence shown in SEQ ID NOs: 1, 6, 7, 8, 9, or 10.

[0023] The variable region may include a light chain variable domain containing an amino acid sequence that is at least 80% identical to that of SEQ ID NOs. 2, 11, 12, 13, 14, or 15, or an amino acid sequence represented by SEQ ID NOs. 2, 11, 12, 13, 14, or 15.

[0024] The variable regions include: a heavy chain variable domain containing SEQ ID NO: 1, and a light chain variable domain SEQ ID NO: 2; a heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 11; a heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 12; a heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 13, a heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 14; a heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 15; a heavy chain variable domain containing SEQ ID NO: 7, and a light chain variable domain containing SEQ ID NO: 11; a heavy chain variable domain containing SEQ ID NO: 7, and a light chain variable domain containing SEQ ID NO: 12; a heavy chain variable domain containing SEQ ID NO: 7, and a light chain variable domain containing SEQ ID NO: 13; a heavy chain variable domain containing SEQ ID NO: 7, and a light chain variable domain containing SEQ ID NO: 14; a heavy chain variable domain containing SEQ ID NO: 7, and a light chain variable domain containing SEQ ID NO: 15; a heavy chain variable domain containing SEQ ID NO: 8, and a light chain variable domain containing SEQ ID NO: 11; a heavy chain variable domain containing SEQ ID NO: 8, and a light chain variable domain containing SEQ ID NO: 12; a heavy chain variable domain containing SEQ ID NO: 8 A variable domain and a light chain variable domain including SEQ ID NO: 13; a heavy chain variable domain including SEQ ID NO: 8 and a light chain variable domain including SEQ ID NO: 14; a heavy chain variable domain including SEQ ID NO: 8 and a light chain variable domain including SEQ ID NO: 15; a heavy chain variable domain including SEQ ID NO: 9 and a light chain variable domain including SEQ ID NO: 11; a heavy chain variable domain including SEQ ID NO: 9 and a light chain variable domain including SEQ ID NO: 12; a heavy chain variable domain including SEQ ID NO: 9 and a light chain variable domain including SEQ ID NO: 13; a heavy chain variable domain including SEQ ID NO: 9 and a light chain variable domain including SEQ ID NO: 14; a heavy chain variable domain including SEQ ID NO: 9 and a light chain variable domain including SEQ ID NO: 15; a heavy chain variable domain including SEQ ID NO: 10 and a light chain variable domain including SEQ ID NO: 11; a heavy chain variable domain including SEQ ID NO: 10 and a light chain variable domain including SEQ ID NO: 12; a heavy chain variable domain including SEQ ID NO: 10 and a light chain variable domain including SEQ ID NO: 13; a heavy chain variable domain including SEQ ID NO: 10 and a light chain variable domain including SEQ ID NO: 14; or a heavy chain variable domain including SEQ ID NO: 10 and a light chain variable domain including SEQ ID NO: 15.

[0025] The antibody products provided herein may include heavy chains comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH).

[0026] The antibody products provided herein may include light chains containing a variable light chain domain (VL) and a human constant light chain domain (CL).

[0027] The antibody products provided herein may include 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).

[0028] The heavy chain constant domain of the antibody product provided herein may include an IgA, IgD, IgE, IgG, or IgM heavy chain constant domain. The heavy chain constant domain may include an IgG1 constant domain, an IgG2 constant domain, or an IgG4 constant domain. The heavy chain constant domain may be an IgG1 constant domain. The heavy chain amino acid sequence may be at least 80% identical to the amino acid sequence of 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 the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. The heavy chain constant domain may be an IgG4 constant domain. The heavy chain amino acid sequence may be at least 80% identical to the amino acid sequence of 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 the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37.

[0029] The antibody products provided herein may include a light chain comprising a variable light chain region (VL) and a human light chain constant region (CL).

[0030] The human light chain constant region (CL) may include the kappa domain or a fragment thereof. The light chain amino acid sequence may be at least 80% identical to the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42, or may include the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42.

[0031] This disclosure provides an antibody product that binds to human LILRB2, the antibody product comprising: an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 1, and a kappa light chain having an amino acid sequence including SEQ ID NO: 2; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 11; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 12; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 13; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 14; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 15; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 7, and a kappa light chain having an amino acid sequence including SEQ ID NO: 11; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 7, and a kappa light chain having an amino acid sequence including SEQ ID NO: 12; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 7, and SEQ ID NO: 13 Kappa light chain having an amino acid sequence including; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 7, and Kappa light chain having an amino acid sequence including SEQ ID NO: 14; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 7, and Kappa light chain having an amino acid sequence including SEQ ID NO: 15; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 8, and Kappa light chain having an amino acid sequence including SEQ ID NO: 11; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 8, and Kappa light chain having an amino acid sequence including SEQ ID NO: 12; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 8, and Kappa light chain having an amino acid sequence including SEQ ID NO: 13; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 8, and Kappa light chain having an amino acid sequence including SEQ ID NO: 14; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 8, and Kappa light chain having an amino acid sequence including SEQ ID NO: 15; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 9, and Kappa light chain having an amino acid sequence including SEQ ID NO: 11; IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 9, and Kappa light chain having an amino acid sequence including SEQ ID NO: 12;A kappa light chain having an amino acid sequence including SEQ ID NO: 9; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 13; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 9; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 14; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 9; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 15; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 10; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 11; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 10; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 12; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 10; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 13; an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 10; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 14; or an IgG1 heavy chain having an amino acid sequence including SEQ ID NO: 10; an IgG1 light chain having an amino acid sequence including SEQ ID NO: 15.

[0032] This disclosure provides an antibody product that binds to human LILRB2, the antibody product comprising: an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 1, and a kappa light chain having an amino acid sequence including SEQ ID NO: 2; an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 11; an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 12; an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 13; an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 14; an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 6, and a kappa light chain having an amino acid sequence including SEQ ID NO: 15; an IgG quadrilateral having an amino acid sequence including SEQ ID NO: 7, and a kappa light chain having an amino acid sequence including SEQ ID NO: 11; an amino acid sequence including SEQ ID NO: 7 A kappa light chain having an amino acid sequence including the sequence of IgG quadrilateral and the sequence of amino acids including SEQ ID NO: 12; an IgG quadrilateral having an amino acid sequence including the sequence of SEQ ID NO: 7 and the sequence of amino acids including the sequence of SEQ ID NO: 13; an IgG quadrilateral having an amino acid sequence including the sequence of amino acids including the sequence of SEQ ID NO: 7 and the sequence of amino acids including the sequence of amino acids including the sequence of SEQ ID NO: 14; an IgG quadrilateral having an amino acid sequence including the sequence of amino acids including the sequence of SEQ ID NO: 7 and the sequence of amino acids including the sequence of SEQ ID NO: 15; an IgG quadrilateral having an amino acid sequence including the sequence of amino acids including the sequence of SEQ ID NO: 8 and the sequence of amino acids including the sequence of SEQ ID NO: 11; an IgG quadrilateral having an amino acid sequence including the sequence of amino acids including the sequence of SEQ ID NO: 8 and the sequence of amino acids including the sequence of SEQ ID NO: 12; an IgG quadrilateral having an amino acid sequence including the sequence of amino acids including the sequence of SEQ ID NO: 8 and the sequence of amino acids including the sequence of SEQ ID NO: 13; or an IgG quadrilateral having an amino acid sequence including the sequence of amino acids including the sequence of SEQ ID NO: 8 and the sequence of amino acids including the sequence of SEQ ID NO: 14.

[0033] The antibody products provided herein may be monoclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, or single-chain antibodies.

[0034] The antibody products provided herein may be monospecific, bispecific, triplicate, or multispecific antibodies.

[0035] The antibody products provided herein can specifically bind to human LILRB2 expressed by bone marrow cells or cancer cells. The antibody products have a K content of less than 1 pM to about 1000 pM. D It can specifically bind to human LILRB2. The antibody product can specifically bind to human LILRB1 expressed by bone marrow cells, lymphocytes, or cancer cells. The antibody product has a K content of approximately 0.1 pM to approximately 300 nM. D It can specifically bind to human LILRB1.

[0036] The antibody products provided herein can bind to human immunosuppressive myeloid cells. The antibody products can bind to human immunosuppressive myeloid cells in the tumor microenvironment. Immunosuppressive myeloid cells may be macrophages, myeloid dendritic cells, or myeloid-derived suppressor cells. Immunosuppressive myeloid cells may be M2a, M2b, M2c, or M2d macrophages. The antibody products have a K content of 0.05 nM to 50 nM. D It can specifically bind to human M2c macrophages. Binding of the antibody product to immunosuppressive myeloid cells may or may not be cytotoxic to the cells, depending on the circumstances.

[0037] The antibody products provided herein can bind to human lymphocytes. The antibody products can bind to human lymphocytes in the tumor microenvironment. The lymphocytes may be human B lymphocytes, human T lymphocytes, or NK cells. The antibody products have a K content of 0.001 nM to 300 nM. D It can specifically bind to human lymphocytes. The binding of the antibody product to lymphocytes may or may not be cytotoxic to macrophages, depending on the circumstances.

[0038] The antibody products provided herein can inhibit the interaction between LILRB1 or LILRB2 expressed in bone marrow cells and HLA-G expressed in cells in the tumor microenvironment, such as tumor cells, cancer-associated fibroblasts, other immunosuppressive bone marrow cells, or lymphocytes.

[0039] The antibody products provided herein can inhibit the immunosuppressive interaction between LILRB1 expressed in lymphocytes and HLA-G expressed in tumor cells, cancer-associated fibroblasts, and immunosuppressive myeloid cells or lymphocytes.

[0040] The antibody products provided herein can inhibit the immunosuppressive interaction between LILRB1 or LILRB2 expressed in bone marrow cells and HLA class I expressed in the same bone marrow cells, or in tumor cells, cancer-associated fibroblasts, other immunosuppressive bone marrow cells, or lymphocytes.

[0041] The antibody products provided herein can inhibit the immunosuppressive interaction between LILRB1 expressed in lymphocytes and HLA-class I expressed in the same lymphocytes, or in tumor cells, cancer-associated fibroblasts, other immunosuppressive myeloid cells, or lymphocytes.

[0042] The antibody products provided herein can be bound by Fc receptors expressed on immunosuppressive macrophages or other myeloid cells or lymphocytes. The antibody products can be bound by CD16(FcγRIIIa), CD32(FcγRII), or CD64(FcγRI) expressed on immunosuppressive macrophages or other myeloid cells or lymphocytes. The antibody products can bind to LILRB1 or LILRB2 expressed on myeloid cells and can be bound by CD16(FcγRIIIa), CD32(FcγRII), or CD64(FcγRI) expressed on the same myeloid cells. The antibody products can bind to LILRB1 expressed on lymphocytes and can be bound by CD16(FcγRIIIa), CD32(FcγRII), or CD64(FcγRI) expressed on the same lymphocytes. The antibody product can bind to LILRB1 or LILRB2 expressed in the first cell, and can also be bound by CD16 (FcγRIIIa), CD32 (FcγRII), or CD64 (FcγRI) expressed in the second cell.

[0043] This disclosure provides a method for subjects requiring cancer immunotherapy, where cancer is associated with the presence of immunosuppressive macrophages, and the method comprises administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody product provided herein. The method may include administering an amount of the pharmaceutical composition effective to inhibit the immunosuppressive interaction between LILRB1 or LILRB2 expressed in myeloid cells and HLA-G or HLA class I expressed in myeloid cells, or in tumor cells, cancer-associated fibroblasts, other immunosuppressive myeloid cells, or lymphocytes. The method may also include administering an amount of the pharmaceutical composition effective to inhibit the immunosuppressive interaction between LILRB1 expressed in lymphocytes and HLA-G or HLA-class I expressed in lymphocytes, or in tumor cells, cancer-associated fibroblasts, other immunosuppressive myeloid cells, or lymphocytes. The antibody product can bind to macrophages, and the binding of the antibody product to macrophages has the following effect: CD4 + T cells, CD8 +Promote the activation of T cells, NK cells, or any combination thereof; CD4 + T cells, CD8 + Promote the proliferation of T cells, NK cells, or any combination thereof; prevent the polarization of macrophages to an immunosuppressive phenotype; and enhance the innate antitumor response, and can result in at least one of these. CD4 + T cells, CD8 + The activation of T cells, NK cells, or any combination thereof can be measured as enhanced levels of IFN-γ, TNF-α, or perforin, or any combination thereof. The binding of the antibody product to macrophages may or may not be cytotoxic to macrophages as required. The binding of the antibody product to macrophages includes the internalization of the antibody product by macrophages; the secretion of TNFα, IL-6, perforin, or any combination thereof; the reduced secretion of IL-10; CD4 + T cells, CD8 + The activation of T cells, NK cells, or any combination thereof; CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof; or the promotion of tumor cell killing in the tumor microenvironment, can be achieved.

[0044] In the cancer immunotherapy methods provided herein, the binding of the antibody product to macrophages can increase the immunostimulatory activity in the tumor microenvironment.

[0045] In the cancer immunotherapy methods provided herein, the binding of the antibody product to macrophages can reduce the immunosuppressive activity of macrophages.

[0046] In the cancer immunotherapy methods provided herein, the binding of the antibody product to macrophages can reduce the tumor-promoting activity of macrophages.

[0047] In the cancer immunotherapy methods provided herein, the binding of the antibody product to macrophages is CD4+ T cell activation, CD4 + T cell proliferation, or CD4 + It can promote both the activation and proliferation of T cells.

[0048] In the cancer immunotherapy methods provided herein, the binding of the antibody product to macrophages is performed on CD8 + T cell activation, CD8 + T cell proliferation, or CD8 + It can promote both the activation and proliferation of T cells.

[0049] In the cancer immunotherapy methods provided herein, the binding of antibody products to macrophages can promote cytotoxic lymphocyte-mediated killing of cancer cells.

[0050] In the cancer immunotherapy methods provided herein, the binding of antibody products to macrophages can promote NK cell-mediated tumor cell killing.

[0051] In the cancer immunotherapy method provided herein, the binding of an antibody product can reduce the suppression of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment.

[0052] In the cancer immunotherapy method provided herein, in which an antibody product binds to lymphocytes, the binding of the antibody product to lymphocytes promotes NK cell-mediated killing of tumor cells, and CD8 + Enhances IFN-γ secretion by T cells and its cytotoxic activity, or enhances NK cells and CD8 + It can either activate T cells or block LILRB1-mediated suppression of lymphocytes.

[0053] In the cancer immunotherapy methods provided herein, the cancer may be, for example, a sarcoma, carcinoma, or hematological cancer. The cancer may be glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, pancreatic adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell carcinoma, gastric cancer, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid cancer, cutaneous squamous cell carcinoma, or ovarian cancer. The cancer may be a B-cell malignancy selected from B-cell lymphoma (diffuse large B-cell lymphoma, small lymphocytic lymphoma or chronic lymphocytic leukemia, non-Hodgkin lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, etc.), B-cell leukemia, acute myeloid leukemia, and multiple myeloma.

[0054] In the cancer immunotherapy method provided herein, cancer cells express LILRB2 or LILRB1. Cancer cells can overexpress LILRB2 or LILRB1.

[0055] A cancer immunotherapy method provided herein may further include administering an effective amount of an anticancer therapeutic product to a subject. The anticancer therapeutic product may include an immune checkpoint inhibitor. The immune checkpoint inhibitor may be administered in an amount effective to reduce T cell immunosuppression. The immune checkpoint inhibitor may be a PD-1 antagonist, a PD-L1 antagonist, or a CLTA-4 antagonist. T cell immunosuppression may include immunosuppression mediated by the interaction between T cells and myeloid cells expressing PD-L1.

[0056] This disclosure provides isolated nucleic acids comprising nucleotide sequences encoding heavy chain variable domains provided herein.

[0057] This disclosure provides isolated nucleic acids comprising nucleotide sequences encoding light chain variable domains provided herein.

[0058] This disclosure provides expression vectors comprising nucleic acids provided herein.

[0059] This disclosure provides host cells comprising nucleic acids or expression vectors provided herein.

[0060] This disclosure provides a method for producing a protein comprising an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain. Such a method may include growing host cells provided herein that encode an antibody product under conditions such that the host cells express a protein comprising an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain, and purifying the protein comprising the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain.

[0061] This disclosure provides a method for producing an antibody product that binds to human LILRB2. Such a method may include growing a host cell containing an expression vector provided herein encoding the antibody product under conditions such that the host cell expresses a protein containing an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain, thereby producing the antibody product, and purifying the antibody product.

[0062] This disclosure provides compositions comprising an antibody product provided herein and an excipient.

[0063] This disclosure provides a product comprising a composition provided herein and a container.

[0064] This disclosure provides the use of antibody products or compositions provided herein for the manufacture of pharmaceuticals for the treatment of cancer in subjects requiring cancer treatment.

[0065] This disclosure provides a pharmaceutical composition comprising an antibody product provided herein and a pharmaceutically acceptable excipient.

[0066] This disclosure provides antibody products or pharmaceutical compositions provided herein for use in treating subjects having cancer expressing LILRB2.

[0067] This disclosure provides the use of antibody products in the manufacture of pharmaceuticals for the treatment of subjects having cancer that expresses LILRB2.

[0068] This disclosure provides a method for detecting LILRB2 in a sample, tissue, or cell using an antibody product provided herein, the method comprising contacting the sample, tissue, or cell with the antibody product and detecting the antibody product.

[0069] This disclosure provides a method for reducing the biological activity of LILRB1 and LILRB2 in subjects requiring such reduction, the method comprising administering a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein. The antibody product can mediate the depletion of at least one cancer cell expressing LILRB2.

[0070] This disclosure provides a method for doing so in subjects requiring enhancement of an immune response, the method comprising administering a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein.

[0071] This disclosure provides a method for a subject requiring cancer immunotherapy, wherein cancer cells express LILRB2, and the method comprises administering a therapeutically effective amount of the antibody product provided herein to the subject.

[0072] 90 methods comprising administering an antibody product in an amount effective to mediate the death of cancer cells via antibody-dependent cytotoxicity. The method may include administering an antibody product in an amount effective to reduce LILRB1- or LILRB2-mediated suppression of T cells in a subject. The method may further include administering to a subject a PD-1 antagonist or a PD-L1 antagonist in an amount sufficient to reduce PD-1 / PD-L1 system-mediated immunosuppression of T cells in the subject, or a CTLA-4 antagonist in an amount sufficient to reduce CTLA-4 system-mediated immunosuppression of T cells in the subject. The PD-1 antagonist may include a PD-1 antibody product. The PD-L1 antagonist may include a PD-L1 antibody product. The CTLA-4 antagonist may include a CTLA-4 antibody product.

[0073] The antibody products provided herein may include detectably labeled or conjugated toxins, drugs, receptors, enzymes, or receptor ligands. The antibody products may include a therapeutic moiety or a cytotoxic moiety.

[0074] The pharmaceutical compositions provided herein may include an antibody product provided herein that reduces or prevents the binding of LILRB1 or LILRB2 to its ligand, and / or reduces or prevents LILRB1- or LILRB2-mediated signaling, and a physiologically acceptable carrier or excipient. The ligand may be, for example, 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 may be expressed on the surface of bone marrow cells, lymphocytes, or tumor cells.

[0075] A method provided herein for treating a subject in need of treatment for cancer or tumor may include administering to the subject an effective amount of a pharmaceutical composition provided herein. The subject may have cancer or tumor containing cells expressing or overexpressing the ligand for LILRB2. The antibody product or antigen-binding fragment thereof in the pharmaceutical composition may increase the immune response, delay or prevent tumor growth, inhibit tumor-mediated immunosuppression, eliminate the tumor, deplete or inhibit the activity of tumor-associated macrophages and alter their activity, reduce tumor-associated macrophage-mediated immunosuppression, reduce or reverse T cell suppression, or a combination thereof. The cancer or tumor may contain macrophages expressing LILRB2. The method may further include administering to the subject a therapeutic amount of a second therapeutic product. The second therapeutic product may include an immune checkpoint inhibitor. The second therapeutic product may include a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

[0076] The antibody products provided herein include a heavy chain containing SEQ ID NO: 1 and a light chain containing SEQ ID NO: 2; a heavy chain containing SEQ ID NO: 6 and a light chain containing SEQ ID NO: 11; a heavy chain containing SEQ ID NO: 6 and a light chain containing SEQ ID NO: 12; a heavy chain containing SEQ ID NO: 6 and a light chain containing SEQ ID NO: 13; a heavy chain containing SEQ ID NO: 6 and a light chain containing SEQ ID NO: 14; a heavy chain containing SEQ ID NO: 6 and a light chain containing SEQ ID NO: 15; a variable heavy chain containing SEQ ID NO: 7 and a variable light chain containing SEQ ID NO: 11; a heavy chain containing SEQ ID NO: 7 and a light chain containing SEQ ID NO: 12; a heavy chain containing SEQ ID NO: 7 and a light chain containing SEQ ID NO: 13; a heavy chain containing SEQ ID NO: 7 and a light chain containing SEQ ID NO: 14; a heavy chain containing SEQ ID NO: 7 and a light chain containing SEQ ID NO: 15; a heavy chain containing SEQ ID NO: 8 and a light chain containing SEQ ID NO: 8 and a light chain containing SEQ ID NO: 12 A chain; a heavy chain containing SEQ ID NO: 8 and a light chain containing SEQ ID NO: 13; a heavy chain containing SEQ ID NO: 8 and a light chain containing SEQ ID NO: 14; a heavy chain containing SEQ ID NO: 8 and a light chain containing SEQ ID NO: 15; a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 11; a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 12; a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 13; a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 15; a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 11; a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 12; a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 13; a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 14; or a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 15.

[0077] The antibody products provided herein include a heavy chain containing SEQ ID NO: 1 and a light chain containing SEQ ID NO: 2; a heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33 and a light chain containing SEQ ID NO: 38; a heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33 and a light chain containing SEQ ID NO: 39; a heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33 and a light chain containing SEQ ID NO: 40; a heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33 and a light chain containing SEQ ID NO: 41; a heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33 and a light chain containing SEQ ID NO: 42; and SEQ ID NO: 29 or SEQ ID NO: 34. Heavy chains containing SEQ ID NO: 38; heavy chains containing SEQ ID NO: 29 or 34, and light chains containing SEQ ID NO: 39; heavy chains containing SEQ ID NO: 29 or 34, and light chains containing SEQ ID NO: 40; heavy chains containing SEQ ID NO: 29 or 34, and light chains containing SEQ ID NO: 41; heavy chains containing SEQ ID NO: 29 or 34, and light chains containing SEQ ID NO: 42; heavy chains containing SEQ ID NO: 30 or 35, and light chains containing SEQ ID NO: 38; heavy chains containing SEQ ID NO: 30 or 35, and light chains containing SEQ ID NO: 39; sequence number Heavy chain containing sequence number 30 or sequence number 35, and light chain containing sequence number 40; heavy chain containing sequence number 30 or sequence number 35, and light chain containing sequence number 41; heavy chain containing sequence number 30 or sequence number 35, and light chain containing sequence number 42; heavy chain containing sequence number 31 or sequence number 36, and light chain containing sequence number 38; heavy chain containing sequence number 31 or sequence number 36, and light chain containing sequence number 39; heavy chain containing sequence number 31 or sequence number 36, and light chain containing sequence number 40; heavy chain containing sequence number 31 or sequence number 36, and A light chain containing sequence number 41; a heavy chain containing sequence number 31 or 36, and a light chain containing sequence number 42; a heavy chain containing sequence number 32 or 37, and a light chain containing sequence number 38; a heavy chain containing sequence number 32 or 37, and a light chain containing sequence number 39; a heavy chain containing sequence number 32 or 37, and a light chain containing sequence number 40; a heavy chain containing sequence number 32 or 37, and a light chain containing sequence number 41; or a heavy chain containing sequence number 32 or 37, and a light chain containing sequence number 42.

[0078] The following drawings and detailed description (including examples) illustrate various non-limiting aspects of the subject matter intended herein. [Brief explanation of the drawing]

[0079] The aspects of this disclosure are shown in the following diagram.

[0080] [Figure 1A-1B] LILRB1-expressing 721.221B lymphoma and LILRB2 / 1 chimeric and humanized mutants that bind to HEK-293 cells expressing LILRB2. [Figure 2A-2B] Blocking of LILRB2-Fc and LILRB1-Fc binding to HLA-G by LILRB2 / 1 chimeric IgG1 and humanized mutants. [Figure 3A-3C] Conjugation of LILRB2 / 1 antibodies to human monocytes, M0, and M2c macrophages. [Figure 4A-4H] LILRB2 / 1 antibody conjugation to human monocytes and lymphocyte populations from PBMCs in three subjects. [Figures 5A-5D] IFN-γ secretion from PBMCs (from 4 subjects) stimulated with LPS after treatment with anti-LILRB2 / 1 chimeric IgG1 and humanized mutants in dose setting. [Figure 6A-6B] Effect of LILRB2 / 1 antibody on TNF-α secretion in CD40-activated macrophages (from two subjects) [Figures 7A-7B] In an M2c and CD8+ T cell co-culture assay, humanized mutants were used to (A) rescue CD8+ T cell proliferation and (B) rescue IFN-γ secretion (from 3 subjects). [Figures 8A-8C] Rescue of IFN-γ release by exhausted T cells from M2c-mediated immunosuppression using LILRB2 / 1 chimeric and humanized mutants (from 3 subjects) [Figure 9A-9B] Enhanced NK cell cytotoxicity by LILRB2 / 1 chimeric IgG1 and humanized mutants [Figure 10A-10H] Induction of LILRB2 / 1 chimeric and humanized mutants of IFN-γ and IL-6 release in whole blood from healthy subjects. [Figure 11A-11C]LILRB2 / 1 antibody conjugation to bone marrow cell subsets and lymphocytes (from 3 subjects) [Figure 12A-12C] Pharmacokinetic profiling of LILRB2 / B1 chimeric and humanized mutants in FcRn mice [Figure 13] Antitumor efficacy of chimeric LILRB2 / 1 antibody in humanized NSG-SGM3 mouse and subcutaneous SK-MEL-5 human melanoma tumor models. [Figures 14A-14B] The LILRB2 / 1 humanized antibody mutant reduces the HLA-G-mediated suppression of tumor cell phagocytosis by M2 macrophages. [Modes for carrying out the invention]

[0081] Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art in the field to which the claimed subject matter pertains.

[0082] Antibody products This disclosure provides antibody products that specifically bind to certain human LILRB proteins, particularly LILRB1 and LILRB2, compared to other LILR family members.

[0083] "LILRB2 / 1 antibody product" means an antibody or fragment thereof that has variable affinity among family members, but specifically binds to LILRB1 and LILRB2 compared to binding to other LILR family members. In some embodiments, LILRB2 / 1 antibody products bind with an affinity that allows the antibody product to block or substantially impair the binding of LILRB1 and LILRB2 to their native ligands, or to modulate the activity mediated by LILRB1 and LILRB2. LILRB2 / 1 antibodies can enable LILRB2 antagonism and LILRB1 antagonism. Therefore, they may be referred to as "LILRB2 / 1 antagonists".

[0084] A "LILRB1 antagonist" means any chemical compound or biological molecule that blocks or substantially impairs the binding of LILRB1 to any of its natural ligands, including HLA-A, -B, -C, -F, and -G, and HLA-B27. A LILRB1 antagonist can block or impair the interaction with a ligand on another cell, thereby blocking or inhibiting the functional activity associated with such a "trans" interaction. A LILRB1 antagonist can block or impair the interaction with a ligand on the same cell, thereby blocking or inhibiting the functional activity associated with such a "cis" interaction. An antibody that specifically binds to LILRB1 can be a LILRB1 antagonist. Therefore, a "LILRB1 antibody" or "LILRB1 antibody product" specifically binds to LILRB1 compared to binding to other LILR family members.

[0085] A "LILRB2 antagonist" means any chemical compound or biological molecule that blocks or substantially impairs the binding of LILRB2 to either its natural ligands, such as members of the human leukocyte antigen family (including HLA-G, HLA-A, HLA-B, and HLA-F) or angiopoietin-like proteins (ANGPTL2, or ANGPTL5, etc.). A LILRB2 antagonist can block or impair the interaction with a ligand on another cell, thereby blocking or inhibiting the functional activity associated with such a "trans" interaction. A LILRB2 antagonist can block or impair the interaction with a ligand on the same cell, thereby blocking or inhibiting the functional activity associated with such a "cis" interaction. An antibody that specifically binds to LILRB2 can be a LILRB2 antagonist. Therefore, a "LILRB2 antibody" or "LILRB2 antibody product" specifically binds to LILRB2 compared to binding to other LILR family members.

[0086] Detailed, reviewed genetic information on human LILRB2 is readily available, for example, at www.ncbi.nlm.nih.gov / gene / 10288.

[0087] Detailed, reviewed genetic information on human LILRB1 is readily available, for example, at www.ncbi.nlm.nih.gov / gene / 10859.

[0088] The terms “polypeptide” and “protein” are used interchangeably herein, in the conventional way, to refer to molecules formed from amino acids. Polypeptides are not limited to any particular length. Peptides are included within polypeptides unless otherwise specified. These terms do not specify or exclude post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., or other modifications known in the art, both naturally occurring and non-natural. Polypeptides of interest in the context of antibodies of this disclosure include, but are not limited to, polypeptide fragments containing CDRs that can bind to LILRB1 or LILRB2 proteins expressed by bone marrow cells, lymphocytes, or cancer cells.

[0089] The term "polypeptide fragment" refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to a full-length native protein. Such fragments may also contain modified amino acids compared to a native protein. Fragments are approximately 5 to 500 amino acid lengths. For example, fragments may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acid lengths. Polypeptide fragments include immunologically functional fragments of an antibody containing a binding domain. In the case of the LILRB2 / 1 antibody disclosed herein, useful fragments include, but are not limited to, a CDR region, a variable domain of the heavy or light chain, a portion of the antibody chain, or only its variable region containing two CDRs.

[0090] As used herein, the term “isolated protein” means that the protein in question is (1) free from at least some other proteins commonly found, (2) essentially free from other proteins from the same source, (3) expressed by cells from different species, (4) isolated from at least about 50 percent of naturally occurring polynucleotides, lipids, carbohydrates, or other substances, (5) operably related (by covalent or non-covalent interactions) with polypeptides not naturally occurring, or (6) not present in nature. Synthetic genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof, can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.

[0091] A "mutant" of a polypeptide (e.g., an antibody) includes an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted compared to another polypeptide sequence. Mutants include fusion proteins.

[0092] A polypeptide "derivative" is a polypeptide (e.g., an antibody) that has been chemically modified in some way other than insertion, deletion, or substitution, for example, by conjugation to another chemical moiety.

[0093] The term "antibody" generally refers to immunologically functional immunoglobulin proteins that consist of one or more polypeptide chains and are specifically bound to an antigen. In humans, an antibody typically consists of four linked polypeptide chains, forming a "tetramer" containing two identical "heavy" chains and two smaller, identical "light" chains. Each of the two heavy chains is linked to one light chain, and they are also linked in parallel to each other. The binding gives the antibody a roughly Y-shaped structure, such that the binding portion of the heavy chains forms the "legs" of a "Y," and each light chain (along with the portion of the heavy chain it is bound to) forms the "arms" of a "Y." Since each arm of the antibody contains an antigen-binding site, a typical antibody can bind to two 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 region and their functional purpose. Some classes of intact antibodies in humans may differ from circulating IgM antibodies, which typically contain a tetrameric "Y" structural unit, for example, five such units linked at their bases in a nearly circular array. Further details on antibody structure and function are provided elsewhere in this specification.

[0094] In a typical antibody, each pair of tetrameric units or caplets contains one full-length "light" chain (approximately 25 kDa) and one full-length "heavy" chain (approximately 50–70 kDa). Each individual immunoglobulin chain consists of several "immunoglobulin domains," each comprising approximately 90–110 amino acids, which exhibit characteristic folding patterns. These domains are the basic units that make up the antibody polypeptide chain. The amino-terminus of each chain typically contains a variable domain involved in antigen recognition. The carboxyl-terminus is evolutionarily more conserved than the amino-terminus of the chain and is called the "constant region" or "C region."

[0095] The term "heavy chain" includes the full-length immunoglobulin heavy chain and its fragments that have variable domain sequences sufficient to confer binding specificity, either alone or together with light chain variable domains. Heavy chains are typically classified as mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. The IgM subtype includes IgM and IgM2. The IgA subtype includes IgA1 and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains, the IgG and IgE isotypes contain two heavy chains and two light chains, and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically contains one or more domains that can perform effector functions. The number of heavy chain constant domains depends on the isotype. For example, each full-length IgG heavy chain contains three C domains known as CH1, CH2, and CH3, with CH3 being the closest to the carboxyl terminus. The antibody product provided may have any of these isotypes and subtypes. For example, a LILRB2 / 1 antibody product can be an intact antibody of the IgG1 or IgG4 subtype.

[0096] The term "light chain" includes full-length immunoglobulin light chains and fragments thereof that have a variable domain sequence sufficient to confer binding specificity, either alone or in conjunction with heavy chain variable domains. Human light chains are generally classified as kappa (κ) or lambda (λ) light chains. Full-length light chains contain an amino-terminal variable domain (VL) and a carboxy-terminal constant domain (CL).

[0097] In the light and heavy chains, the variable and constant domains are spontaneously linked by "J" regions of approximately 12 or more amino acids, and the heavy chain further contains "D" regions of approximately 10 or more amino acids. See, for example, Fundamental Immunology, 2nd ed., Ch.7 (Paul, ed.) 1989, New York: Raven Press.

[0098] The variable domains of immunoglobulin chains generally exhibit the same overall structure, including a relatively conserved framework region (FR) linked by three hypervariable regions, more frequently called “complementarity-determining regions” or CDRs. The CDRs from the two chains of each heavy / light chain pair typically align with the framework region to form a structure that specifically binds to a particular epitope on a target protein (e.g., LILRB1 or LILRB2). From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically follow the following order of elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids occupying each position in these domains.

[0099] Current technologies utilize various numbering schemes with different definitions of CDR length and position. For example, the Kabat numbering scheme is sequence alignment based and uses a "variability parameter" (the number of different amino acids at a given position divided by the frequency of the most abundant amino acid at that position) to predict the CDR [Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Pub. no. 91-3242 (1991)]. On the other hand, the Chothia numbering scheme is a structure-based numbering scheme that aligns antibody crystal structures so that loop structures are defined 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, unlike conventional methods [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 sequence alignment from a complete reference gene database including the whole immunoglobulin superfamily [Lefranc et al., Dev Comp Immunol. 2003 27(1):55-77; (www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html)].The Honneger numbering scheme (AHo) is based on the structural alignment of the 3D structure of the variable region and estimates the lengths of the framework and CDR using structurally conserved Cα positions [Honegger et al., J Mol Biol. 2001 309(3):657-70]. Those skilled in the art will understand that the definition of CDR varies depending on the method used.

[0100] Some of the antibody products offered typically have structures related to naturally occurring antibodies. Therefore, the term “antibody product” includes any class or subclass of intact antibody or fragment thereof that can compete with an intact antibody for specific binding to a target antigen, including chimeric, humanized, fully human, and bispecific antibodies, as well as other forms. As mentioned above, intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some examples, they may contain fewer chains, e.g., antibodies naturally occurring in camelids that may contain only heavy chains, and a shark-derived V NAR domain. Antibody products may originate from a single source or they may be “chimeric,” meaning that different parts of the antibody may originate from two different antibodies. For example, the complementarity-determining region that confers the antibody’s binding specificity may originate from a rat or mouse source, while the framework portion of the variable region may originate from a different species source, such as human. In other chimeric forms, the light and heavy variable domains (which may have constant domains) may originate from one or more constant domains from one species and another. See, for example, U.S. Patent No. 11352444. The antibody products offered 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 containing two full-length heavy chains and two full-length light chains (such as IgG antibodies), as well as other isotypes of antibodies, their derivatives, variants, and fragments. The antibody products offered include, but are not limited to, monoclonal antibodies, human antibodies, chimeric antibodies, and humanized antibodies. The immunologically functional antibody fragments offered include, but are not limited to, scFv, Fab, Fab', F(ab')2, and domain antibody products.

[0101] As used herein, an immunofunctional fragment of an immunoglobulin (or simply a "fragment") includes a light chain or a heavy chain (or both) that can specifically bind to an antigen, wherein the light chain or heavy chain (or both) refers to a portion of an antibody lacking at least some of the amino acids present in the full-length chain. Such fragments are biologically active in that they can specifically bind to a target antigen and compete with an intact antibody for specific binding to a given epitope. Such fragments may retain at least one CDR present in the full-length light chain or heavy chain and may include a single heavy chain and / or a light chain or a portion thereof. These biologically active fragments may be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Examples of immunofunctional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and may originate from any mammalian source, including, but are not limited to, humans, mice, rats, camelids, or rabbits. It is further intended that the functional portion of the antibody of the present invention, for example, one or more CDRs, can be covalently bound to a second protein or small molecule to create a therapeutic agent that is directed to a specific target in the body, has bifunctional therapeutic properties, or has a long serum half-life.

[0102] A "Fab fragment" consists of one light chain (VL+CL) and a portion of a heavy chain (VH+CH1) containing a variable domain and a CH1 domain. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0103] The "Fc" region contains two heavy chain fragments, each containing the antibody's CH2 and CH3 domains, and possibly a lower hinge region. The two heavy chain fragments are held together by two or more disulfide bonds (typically within the hinge region) and hydrophobic interactions of the CH3 domain.

[0104] A "Fab' fragment" comprises one light chain and a portion of a heavy chain that includes a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains. Two Fab' fragments can form an interchain disulfide bond between their two heavy chains to form an F(ab')2 molecule.

[0105] The "F(ab')2 fragment" consists of 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, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.

[0106] The "Fv region" contains variable domains from both the heavy and light chains, but lacks a constant domain.

[0107] A "single-chain antibody" is an Fv molecule in which a heavy-chain variable domain and a light-chain variable domain are linked by a flexible linker to form a single polypeptide chain that forms an antigen-binding region. Single-chain antibodies are discussed in detail, for example, in PCT Publication No. 88 / 01649, and U.S. Patents No. 4,946,778 and No. 5,260,203.

[0108] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only a variable domain of the heavy chain or a variable domain of the light chain. In some cases, two or more VH domains are covalently linked to a peptide linker to produce a bivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0109] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific (see below).

[0110] A "multispecific antibody" is one that targets two or more antigens or epitopes.

[0111] A "bispecific (dual-specific)" or "bifunctional" antibody is a hybrid antibody that has two different antigen-binding sites. Bispecific antibodies are a type of multispecific antibody and can be produced by various methods, including but not limited to hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai and Lachmann, Clin Exp Immunol. 1990 79:315-21 and Kostelny et al., J Immunol. 1992 148:1547-53. The two binding sites of a bispecific antibody bind to two different epitopes that may be present on the same or different protein targets. A "triple-specific" antibody has three different antigen-binding sites. See also Merchant et al., Nat Biotechnol. 1998 16:677-81.

[0112] The term "neutralizing antibody" refers to an antibody that binds to a ligand, preventing the ligand from binding to its binding partner, and otherwise interrupting the biological response resulting from the ligand's binding to its binding partner. When evaluating the binding and specificity of an antibody or its immunologically functional fragment, the antibody or fragment substantially inhibits ligand binding to its binding partner if the excess antibody reduces the amount of the 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 by an in vitro competitive binding assay). In the case of an antibody product that binds to the LILRB1 protein or the LILRB2 protein, the neutralizing antibody product reduces the ability of the LILRB1 protein or the LILRB2 protein (or both) to bind to one or more of its ligands, thereby inhibiting LILRB-mediated activity (as shown, for example, in the examples herein).

[0113] When used in the context of competing antibody products for the same epitope, the term "competition" means that competition between antibodies is determined by an assay in which the antibody products under test prevent or inhibit the specific binding of the reference antibody product to a common antigen (e.g., LILRB2 or LILRB1, or a fragment thereof). Numerous types of competitive binding assays: e.g., solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), 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 labeling assays, solid-phase direct labeling sandwich assays [e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)]; solid-phase direct labeling RIA using I-125 labeling [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 directly labeled RIA [Moldenhauer et al., Scand J Immunol. 1990 32:77-82] can be used. Typically, such assays involve the use of purified antigen bound to a solid surface or cells having 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. Usually, the test antibody is present in excess. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitopes as the reference antibody, and antibodies that bind to epitopes sufficiently proximal to the epitope bound by the reference antibody to cause steric hindrance. Usually, when competitive antibodies are present in excess, they inhibit the specific binding of the reference antibody to the common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.In some cases, binding can be inhibited by selective binders such as antibodies by at least 80%, 85%, 90%, 95%, or 97% or more, and can be used in animals to produce antibodies that can further bind to the antigen. The antigen may have one or more epitopes that can interact with different antibodies.

[0114] The term "epitope" includes any determinant that can specifically bind to an antibody or T cell receptor. An epitope is a region of an antigen that is bound by an antibody that specifically targets that antigen, and if the antigen is a protein, it includes a specific amino acid that comes into direct contact with the antibody. In most cases, epitopes are located on proteins, but in some examples they can be located on other types of molecules, such as nucleic acids. Epitope determinants 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 properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on the target antigen in complex mixtures of proteins and / or macromolecules.

[0115] Dissociation constant (K d If the K content is less than 100 nM, the antibody product "specifically binds" to its target antigen. Antibodies are K d If the amount is less than 10 nM, it is considered "high affinity". d When the K content is less than 0.5 nM, it binds specifically to the antigen with "ultra-high affinity". The antibody product has a K content in the range of approximately 0.5 nM to approximately 500 nM. d The antibody product may have a K content in the range of approximately 100 to approximately 500 nM. d Those skilled in the art will recognize that specific bonding does not mean exclusive bonding, but rather allows for a degree of nonspecific bonding, as is typical in biological reactions between groups that have affinity for one another.

[0116] As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two drugs, and the equilibrium dissociation constant KD , the calculated ratio (K) between the dissociation constant and association constant between the antibody and its antigen off / K on It can be expressed as ). Affinity is K D The association constant K is the reciprocal of association constant. A It can also be expressed as follows. The antibody products disclosed herein are K for human LILRB2 and human LILRB1. D The binding affinity measured by 10 -4 M or less, or 10 -16 Shown within the range of M or less (for example, approximately 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 (M or less). The antibodies described herein are 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 It can specifically bind to human LILRB2 polypeptide or human LILRB1 polypeptide. Methods for determining the affinity of the two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (SPR), and biolayer interferometry (BLI).

[0117] As used herein, an antibody product is said to be "immunospecific," "specific," or "specifically binding" if it forms a complex with an antigen that is relatively stable under physiological conditions. The terms "preferentially binding" or "specifically binding" mean that the antibody or fragment thereof binds to an epitope with higher affinity than it would to an unrelated amino acid sequence, is cross-reactive with other polypeptides containing the epitope, and is not toxic at a level suitable for formulation for use in administration to human subjects. This term is also applicable, for example, if an antibody product is specific to a particular epitope supported by two or more antigens, in which case the antibody or its antigen-binding fragment carrying the antigen-binding domain can specifically bind to the epitope found on different antigens. Relative affinity measured between one antibody and another against one epitope may differ between comparators, between variants of the same comparator, or across experimental conditions. The LILRB2 / 1 antibodies disclosed herein preferentially bind to LILRB1 and LILRB2 compared to other LILRA and LILRB family members. Such relative affinity can be at least 1x greater, at least 2x greater, at least 3x greater, at least 4x greater, at least 5x greater, at least 6x greater, at least 7x greater, at least 8x greater, at least 9x greater, 10x greater, at least 20x greater, at least 30x greater, at least 40x greater, at least 50x greater, at least 60x greater, at least 70x greater, at least 80x greater, at least 90x greater, at least 100x greater, at least 200x greater, at least 250x greater, at least 500x greater, or at least 1000x greater than the affinity of the antibody product to amino acid sequences derived from other LILR family members.

[0118] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. "Identity percentage" means the percentage of identical residues between amino acids or nucleotides in the molecules being compared, and is calculated based on the smallest size of the molecules being compared. For these calculations, any gaps in the alignment 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; and Carillo et al., SIAM J Applied Math. 1988 48(5):1073-82.

[0119] When calculating identity percentage, the sequences being compared are aligned to yield the greatest possible match between them. An exemplary computer program used to determine identity percentage is the GCG program package, which includes GAP (Devereux et al., Nucl Acid Res. 1984 12:387-95; Genetics Computer Group, University of Wisconsin, Madison, Wisc.). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percentage sequence identity is determined. The sequences are aligned for the best possible matching of their respective amino acids or nucleotides ("matched span" determined by the algorithm). A gap opening penalty (calculated as 3 times the mean diagonal (3×), where the "mean diagonal" is the average of the diagonals of the comparison matrix used); where the "diagonal" is the score or number assigned to each complete amino acid match by a particular comparison matrix) and a gap expansion penalty (usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used with the algorithm. Standard comparison matrices [for example, Dayhoff et al., Atlas of Protein Sequence and Structure, 5:345-352 (1978) for the PAM 250 comparison matrix, and 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.

[0120] The recommended parameters for determining the identity percentage 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: Henikoff et al., 1992, BLOSUM 62 from the same; Gap penalty: 12 (but no penalty for terminal gaps); Gap length penalty: 4; Similarity threshold: 0.

[0121] A specific alignment scheme for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this small aligned region 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 across at least 50 consecutive amino acids of the target polypeptide.

[0122] 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), and gap 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)).

[0123] "Amino acid" includes its usual meaning in this 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- and α-disubstituted amino acids, N-alkyl amino 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, following standard usage and convention, the left direction is towards the amino terminus and the right direction is towards the carboxyl terminus.

[0124] Table 1 below shows the heavy chain variable domain and light chain variable domain of the LILRB-specific antibody named B21A, as well as the corresponding full-length heavy chain (hIgG1 and hIgG4) and light (kappa) chain provided herein. The CDRs for the heavy chain variable (VH) domain (SEQ ID NO: 1) and light chain variable (VL) domain (SEQ ID NO: 2) are shown, with IMGTs double-underlined and Kabats in bold. The IMGTs and Kabat CDRs for the heavy chain (H) and light chain (L) are shown in Table 2.

[0125] [Table 1]

[0126] [Table 2]

[0127] Humanized variants of the B21A antibody were prepared as described in the examples. Representative variable domains of the humanized heavy and light chains are shown in Table 3.

[0128] [Table 3]

[0129] Those skilled in the art will recognize that antibody products such as full-length intact LILRB2 / 1 antibodies and LILRB2- and LILRB1-conjugated antibody fragments can be prepared based on the heavy-chain variable domains and light-chain variable domains shown in Tables 1 and 3, or based on the CDRs shown in Table 2. As described in the Examples, in addition to the full-length antibody chains shown in Table 1, full-length IgG1 or IgG4 heavy chains containing the VH variable domains (SEQ ID NOs. 6-10) shown in Table 3 were prepared in combination with kappa light chains containing the VL domains (SEQ ID NOs. 11-15) shown in Table 3. The full-length IgG4 heavy chain sequences are shown as SEQ ID NOs. 28-32. The full-length IgG1 heavy chain sequences are shown as SEQ ID NOs. 33-37. The full-length kappa light chain sequences are shown as SEQ ID NOs. 38-42. LILRB2 / 1 conjugated antibody products were prepared using all combinations of heavy and light chains, and some of these tests in various nonclinical assays are described in the Examples.

[0130] The antibody product may contain a light chain variable domain comprising an amino acid sequence that differs from the sequence of the light chain variable domain described herein by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, such a difference in each sequence being independently either a deletion, insertion, or substitution of one amino acid. The light chain variable region in some antibodies comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the light chain variable region in Table 1 or Table 3.

[0131] The antibody product may contain a heavy chain variable domain containing an amino acid sequence in which only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues differ from the sequence of the heavy chain variable domain provided herein, and each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid. The heavy chain variable region in some antibodies contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the heavy chain variable region in Table 1 or Table 3, and furthermore, other antibody products may contain variant light chains and variant heavy chains as described herein.

[0132] The antibody product provided may contain all 1, 2, 3, 4, 5, or 6 CDRs. Some antibody products contain both light chain CDR3 and heavy chain CDR3. Certain antibody products have variant forms of CDRs in which one or more CDRs (i.e., 2, 3, 4, 5, or 6) have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the respective CDR sequence. For example, an antibody product may contain both light chain CDR3 and heavy chain CDR3 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the light chain CDR3 sequence and heavy chain CDR3, respectively. The CDR sequences of the provided antibody products may also differ from the CDR sequences in Table 1 or Table 3, such that the amino acid sequence of any given CDR differs from the sequences listed in Table 11 or Table 3 by only one, two, three, four, or five or fewer amino acid residues. The differences from the listed sequences are conservative substitutions.

[0133] When an antibody product is said to bind to an epitope within LILRB2, it means that the antibody product specifically binds to a polypeptide composed of residues identified by a specific residue (e.g., a specific segment of LILRB2). Such an antibody does not necessarily come into contact with all residues within LILRB2. Furthermore, not all single amino acid substitutions or deletions within the LILRB2 peptide significantly affect the binding affinity. The precise epitope specificity of an antibody can be determined in various ways. One approach involves, for example, testing a collection of approximately 15-amino acid duplicate peptides across the LILRB2 sequence, differing by a small number of amino acid increments (e.g., three amino acids). The peptides are immobilized in wells of a microtiter dish. Immobilization can be achieved by biotinylation of one end of the peptide. In some cases, different samples of the same peptide can be biotinylated at the N-terminus and C-terminus and immobilized in separate wells for comparison. This is useful for identifying end-specific antibodies. In some cases, additional peptides terminating at the specific amino acid of interest can be included. This approach is useful for identifying terminal-specific antibodies against internal fragments of LILRB2. Antibody products are screened for specific binding to various peptides. An epitope is defined as a segment of amino acids common to all peptides to which the antibody specifically binds.

[0134] Antibody products that compete with one of the exemplified antibodies for specific binding to LILRB2 are also provided. Such antibody products can 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 antibody or fragment are expected to exhibit similar functional properties. The exemplified antibody products include those having heavy and light chains, variable domains, and CDRs, as provided in Table 1, Table 2, or Table 3. Competing antibody products may include those that bind to the epitopes described in the Antibody and Epitope sections above.

[0135] The antibody products provided include monoclonal antibodies that bind to LILRB2. Monoclonal antibodies can be produced, for example, by immortalizing spleen cells isolated from transgenic animals after completion of an immunization schedule, using any technique known in the art. Spleen cells can be immortalized, for example, by fusing them with myeloma cells to produce hybridomas, using any technique known in the art. Myeloma cells for use in hybridoma-producing fusion procedures are preferably non-antibody-producing, have high fusion efficiency, and have enzyme deficiencies that prevent them from growing in specific selective media that support the proliferation of only the desired fusion cells (hybridoms). Examples of cell lines suitable for mouse 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 for rat 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.

[0136] In some cases, hybridoma cell lines are produced by immunizing animals (e.g., transgenic animals having human immunoglobulin sequences) with the LILRB2 immunogen; collecting spleen cells from the immunized animals; fusing the collected spleen cells with myeloma cell lines to generate hybridoma cells; and establishing hybridoma cell lines from the hybridoma cells and identifying hybridoma cell lines that produce antibodies binding to the LILRB2 polypeptide. Hybridoma cell lines and LILRB2 monoclonal antibodies produced therefrom are provided herein.

[0137] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any useful techniques known in the field of antibody technology. Hybridoma or monoclonal antibodies can be further screened to identify monoclonal antibodies with specific properties. Examples of such screening are provided in the following examples.

[0138] Chimeric and humanized antibodies based on the aforementioned sequences are also provided. Monoclonal antibodies for therapeutic use 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 its immunologically functional portion. Generally, part of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, and the rest of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass. For methods relating 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 U.S. Patents 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101.

[0139] Generally, the purpose of creating chimeric antibodies is to produce a chimera in which the number of amino acids derived from the intended patient species is maximized. One example is a "CDR-implanted" antibody, in which the antibody contains one or more complementarity-determining regions (CDRs) from a specific species or belonging to a specific antibody class or subclass, and the rest of the antibody chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass. For use in humans, the V region or selected CDR from rodent antibodies is often implanted into human antibodies, replacing the naturally occurring V region or CDR in the human antibody.

[0140] Humanized antibody products are provided. Generally, humanized antibodies are produced from monoclonal antibodies initially created in non-human animals. In these monoclonal antibodies, specific amino acid residues, typically derived from the non-antigen recognition region of the antibody, are modified to be homologous to the corresponding residues in the corresponding isotype of human antibody. Humanization can be carried out using various methods, for example, by substituting at least a portion of the rodent variable region with the corresponding region of a human antibody [e.g., U.S. Patent Nos. 5,585,089 and 5,693,762, Jones et al., Nature. 1986 321:522-5, Riechmann et al., Nature. 1988 332:323-7, Verhoeyen et al., Science. 1988 239:1534-6]. Hybrid antibodies can be produced by using a constant region from a non-human species together with the human variable region.

[0141] Fully human antibodies are also available. Methods are known for producing fully human antibodies specific to a given antigen without exposing humans to the antigen ("fully human antibodies"). One means of carrying out the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introducing a human immunoglobulin (Ig) locus into mice in which the endogenous Ig gene has been inactivated is one means of producing fully human monoclonal antibodies (MAb) in mice, which are animals that can be immunized with any desired antigen. Using fully human antibodies can minimize the immunogenicity and allergic responses that may occasionally be caused by administering mouse or mouse-derived monoclonal antibodies as therapeutic agents to humans.

[0142] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that can produce 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 sometimes 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, the transgenic animal is prepared by inactivating the endogenous mouse immunoglobulin loci that encode the mouse heavy and light chain immunoglobulin chains within it, and inserting a large fragment of human genomic DNA containing loci that encode human heavy and light chain proteins into the mouse genome. Next, partially modified animals having human immunoglobulin loci lacking complete complement are crossbred to obtain animals possessing all of the desired immune system modifications. Upon administration of the immunogen, these transgenic animals produce antibodies that are immune-specific to the immunogen but have human amino acid sequences rather than mouse amino acid sequences containing variable regions. For further details of such methods, see, for example, WO96 / 33735 and WO94 / 02602. Additional methods relating to transgenic mice for producing human antibodies are described in U.S. Patents 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, PCT Publications Nos. 91 / 10741 and 90 / 04036, and EPO Publication No. 546073B1.Transgenic mice referred to herein as "HuMab" mice contain human immunoglobulin gene miniloci encoding unreorganized human heavy chain (g and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous gamma and kappa chain loci (Lonberg et al., Nature. 1994 368:856-9). Thus, the aforementioned 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 mutations to produce high-affinity human IgG kappa monoclonal antibodies. An exemplary mouse possessing the entire human immunoglobulin locus in the germline is the XenoMouse (Abgenix). Another is the VelociImmune mouse (Regeneron Pharmaceuticals). Other mice include RenMab mice and RenLite mice (Biocytogen), and more recently, AlivaMab mice (Ablexis) and ATX-GX mice (Alloy Therapeutics).

[0143] Using hybridoma technology, antigen-specific human monoclonal antibodies with desired specificity can be produced and selected from transgenic mice as described above. Such antibodies can be cloned and expressed using suitable vectors and host cells, or the antibodies can be recovered from cultured hybridoma cells.

[0144] Fully human antibodies can also be derived from phage display libraries (as disclosed in Hoogenboom and Winter, J Mol Biol. 1992 227(2):381-8, and Marks et al., J Mol Biol. 1991 222:581-97). Phage display techniques mimic immunoselection by displaying an antibody repertoire on the surface of filamentous bacteriophages, and subsequent phage selection by binding to selected antigens. One such technique is described in PCT Publication 99 / 10494, which describes the isolation of high-affinity and functional agonist antibodies against MPL- and msk- receptors using such an approach.

[0145] Single-chain antibodies are provided. Single-chain antibodies are formed by linking heavy chain variable domain (Fv region) fragments and light chain variable domain (Fv region) fragments (such as those shown in Table 1 or Table 3) via amino acid crosslinks (short peptide linkers) to obtain a single polypeptide chain. Such single-chain Fv (scFv) can be prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). Depending on the length of the flexible linker between the two variable domains, the resulting polypeptide can fold itself to form an antigen-binding monomer or form a multimer (e.g., a dimer, trimer, or tetramer). Techniques developed for the production of single-chain antibodies include those described in U.S. Patent 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. A "diabody" is a dimer of scFV.

[0146] Antibodies provided herein, which belong to one subclass, can be modified to antibodies from different subclasses using subclass switching techniques. For example, the variable domains shown in Table 1 or Table 3 can be bound to the constant domain of any desired Ig subtype. Such techniques enable the preparation of new antibodies that possess the antigen-binding properties of a given antibody (parent antibody) but also exhibit biological properties associated with a different antibody isotype or subclass than those of the parent antibody. Recombinant DNA techniques can be used. Cloned DNA encoding a specific antibody polypeptide, for example, DNA encoding the constant domain of an antibody of a desired isotype, can be used in such a procedure. See, for example, Lantto et al., Methods Mol Biol. 2002 178:303-16. Thus, the antibodies provided include the desired isotypes (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE, and IgD).

[0147] The antibody product provided may include one or more of the following heavy chain variable domains CDRs (such CDRs are determined according to IMGT, Kabat, or other methods): (i) CDR-H1 having at least 80% sequence identity with CDR-H1 of SEQ ID NO: 1, 6, 7, 8, 9, or 10; (ii) CDR-H2 having at least 80% sequence identity with CDR-H2 of SEQ ID NO: 1, 6, 7, 8, 9, or 10; and (iii) CDR-H3 having at least 80% sequence identity with CDR-H3 of SEQ ID NO: 1, 6, 7, 8, 9, or 10. The antibody product provided may include one or more of the following CDRs from among the light chain variable domains exemplified herein (such CDRs are determined according to IMGT, Kabat, or other methods): (i) CDR-L1 having at least 80% sequence identity with CDR-L1 of SEQ ID NO: 2, 11, 12, 13, 14, or 15; (ii) CDR-L2 having at least 80% sequence identity with CDR-L2 of SEQ ID NO: 2, 11, 12, 13, 14, or 15; and (iii) 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 with the determined CDR sequence. The antibody product may contain one, two, three, four, five, or all six of the aforementioned CDRs, as long as they specifically bind to hLILRB2.

[0148] The antibody product provided may include one or more of the following exemplary heavy-chain IMGT CDRs: (i) CDR-H1 having at least 80% sequence identity with SEQ ID NO: 16, (ii) CDR-H2 having at least 80% sequence identity with SEQ ID NO: 17, and (iii) CDR-H3 having at least 80% sequence identity with SEQ ID NO: 18. The antibody product provided may also include one or more of the following light-chain CDRs: (i) CDR-L1 having at least 80% sequence identity with SEQ ID NO: 19, (ii) CDR-L2 having at least 80% sequence identity with SEQ ID NO: 20, and (iii) CDR-L3 having at least 80% sequence identity with SEQ ID NO: 21. In some embodiments, the CDR has at least 85%, at least 90%, at least 95%, or at least 99% identity with a given CDR sequence. The antibody product may contain one, two, three, four, five, or all six of the aforementioned CDRs, as long as they specifically bind to hLILRB2.

[0149] The antibody product provided may include one or more of the following exemplary heavy chain Kabat CDRs: (i) CDR-H1 having at least 80% sequence identity with SEQ ID NO: 22, (ii) CDR-H2 having at least 80% sequence identity with SEQ ID NO: 23, and (iii) CDR-H3 having at least 80% sequence identity with SEQ ID NO: 24. The antibody product provided may also include one or more of the following light chain CDRs: (i) CDR-L1 having at least 80% sequence identity with SEQ ID NO: 25, (ii) CDR-L2 having at least 80% sequence identity with SEQ ID NO: 26, and (iii) CDR-L3 having at least 80% sequence identity with SEQ ID NO: 27. In some embodiments, the CDR has at least 85%, at least 90%, at least 95%, or at least 99% identity with a given CDR sequence. The antibody product may contain one, two, three, four, five, or all six of the aforementioned CDRs, as long as they specifically bind to hLILRB2.

[0150] The antibody product provided may include (a) a heavy chain variable region having 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NOs: 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 with SEQ ID NOs: 2, 11, 12, 13, 14, or 15; or (c) the heavy chain variable region of (a) and the light chain variable region of (b).

[0151] Other antibody products offered compete with the antibodies described above for specific binding to the LILRB2 polypeptide. For example, antibody products are offered that compete with antibodies consisting of two identical heavy chains and two identical light chains, where the heavy chains include SEQ ID NOs: 1, 6, 7, 8, 9, or 10, and the light chains include SEQ ID NOs: 2, 11, 12, 13, 14, or 15.

[0152] A LILRB2 / 1 antibody product having a half-life of at least 1 day in vitro or in vivo is provided (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.

[0153] mutant This specification provides mutants of LILRB2 / 1 antibody products (for example, mutants of antibody products having sequences listed in Tables 10 and 12). For example, an antibody product may have one or more conservative amino acid substitutions in one or more of the heavy chain variable regions, light chain variable regions, or CDRs listed in Tables 10 and 12.

[0154] Naturally occurring amino acids can be classified 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; 5) Residues affecting chain orientation: Gly, Pro; and 6) Aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions may involve exchanging one member of one of these classes with another member of the same class. Conservative amino acid substitutions can also involve amino acid residues that do not exist naturally, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptide mimes and other inverted or reversed forms of amino acid moieties.

[0155] Non-conservative substitutions may involve exchanging a member of one of the above classes with a member of another class. Such substituted residues can be introduced into regions of antibody products homologous to human antibodies, or into non-homologous regions of molecules.

[0156] When making such changes, the hydroxyl index of amino acids can be taken into consideration. The hydroxyl profile of a protein is calculated by assigning a numerical "hydrophilic index" to each amino acid and then repeatedly averaging these values ​​along the peptide chain. Each amino acid is assigned a hydroxyl index based on its hydrophobic and charge properties. These are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0157] The importance of hydrophobic profiles in conferring interactive biological functions to proteins is well understood in the art [e.g., Kyte and Doolittle, J Mol Biol, 157(1):105-132 (1982)]. It is known that certain amino acids can be used as substitutes for other amino acids with similar hydropathic indices or scores, while still retaining similar biological activity. When making changes based on hydropathic indices, substitutions of amino acids with hydropathic indices within ±0.2 are permitted. Substitutions of amino acids with hydropathic indices within ±0.1 are also permitted. Substitutions of amino acids with hydropathic indices within ±0.5 are also permitted.

[0158] Similar residue substitutions in amino acid sequences can be effectively carried out based on the relative hydrophilicity or hydrophobicity of the residues, and are understood to be particularly effective when the resulting biologically functional proteins or peptides are intended for use in immunological molecules, as in this case. The maximum local mean hydrophobicity of a protein, governed by the hydrophilicity of its adjacent amino acids, can correlate with its immunogenicity and antigen-binding or immunogenicity, i.e., the biological properties of the protein.

[0159] Various methods are known for estimating the hydrophilicity or hydrophobicity of amino acid residues in proteins. A comparative study of such methods can be found in Biswas et al., J Chromatogr A.1000(1-2):637-55. Hopp and Woods (Mol Immunol. 1983 20(4):483-9) assigned the following hydrophilicity values ​​to amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±0.1), glutamic acid (+3.0±0.1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5±0.1), alanine (-0.5), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4). In this ranking system, more hydrophilic residues are assigned positive values, and fewer hydrophilic residues are assigned negative values. When modifications are made based on similar hydrophilic values, amino acid substitutions with a hydrophilic value of ±0.2 are included; otherwise, amino acid substitutions with a hydrophilic value of ±0.1 or ±0.5 are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also called "epitope core regions."

[0160] Those skilled in the art will be able to determine suitable variants of the polypeptides described herein using well-known techniques. Those skilled in the art will also be able to identify suitable regions of the molecule that can be altered without disrupting its activity by targeting regions not considered important for activity. Furthermore, those skilled in the art will be able to identify conserved residues and portions of molecules among similar polypeptides. Even regions that may be important for biological activity or structure can undergo conserved amino acid substitutions without disrupting biological activity or adversely affecting the polypeptide structure.

[0161] Furthermore, those skilled in the art can outline structure-function studies to identify residues in similar polypeptides that are important for activity or structure. Considering such comparisons, the importance of amino acid residues in a protein corresponding to amino acid residues important for the activity or structure of similar proteins can be predicted. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0162] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence associated with that structure in similar polypeptides. Considering such information, those skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. Since such residues may be involved in important interactions with other molecules, those skilled in the art can choose not to induce radical changes in amino acid residues predicted to be on the protein surface. Furthermore, those skilled in the art can construct test mutants containing a single amino acid substitution at each desired amino acid residue. These mutants can then be screened using assays for LILRB1 and / or LILRB2 binding activity (see examples below), thus providing information on which amino acids can be modified and which should not. In other words, based on 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.

[0163] Substantial alterations to the functional and / or biochemical characteristics of antibody products described herein can be achieved by creating substitutions in the heavy and light chain amino acid sequences that are significantly different in (a) the structure of the molecular skeleton in the region of substitution, e.g., sheet-like or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulkiness of the side chains. "Conservative amino acid substitution" may include substituting 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. Furthermore, any native residue in the polypeptide can also be substituted with alanine, as previously described for alanine scanning mutagenesis.

[0164] Amino acid substitutions (whether conserved or non-conserved) of antibodies in question can be carried out 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 protein complex formation, (4) alter ligand or antigen binding affinity, and / or (4) confer or modify other physicochemical or functional properties to such polypeptides. For example, one or more amino acid substitutions (e.g., conserved amino acid substitutions) can be made in naturally occurring sequences. The substitutions can be made in portions of the antibody outside of domains that form intermolecular contacts. Conserved amino acid substitutions can be used that do not substantially alter the structural characteristics of the parent sequence (e.g., one or more substituted amino acids that do not disrupt the secondary structure characterizing the parent antibody or natural antibody). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, ed.), 1984, New York: WH 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, which are each incorporated herein by reference.

[0165] Glycosylation variants of antibody products are provided in which the number and / or type of glycosylation sites 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. The N-linked glycosylation sites are characterized by the sequence:Asn-X-Ser or Asn-X-Thr, where the amino acid residue indicated as X can be any amino acid residue except proline. Substitutions of amino acid residues to create this sequence provide potential new sites for the addition of N-linked carbohydrate chains. Alternatively, substitutions that eliminate or modify this sequence prevent the addition of N-linked carbohydrate chains present in the native polypeptide. For example, glycosylation may be reduced by the deletion of Asn or by substituting Asn with a different amino acid. For example, one or more new N-linked sites may be created. Antibodies typically have N-linked glycosylation sites in the Fc region.

[0166] Further antibody product variants include cysteine ​​variants in which one or more cysteine ​​residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine ​​variants are particularly useful when the antibody needs to be refolded into a biologically active conformation. Cysteine ​​variants may have fewer cysteine ​​residues than the native antibody, typically having an even number to minimize interactions caused by unpaired cysteine.

[0167] Effector function Antibody structure influences the role an antibody plays in the immune system and the effects it can induce or exert. 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 the binding of the Fc portion of an antibody to a specific receptor molecule, "Fc receptor" or "FcR," expressed by the cells whose function is affected.

[0168] IgG is considered the most versatile immunoglobulin because, in some embodiments, it performs all the functions of an immunoglobulin molecule. IgG is the primary Ig in serum and the only class of Ig that crosses the placenta. IgG also fixes complement, although 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, and IgG2 and IgG4 do not bind to the Fc receptor. As a result of binding to the Fc receptor on PMNs, monocytes, and macrophages, cells now internalize antigens more effectively in some cases. IgG is an opsonin that enhances phagocytosis. Binding of IgG to the Fc receptor on other types of cells results in the activation of other functions.

[0169] In certain embodiments, FcR is a naturally occurring human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma ("γ") receptors) and include allele variants of the FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16) subclass receptors, which are alternatively spliced ​​forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have different but similar amino acid sequences, primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain.

[0170] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcR) present on specific cytotoxic cells (e.g., natural killer (NK) cells (also known as large granular lymphocytes (LGLs)), neutrophils, and macrophages) causes these cytotoxic effector cells to specifically bind to antigen-carrying target cells, subsequently killing the target cells with cytotoxicity. Antibodies "arm" the cytotoxic cells and are necessary for such death. Primary cells mediating ADCC by NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In some embodiments, an in vitro ADCC assay is performed to evaluate the ADCC activity of a molecule of interest. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0171] Alternatively, or further, in some embodiments, the ADCC activity of the molecule of interest is evaluated in vivo, for example, in an animal model.

[0172] In certain therapeutic applications, the internalization process is used to kill or reduce the activity or proliferation of target cells expressing the LILRB2 protein. The number of internalized antibody molecules may be sufficient or appropriate to kill cells or inhibit their growth. Depending on the potency of the antibody or antibody conjugate, in some examples, 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 cells that the internalization of one molecule of the toxin conjugated to an antibody is sufficient to kill the target cell.

[0173] In some embodiments, the LILRB2 / 1 antibody or antigen-binding fragment provided herein is conjugated or linked to a therapeutic portion, an imaging portion, a detectable portion, or an affinity tag. Methods for conjugating or linking polypeptides are well known in the art. Association (binding) between the compound and the label includes, but is not limited to, any means known in the art, including, covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the antibody or its antigen-binding fragment is conjugated to an affinity tag (e.g., a purification tag) or recombinantly operated with an affinity tag. For example, affinity tags such as polyhistidine (e.g., His6) tags are conventionally available in the art.

[0174] In some embodiments, the LILRB2 / 1 antibody or antigen-binding fragment further comprises a detectable portion. Detection is achieved, for example, in vitro, in vivo, or ex vivo. For example, in vitro assays for the detection and / or determination (quantification, identification, etc.) of the hLILRB2 protein expressed by macrophages using an antibody or its antigen-binding fragment include, but are not limited to, ELISA, RIA, and Western blotting. In some embodiments, in vitro detection, diagnosis, or monitoring of the antigen of an antibody is performed by obtaining a sample (e.g., a blood sample) from a subject and testing the sample, for example, with a standard ELISA assay.

[0175] derivative Derivatives of the LILRB2 / 1 antibody product described herein are also provided. Derivatized antibody products may contain any molecules or substances that impart desired properties to the antibody product, such as an extension of the half-life in a particular use. Derivatized antibody products may include, for example, a detectable (or labeled) moiety (e.g., a radioactive molecule, a colorimetric molecule, an antigenic molecule or an 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 may be used to derivatize antibody products include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-bound and PEGylated derivatives of antibody products can be prepared using techniques well known in the art. Antibodies can be conjugated to or otherwise linked to transthyretin (TTR) or TTR variants. TTR or TTR variants can be chemically modified with chemicals selected from the group consisting of, for example, dextran, poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol.

[0176] Other derivatives include covalent or aggregated conjugates of the LILRB2 / 1 antibody product with other proteins or polypeptides, such as the expression of a recombinant fusion protein containing a heterologous polypeptide fused to the N-terminus or C-terminus of the LILRB2 / 1 antibody product. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, such as a yeast alpha-factor leader or an epitope tag. The LILRB2 / 1 antibody product-containing fusion protein may include peptides added to facilitate the purification or identification of the LILRB2 / 1 antibody product (e.g., poly-His). The LILRB2 / 1 antibody product can also be conjugated to a FLAG peptide, as described in Hopp et al., Bio / Technology 1988 6:1204-10 and U.S. Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly conjugated by a specific monoclonal antibody (mAb), enabling rapid assay and easy purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which a FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, Mo.).

[0177] Oligomers containing one or more LILRB2 / 1 antibody products can be used as LILRB2 antagonists. The oligomers can be covalent or non-covalent dimers, trimers, or more. Oligomers containing two or more LILRB2 / 1 antibody products are intended for use, one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, and heterotetramers.

[0178] The oligomer may contain multiple LILRB2 / 1 antibody products bound via covalent or non-covalent interactions between peptide portions fused to the LILRB2 / 1 antibody polypeptide. Such peptides may be peptide linkers (spacers) or peptides that have properties that promote oligomerization. Certain polypeptides derived from leucine zippers and antibodies are among the peptides that can promote the oligomerization of the LILRB2 / 1 antibody product bound to them, as will be described in more detail below.

[0179] The oligomer may contain 2 to 4 LILRB2 / 1 antibody products. The LILRB2 product portion of the oligomer may be any of the above forms, for example, a variant or a fragment. The oligomer contains a LILRB2 / 1 antibody product having LILRB2 binding activity.

[0180] The preparation of fusion proteins containing heterologous polypeptides fused to various parts of antibody-derived polypeptides (including the Fc domain) is described, for example, by Ashkenazi et al., Proc Natl Acad Sci USA. 1991 88(23):10535-9, Byrn et al., Nature. 1990 344(6267):667-70, and Hollenbaugh and Aruffo, Curr Protoc Immunol. 2002 48(1):4:10.19.1-10.19.11.

[0181] A dimer containing two fusion proteins is provided, which are produced by fusing the LILRB2 binding fragment of a LILRB2 / 1 antibody to the Fc region of the antibody. The dimer can be produced, for example, by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in host cells transformed with a recombinant expression vector, and assembling the expressed fusion protein into a very similar antibody molecule, in which case an interchain disulfide bond is formed between the Fc portions to obtain the dimer.

[0182] As used herein, the term “Fc polypeptide” includes both native and mutaine forms of polypeptides derived from the Fc region of an antibody. It also includes cleavage forms of such polypeptides that include a hinge region that promotes dimerization. Fusion proteins containing the Fc portion (and the oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography on a protein A or protein G column.

[0183] One exemplary Fc polypeptide described in PCT Publication No. 93 / 10151 and U.S. Patents No. 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 natural C-terminus of the Fc region. Another exemplary Fc polypeptide is Fc mutein, described in U.S. Patent No. 5,457,035 and Baum et al., EMBO J.1994 13:3992-4001. The amino acid sequence of this mutein is identical to that of the natural Fc sequence shown in PCT Publication No. 93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. Mutein exhibits reduced affinity for the Fc receptor.

[0184] Alternatively, the oligomer is a fusion protein containing multiple LILRB2 / 1 antibody polypeptides, with or without a peptide linker (spacer peptide). Among the preferred peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233.

[0185] Another method for preparing oligomeric LILRB2 / 1 antibody product derivatives involves the use of leucine zippers. Leucine zipper domains are peptides that promote the 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 94 / 10308, and leucine zippers derived from lung surfactant protein D (SPD) are described in Hoppe et al., FEBS Lett. 1994 344:191-5. The use of modified leucine zippers that enable stable trimerization of heterologous proteins fused to them is described in Fanslow et al., Semin Immunol. 1994 6:267-78. Generally, recombinant fusion proteins containing LILRB2 / 1 antibody fragments fused to leucine zipper peptides are expressed in suitable host cells, and the resulting soluble oligomeric LILRB2 / 1 antibody product is recovered from the culture supernatant.

[0186] The LILRB2 / 1 antibody products described herein may also be derivatized or modified so that the product has a longer half-life compared to underivated or unmodified antibodies. For example, the antibody product may contain point mutations that increase the serum half-life, as described in PCT Publication No. 00 / 09560.

[0187] Nucleic acids and cells Also provided herein are nucleic acids encoding one or more chains of the antibody product, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting polynucleotide expression, and the aforementioned complementary sequences.

[0188] The nucleic acids provided encode antibody products disclosed herein, for example, the light chain variable regions shown in Table 1 or Table 3, and / or the heavy chain variable regions shown in Table 1 or Table 3. Due to the degeneracy of the genetic code, each polypeptide sequence listed in Table 1 or Table 3 also encodes other nucleic acid sequences not listed in Table 1 or Table 3. This disclosure provides each degenerate nucleotide sequence encoding each antibody product.

[0189] The terms "polynucleotide" or "nucleic acid" refer to single-stranded or double-stranded polymers. Nucleotides containing polynucleotides may be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. These modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2'',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates. The term encompasses both single-stranded and double-stranded forms.

[0190] "Isolated nucleic acid molecule" means a genome, mRNA, cDNA, or DNA or RNA of synthetic origin, or any combination thereof, in which the isolated polynucleotide is not related to any polynucleotide found in nature, or is linked to a polynucleotide that is not linked in nature. For the purposes of this disclosure, it should be understood that a "nucleic acid molecule containing" a particular nucleotide sequence does not include an intact chromosome. An isolated nucleic acid molecule "containing" a particular nucleic acid sequence may, in addition to the particular sequence, contain up to 10, and even up to 20, other protein or part thereof coding sequences, or may contain operably linked regulatory sequences that control the expression of the coding region of the listed nucleic acid sequence, and / or may contain vector sequences.

[0191] 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 called the 5' direction. The direction of addition from 5' to 3' in a nascent RNA transcript is called the transcription direction. The sequence region on the DNA strand that has the same sequence as the RNA transcript, from 5' end to 5' end of the RNA transcript, is called the "upstream sequence." The sequence region on the DNA strand that has the same sequence as the RNA transcript, from 3' end to 3' end of the RNA transcript, is called the "downstream sequence."

[0192] The term "regulatory sequence" refers to a polynucleotide sequence that can influence the expression and processing of the coding sequence to which it is ligated. The properties of such a regulatory sequence can be host organism-dependent. For example, a eukaryotic regulatory sequence may include a promoter containing one or more recognition sites for a transcription factor, a transcription enhancer sequence, and a transcription termination sequence. A "regulatory sequence" may also include a leader sequence and / or a fusion partner sequence.

[0193] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0194] The terms “expression vector” or “expression construct” refer to a vector comprising nucleic acid sequences suitable for transforming host cells and which induce and / or control the expression of one or more heterogeneous coding regions operably ligated thereto (in conjunction with the host cell). Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, where introns are present, affect RNA splicing of coding regions operably ligated thereto.

[0195] As used herein, “operably linked” means that the components to which this term applies are in a relationship that enables them to perform their inherent functions under favorable conditions. For example, a control sequence in a vector “operably linked” to a protein-coding sequence is linked to it such that the expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0196] The term "host cell" means a cell that has been transformed with a nucleic acid sequence, or can be transformed, to express the gene of interest. This term includes offspring of a parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the gene of interest is present.

[0197] DNA encoding antibody polypeptides (e.g., heavy chain or light chain, variable domain only, or full length) can be isolated from mouse B cells immunized with LILRB2 or its immunogenic fragments. The DNA can be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another known technique that allows for the selection of nucleotide sequences encoding antibody polypeptides.

[0198] Nucleic acids are provided that hybridize with other nucleic acids under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. As defined herein, moderately stringent hybridization conditions use a pre-washing solution containing 5× sodium chloride / sodium citrate (SSC), 0.5% SDS, and 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6× SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions such as one containing about 50% formamide at a hybridization temperature of 42°C), as well as washing conditions of 60°C in 0.5× SSC and 0.1% SDS. Stringent hybridization conditions involve hybridizing in 6× SSC at 45°C, followed by one or more washes at 68°C in 0.1× SSC and 0.2% SDS. Those skilled in the art can manipulate hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to one another typically remain hybridized to one another.

[0199] Basic parameters influencing the selection of hybridization conditions, and guidance for devising suitable conditions, are shown, 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 those skilled in the art, for example, based on DNA length and / or base composition.

[0200] Changes can be introduced by mutations in nucleic acids, thereby resulting in alterations to the amino acid sequence of the polypeptide it encodes (e.g., an antibody or antibody derivative). Mutations can be introduced using any technique known in the art. One or more specific amino acid residues can be modified, for example, using site-directed mutagenesis protocols. One or more randomly selected residues can be modified, for example, using random mutagenesis protocols. However, once this is produced, the mutant polypeptide can be expressed and screened for desired properties.

[0201] The polypeptide, which is a component of the target antibody product, is expressed in any suitable recombinant expression system.

[0202] An expression vector containing a nucleic acid encoding a LILRB2 / 1 antibody product is provided. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0203] Typically, an expression vector used in any host cell contains a sequence for maintaining a plasmid or virus, as well as a sequence for cloning and expressing an exogenous nucleotide sequence. Collectively called “adjacent sequences,” such sequences typically include one or more operablely linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including a donor splice site and an acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and one or more selectable marker elements.

[0204] Optionally, the vector may contain a sequence encoding a “tag,” i.e., an oligonucleotide molecule located at the 5' or 3' end of the encoding sequence, an oligonucleotide sequence encoding polyHis (e.g., hexaHis), or another “tag” on which a commercially available antibody such as FLAG, HA (hemagglutinin from influenza virus), or myc exists. The tag is typically fused to the antibody protein during expression and can function as a means for affinity purification of the antibody from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as the affinity matrix. In some cases, the tag can then be removed from the purified antibody polypeptide by various means, such as using a specific peptidase for cleavage.

[0205] The adjacent sequences in the expression vector may 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 adjacent sequences from two or more sources), synthetic, or native. Therefore, the sources of adjacent sequences may be any prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, as long as the adjacent sequences are functional in the host cellular mechanism and can be activated by the host cellular mechanism.

[0206] Expression vectors and cloning vectors typically contain a promoter operably ligated to a nucleic acid that is recognized by a host organism and encodes a LILRB2 / 1 antibody product. Promoters are conventionally classified into two classes: inductive promoters and constitutive promoters. Inductive promoters initiate an increase in transcription levels from DNA under their control in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, initiate continuous gene product production; that is, there is little or no experimental control over gene expression. Numerous promoters recognized by various potential host cells are well known. A suitable promoter is operably ligated to the DNA encoding the LILRB2 / 1 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.

[0207] Suitable promoters for use with mammalian host cells are well known and not limited to, but include promoters derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (such as adenovirus 2), bovine papillomavirus, 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.

[0208] Examples of useful promoters 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 sequence of Roussarcoma virus (Yamamoto, et al., Cell. 1980 22:787-97), the herpesthymidine kinase promoter (Wagner et al., Proc Natl Acad Sci USA. 1981 78:1444-5), the regulatory sequence of the metallothione 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 Examples include, but are not limited to, Sci USA. 1983 80:21-5). Furthermore, the following animal transcriptional regulatory regions exhibit tissue specificity and are utilized in transgenic animals: the elastase I gene regulatory 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 regulatory region, which is active in pancreatic beta cells (Hanahan, Nature. 1985 315:115-22); the mouse mammary cancer virus regulatory region, which is active in testicular cells, mammary cells, lymphocytes, and mast cells (Leder et al., Cell. 1986 45:485-95); and the albumin gene regulatory region, which is active in the liver (Pinkert et al., Genes Dev 1987 1:268-76), the alpha-fetoprotein gene regulatory region that is active in the liver (Krumlauf et al., Mol Cell Biol. 1985 5:1639-48, Hammer et al., Science.The alpha-1 antitrypsin gene regulatory region is active in the liver (1987 235:53-8), the beta-globin gene regulatory region 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 regulatory region is active in oligodendrocytes in the brain (Readhead et al., Cell. 1987 48:703-12), the myosin light chain-2 gene regulatory region is active in skeletal muscle (Sani, Nature. 1985 314:283-6), and the gonadotropin-releasing hormone gene regulatory region is active in the hypothalamus (Mason et al., Science. 1986 The immunoglobulin gene regulatory regions (234:1372-8), and particularly active in lymphocytes (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) are available for use.

[0209] Enhancer sequences can be inserted into vectors to increase transcription in higher eukaryotes of nucleic acids encoding the LILRB2 / 1 antibody products described herein. Various enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). Viral-derived enhancer sequences can also be used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancing elements for the activation of eukaryotic promoters. Enhancers can be spliced ​​into vectors at the 5' or 3' position relative to the nucleic acid molecule, but they are typically positioned at the 5' position relative to the promoter.

[0210] In an expression vector, the transcription termination sequence typically lies 3' of the end of the polypeptide coding region and serves to terminate transcription. Transcription termination sequences used for expression in prokaryotic cells are typically G-C rich fragments followed by a poly T sequence. The sequences can be easily cloned from a library or even commercially purchased as part of a vector, but can also be readily synthesized using methods for nucleic acid synthesis as described herein.

[0211] Selectable genes can be used to amplify the genes being expressed. Amplification is a process in which genes that cannot be expressed at a single copy are expressed at a high level sufficient to allow cell survival and growth under specific selection conditions and are tandemly repeated within the chromosomes of successive generations of recombinant cells. Examples of suitable amplifiable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase. In the use of these markers, mammalian cell transformants are placed under a selection pressure uniquely adapted such that only the transformants survive by the selectable gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions where the concentration of the selective agent in the medium is continuously increased, thereby allowing only the survival of cells in which the selectable gene has been amplified. Under these circumstances, DNA adjacent to the selectable gene, such as DNA encoding an antibody, is co-amplified with the selectable gene. As a result, a greater amount of the LILRB2 polypeptide is synthesized from the amplified DNA.

[0212] The ribosome binding site is usually required for the initiation of mRNA translation and is characterized by the Shine-Dalgarno sequence (for prokaryotes) or the Kozak sequence (for eukaryotes). The element typically lies 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

[0213] 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 specific 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 associated with expression that may not be completely removed at the position of -1 relative to the first amino acid of the mature protein. 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 cleaved but active form of the desired polypeptide when the enzyme cleaves at such regions within the mature polypeptide.

[0214] If a commercially available expression vector lacks a portion of the desired flanking sequences described above, the vector can be modified by ligating these sequences into the vector individually. After selecting and modifying the vector as desired, a nucleic acid molecule encoding the LILRB2 / 1 antibody product is inserted into an appropriate site of the vector.

[0215] The completed vector containing the sequence encoding the antibody product is inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the selected host cell with the expression vector for the LILRB2 / 1 antibody product can be achieved by well-known methods including transfection, infection, calcium chloride, electroporation, microinjection, lipofection, the DEAE-dextran method, or other known techniques. The method selected will function, in part, with the type of host cell used. These methods and other suitable methods are well known to those skilled in the art.

[0216] Antibodies can be expressed in hybridoma cell lines or non-hybridoma cell lines. Mammalian, insect, or microbial host cells can be transformed using expression constructs encoding antibodies. Transformation can be carried out using any known method for introducing polynucleotides into host cells, including, for example, packaging polynucleotides in a virus or bacteriophage and transfecting the construct into host cells by transfection procedures known in the art, as exemplified by U.S. Patents 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, polynucleotide encapsulation in liposomes, mixing of nucleic acids with positively charged lipids, and direct microinjection of DNA into the nucleus.

[0217] Transformed host cells, when cultured under appropriate conditions, synthesize the LILRB2 / 1 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 producing it (if not secreted). The selection of appropriate host cells depends on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (e.g., glycosylation or phosphorylation), and the ease of folding into biologically active molecules.

[0218] Mammalian cell lines available as hosts for expression are well known in the art, including, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human fetal kidney cells (HEK) (e.g., HEK-293), 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.

[0219] 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 the 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 the active portion of the polypeptide (e.g., the LILRB2 binding portion).

[0220] composition Compositions containing LILRB2 / 1 antibody products are also provided. Pharmaceutical compositions typically include one or more buffers, pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, and preservatives. Use of the aforementioned antibody products in the preparation of pharmaceutical compositions or pharmaceuticals is also provided.

[0221] The acceptable formulation components of a pharmaceutical product are non-toxic to the recipient at the dosage and concentration used. In addition to the antibody products provided herein, compositions may contain components for modifying, maintaining, or preserving, for example, the pH, osmolality by weight, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or osmosis 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); buffering agents (such as acetic acid, boric acid, bicarbonate, tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; and salt-forming ions. Examples of additives include, but are not limited to, sodium (etc.); preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (glycerin, propylene glycol, or polyethylene glycol, etc.); sugar alcohols (mannitol or sorbitol, etc.); suspending agents; surfactants or wetting agents (pluronic acid, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (sucrose or sorbitol, etc.); isotonic agents (alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants.(See Remington's Pharmaceutical Sciences, 23rd ed., (Adejare, ed.), 2020, Elsevier Academic Press).

[0222] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous. Suitable vehicles or carriers for such compositions include water for injection, physiological saline, or artificial cerebrospinal fluid, and other materials common to parenteral administration compositions may be supplemented. Neutral buffered saline or physiological saline mixed with serum albumin are further exemplary vehicles. Compositions containing the LILRB2 / 1 antibody product can be prepared for storage by mixing a selected composition of desired purity with an optional formulation agent in the form of a lyophilized cake or aqueous solution. Furthermore, the LILRB2 / 1 antibody product can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0223] The formulation components are present at the administration site at an acceptable concentration. The buffer is advantageously used to maintain the composition within a pH range of physiological pH or slightly lower, typically about 4.0 to about 8.5, or alternatively, about 5.0 to 8.0. The pharmaceutical composition may contain a TRIS buffer at about pH 6.5 to 8.5, or an acetate buffer at about pH 4.0 to 5.5, which may further contain sorbitol or a preferred alternative thereof.

[0224] Further pharmaceutical compositions may be in the form of sustained-delivery or controlled-delivery formulations. Various techniques can be used to formulate liposome carriers, bio-erosive microparticles or porous beads, and other sustained- or controlled-delivery means such as depot injections (see, for example, PCT Publication 93 / 15722A1, which describes controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions). The sustained-release preparation may include a semipermeable polymer matrix in the form of a molded article, such as a film or microcapsules, polyester, hydrogel, polylactide (U.S. Patent No. 3,773,919 and EP058,481), copolymer of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers. 1983 22:547-56), poly(2-hydroxyethyl methacrylate) (Langer et al., J Biomed Mater Res. 1981 15:167-277 and Langer, Chem Tech. 1982 12:98-105), ethylene vinyl acetate (Langer et al., ibid.), or poly-D(-)-3-hydroxybutyrate (EP133,988). The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art. For example, see Eppstein et al., Proc Natl Acad Sci USA. 1985 82:3688-92, EPO Publication Nos. 036676, 088046, and 143949.

[0225] Once a pharmaceutical composition is 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 that is reconstituted before administration (e.g., lyophilized).

[0226] The components used to formulate the pharmaceutical composition are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually sterile. Unless a given compound is synthesized before 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. The composition for parental administration is also sterile, substantially isotonic, and prepared under GMP conditions.

[0227] The kit is provided for either multiple dose units or single dose units. For example, each kit may include both a first container containing a dry protein and a second container containing an aqueous diluent, including, for example, single and multi-chamber prefilled syringes (e.g., liquid syringes, frozen syringes, or needleless syringes).

[0228] Pharmaceutical compositions can be delivered parenterally, typically by injection. Injections may be intraocular, intraperitoneal, intraportal, intramuscular, intravenous, intrathecal, intracerebral (intraparum), intraventricular, intraarterial, intralesional, perilesional, or subcutaneous. Eye drops can be used for intraocular administration. In some cases, injections can be localized near one or more specific bones targeted by the treatment. For parenteral administration, antibodies can be administered in a pyrogen-free, parenterally acceptable aqueous solution containing the desired LILRB2 / 1 antibody product in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the LILRB2 / 1 antibody product is formulated as a sterile isotonic solution and properly stored.

[0229] Pharmaceutical compositions containing the target LILRB2 / 1 antibody product can be administered continuously by bolus injection, infusion, implantable device, sustained-release system, or other means to achieve sustained release. Pharmaceutical compositions can also be administered topically via implantation of a membrane, sponge, or other suitable material into which the desired molecule is absorbed or encapsulated. When an implantable device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be by diffusion, sustained-release bolus, or continuous release. Preparations can be formulated using beads or liposomes, which can provide controlled or sustained release of the drug, e.g., injectable microspheres, bio-erosive particles, polymer compounds (e.g., polylactic acid; polyglycolic acid; or copro(lactic acid / glycolic acid) (PLGA)), and then delivered via depot injection. Formulation with hyaluronic acid has the effect of promoting duration in circulation.

[0230] The target composition containing the LILRB2 / 1 antibody product can also be used ex vivo. In such cases, cells, tissues, or organs taken from a patient are exposed to or cultured with the LILRB2 / 1 antibody product. The cultured cells can then be re-implanted in the patient or another patient, or used for other purposes.

[0231] LILRB2 / 1 antibody products can be delivered by transplanting specific genetically engineered cells using methods such as those described herein to express and secrete polypeptides. Such cells may be animal or human cells and may be autologous, xenogeneic, or interspecies, or may be immortalized. To reduce the likelihood of an immunological response, cells may be encapsulated to avoid invasion of surrounding tissues. The encapsulation material is typically a biocompatible, semipermeable polymer inclusion or membrane that allows for the release of the protein product but prevents cell destruction by the patient's immune system or other harmful factors from surrounding tissues.

[0232] As used herein, "substantially pure" means that the stated molecular species is the predominant species present, i.e., it is more abundant than any other individual species in the same mixture on a molar basis. A substantially pure molecule can be a composition that contains at least 50% (on a molar basis) of all the polymeric species in which the target species is present. A substantially pure composition can contain at least 80%, 85%, 90%, 95%, or 99% of all the polymeric species present in the composition. The target species can also not be detected as a contaminating species in the composition by conventional detection methods, and thus the composition can be purified to an essential homogeneity consisting of a single detectable polymeric species.

[0233] Dosage The pharmaceutical composition provided can be administered for prophylactic and / or therapeutic treatment.

[0234] As used herein, terms such as “treatment / procedure” and “to treat / procedure” mean administering a drug or performing a procedure for the purpose of obtaining an effect. An effect is prophylactic in that it completely or partially prevents a disease or its symptoms, and / or results in a partial or complete cure of the disease and / or its symptoms. “Treatment” as used herein includes the treatment of a disease or disorder (e.g., cancer) in mammals, in particular humans, and includes (a) preventing the occurrence of a disease or its symptoms in a subject that is predisposed to the disease but has not yet been diagnosed with it (e.g., including diseases associated with or caused by primary diseases), (b) inhibiting the disease, i.e., stopping its development, and (c) reducing the disease, i.e., causing regression of the disease. Treatment means any clinical sign of success in treatment, improvement, or prevention, any objective or subjective parameter such as reduction, remission, reduction of symptoms, or increased tolerance of the diseased state to the patient, slowing the rate of degeneration or debilitation, or reduction of debilitation in the final stages of degeneration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Therefore, the term “to treat” includes the administration of the compounds or agents of this disclosure to prevent or delay, alleviate, or halt 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, symptoms of a disease, or side effects of a disease in a subject. For example, if, after receiving a therapeutic dose of a combination of LILRB2 / 1 antibody products provided herein, a patient exhibits one or more observable and / or measurable changes in endpoints or symptoms of a disease condition, the subject is “treated” for the disease or disorder.

[0235] As defined in this disclosure, “effective response” is achieved when the subject experiences partial or complete relief or reduction of the signs or symptoms of the disease, and in the case of cancer treatment, this includes, but is not limited to, improvement of symptoms, delay of progression, cure, remission, extended survival, or other objective responses. Predicted progression-free survival can be measured over several months to several years, depending on prognostic factors including the number of relapses, the stage of the disease, and other factors. Extended survival includes, but is not limited to, periods of at least one month, approximately two months or more, approximately three months or more, approximately four months or more, approximately six months or more, approximately one year or more, approximately two years or more, approximately three years or more, etc. Overall survival can also be measured over several months to several years, for example. Alternatively, an effective response may be that the subject’s symptoms remain static.

[0236] In preventative measures, the administration of therapeutic agents is performed before the symptoms of an undesirable disease or disorder appear, resulting in the prevention of the disease or disorder or, alternatively, the delay of its progression. Therefore, when used in combination with preventative measures, the term "therapeutably effective" means that, on average, fewer subjects develop the undesirable disease or disorder or experience a progression in the severity of symptoms after treatment.

[0237] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, particularly humans, for which diagnosis, treatment, or treatment is desired. “Mammal” for therapeutic purposes refers to humans, livestock and farm animals, and any animal classified as a mammal, including experimental, zoo, sport, or companion animals such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. A mammal may be a human.

[0238] Generally, the toxicity and therapeutic efficacy of antibody products can be determined by standard pharmaceutical procedures in cell culture and / or experimental animals, for example, LD 50 (A 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 the toxic effect and the therapeutic effect is the therapeutic index, or the ratio LD50. 50 / ED 50 It can be expressed as follows. Compositions exhibiting a large therapeutic index are preferred.

[0239] Data obtained from cell cultures and / or animal studies can be used to formulate a range of dosages for human use. The dosage of the active ingredient is typically ED 50 The circulating concentration is within the range of [the substance], and the toxicity is little to no. The dosage may vary within this range depending on the dosage form used and the route of administration utilized.

[0240] The effective amount of a pharmaceutical composition containing a LILRB2 / 1 antibody product used therapeutically or prophylactically depends, for example, on the context and purpose of treatment. Therefore, it will be understood by those skilled in the art that the appropriate dose level for treatment varies in part depending on the molecule being delivered, the indication for which the LILRB2 / 1 antibody is used, the route of administration, and the patient's size (body weight, body surface or organ size) and / or condition (age and overall health). Clinicians can titrate the dose and modify the route of administration to obtain the optimal therapeutic effect. Typical doses range from approximately 1 mg / kg to a maximum of approximately 1600 mg / kg or more, depending on the factors mentioned above. Dosages can range from 1 mg / kg to a maximum of approximately 200 mg / kg, or 1 mg / kg to a maximum of approximately 1200 mg / kg, or 1 μg / kg to a maximum of approximately 1600 mg / kg.

[0241] The frequency of administration depends on the pharmacokinetic parameters of the LILRB2 / 1 antibody product in the formulation. For example, a clinician administers the composition until the desired effect is achieved. Therefore, the composition can be administered as a single dose, or as two or more doses over time (which may contain equal amounts of the desired molecule), or as a continuous infusion via an implantable device or catheter. The treatment may be continuous or intermittent over time. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routine practice. The appropriate dose can be confirmed by the use of appropriate dose-response data. An exemplary administration schedule is every 2-3 weeks.

[0242] To treat a disease state by targeting LILRB2, a composition containing a LILRB2 / 1 antibody product is administered to the patient in an amount and for a duration sufficient to induce sustained improvement in at least one indicator reflecting the severity of the disorder. Improvement is considered "sustained" if the patient shows improvement at least twice, with intervals of at least 1 to 7 days, and possibly 1 to 6 weeks. The appropriate interval depends to some extent on which disease state is being treated. Determining the appropriate interval for determining whether improvement is sustained is within the scope of the knowledge of those skilled in the art. The degree of improvement is determined based on signs or symptoms, and may also be determined using questionnaires administered to the patient, such as quality of life questionnaires.

[0243] To determine whether the amount and duration of treatment are sufficient, various indicators reflecting the severity of the patient's illness can be evaluated. Baseline values ​​for one or more selected indicators are established by examining the patient before the first dose of the antibody is administered. Preferably, the baseline examination is performed within approximately 60 days after the first dose is administered. If the antibody is administered to treat acute symptoms, for example, to treat fractured bone, the first dose should be administered as soon as substantially possible after the injury occurs.

[0244] Improvement is induced by administering the LILRB2 / 1 antibody product until the patient shows improvement above baseline in one or more selected indicators. In the treatment of chronic conditions, this degree of improvement can be achieved by repeatedly administering the drug for at least one month or longer, for example, one, two, or three months or longer, or indefinitely. A period of 1 to 6 weeks, or even a single dose, is often sufficient to treat acute conditions. In cases of injury or acute conditions, a single dose may be sufficient.

[0245] The severity of a patient's illness after treatment may appear to improve according to one or more indicators, but treatment can be continued indefinitely at the same level, or at a reduced dose or frequency. Once treatment has been reduced or discontinued, it can be resumed at the original level if symptoms reappear.

[0246] How to use The LILRB2 / 1 antibody products disclosed herein have various uses. 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.

[0247] LILRB2 / 1 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 that serve as diagnostic tools for detecting pathological conditions in which LILRB2 is overproduced or underproduced. Potent expression of LILRB2 by macrophages, osteoclasts, and other myeloid cells can be markers of the activity of those cells, and detection of LILRB2 expression on myeloid cells can be used as a marker of disease or disorder characterized by the cell type in question. Similarly, detection of LILRB2 expression by cancer cells can be used to identify subjects in which cancer may be suitable for treatment by the therapeutic LILRB2 / 1 antibody product methods disclosed herein.

[0248] Accordingly, a method is provided 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 from a subject, comprising contacting cells expressing LILRB1 and / or LILRB2 with a LILRB2 / 1 antibody product provided herein. The LILRB2 / 1 antibody product can be conjugated to a detectable portion, and the method includes directly detecting that portion. The method may also include indirectly detecting the binding of the LILRB2 / 1 antibody product to cells by the detectable portion that binds to the antibody. For example, an IgG antibody conjugated to a detectable portion can be used to bind to a LILRB2 / 1 antibody presented as an IgG isotype. The cells may be tumor cells. The cells may be myeloid cells (e.g., monocytes, dendritic cells, macrophages, myeloid-derived suppressor cells, tumor-associated macrophages, immunosuppressive macrophages, or M2-like macrophages) or osteoclasts.

[0249] The provided antibody products can also be used in methods for screening 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 / 1 antibody product binds is brought into contact 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 is an indicator that the candidate molecule binds to LILRB2. Binding of the antibody product can be detected by various methods, such as ELISA. Detection of binding between the LILRB2 / 1 antibody product and LILRB2 can be simplified by labeling the antibody in a detectable manner. In some methods, molecules that show binding in the initial screening are further analyzed to determine whether they inhibit or modulate LILRB2 activity.

[0250] The LILRB2 / 1 antibody products provided herein are useful for the treatment of human diseases, including cancer.

[0251] The LILRB2 / 1 antibody products described herein can be used alone or in combination with other anticancer agents for the treatment of cancer. The cancers to be treated are types of cancer in which cancer cells are known to express LILRB2 or have been previously observed to express LILRB2. Certain cancers that are EGFR variants have been found to express LILRB2 more highly and are therefore targeted for treatment using 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 targeted when PD-L1 expression by cancer cells is not observed. Such treatments are indicated when therapeutic interventions along the PD-1 / PD-L1 axis are ineffective or are expected to be ineffective.

[0252] A method for treating patients with cancer is provided herein, in which a LILRB2 / 1 antibody product mediates the death of cancer cells.

[0253] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and hematological tumors. The term "cancer" also includes diseases of the skin, tissues, organs, bones, cartilage, blood vessels, and vascular tissues. The term "cancer" further encompasses both primary and metastatic cancers.

[0254] Examples of cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cancers of the bladder, blood, bone, bone marrow, brain, breast, cervix, colon, esophagus, gastrointestinal tract, rectum, head and neck, kidney, larynx, liver, lung, oropharynx, neck, ovaries, pancreas, prostate, skin, stomach, testes, thyroid, tongue, and uterus.

[0255] Cancers 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, B-cell 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 cancer, neuroblastoma, glioblastoma, pancreatic cancer, multiple myeloma, prostate cancer, small cell lung cancer, non-small cell lung cancer, and metastatic cancers. Cancers treated include, for example, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell carcinoma, gastric cancer, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid cancer, cutaneous squamous cell carcinoma, or ovarian cancer.

[0256] In addition, cancer can be specifically classified into the following histological types: neoplasms, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; piloma cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combination of hepatocellular carcinoma and cholangiocarcinoma; tubular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; familial adenomatous polyposis; solid tumors; carcinoid tumors, malignant; bronchiolalveolar adenocarcinoma; papillary adenocarcinoma; pigmentophilic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic Clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; 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; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; cystic tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cells Cancer; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Gromangios sarcoma; Malignant melanoma; Apigmented melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevi; Epithelioid sarcoma; Malignant cellular blue nevus; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant Sex; undifferentiated germ cell tumor; fetal cancer; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastoma; ameloblastoma, malignant; ameloblastoma; pineal glandoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal tumor;Cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell type, diffuse; malignant lymphoma, follicular; mycosis fungoides or cutaneous T-cell lymphoma; B-cell lymphoma or other specific non- Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel 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 pilocytic cell leukemia, but may or may not be limited to these.

[0257] As an example, methods for treating subjects suffering from melanoma are provided. As used herein, “melanoma” refers to a condition characterized by the growth of tumors arising from the melanocyte system of the skin and other organs. Most melanocytes are 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 eyes 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.

[0258] Cancer cells treated by the methods provided herein may express LILRB1 and LILRB2. Cancer cells may overexpress LILRB1 and LILRB2.

[0259] Some cytomegalovirus (CMV) infections are thought to be a contributing factor in tumorigenesis. CMV can cause cells to express UL18, a cytomegalovirus MHC class I homolog that binds to LILRB1 (Yang and Bjorkman, Proc Natl Acad Sci USA. 2008 105(29):10095-100). Antibodies can be used in cancer treatment methods for patients with CMV infection in which HCMV DNA is detectable in tumor tissue such as Hodgkin lymphoma and non-Hodgkin lymphoma, colorectal cancer, cervical cancer, and breast cancer. CMV infection that produces HLA-G mimes may contribute to immunosuppression in TMEs through binding to LILRB1 and LILRB2. LILRB2 / 1 antibody products can block these interactions and limit or reduce, or reverse or prevent immunosuppression.

[0260] The way in which LILRB2 / 1 antibody products can mediate cancer cell death is via antibody-dependent cytotoxicity ("ADCC"). ADCC is a process in which an antibody coats target cells (e.g., cancer cells or bacterial cells) and recruits effector cells via a non-phagocytic mechanism to induce target cell death.

[0261] As described above, in the treatment method, the LILRB2 / 1 antibody product provided herein can be used as monotherapy or in combination therapy. "Combination therapy" refers to the administration of one therapeutic agent before, during, or after the administration of other therapeutic agents to the subject.

[0262] In combination therapy for cancer, LILRB2 / 1 antibody products are used in combination with one or more other anti-cancer modalities for the treatment of cancer in a subject. As is well understood in this field, such anti-cancer modalities may include surgery, radiotherapy, chemotherapy, and immunotherapy. In some embodiments, the anti-cancer modality may be a chemotherapeutic agent or a biological molecule. In some embodiments, the anti-cancer modality may be an immunotherapy agent molecule. In some embodiments, the immunotherapy agent may be a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor may be a PD-1 antagonist. In some embodiments, the checkpoint inhibitor may be a PD-L1 antagonist.

[0263] This specification provides a method for treating a patient having cancer, the method comprising administering to the patient a LILRB2 / 1 antibody product described herein (e.g., as a pharmaceutical composition thereof) in combination with at least one other anticancer product (e.g., as a pharmaceutical composition comprising chemotherapy or immunotherapy). In some embodiments, such combination use may include administering an effective amount of the LILRB2 / 1 antibody product and an effective amount of at least one other anticancer product. In some embodiments, such combination use may include administering an effective combination of the LILRB2 / 1 antibody product and at least one other anticancer product. In this case, the LILRB2 / 1 antibody product and at least one other anticancer product may be therapeutically complementary to each other, and one or both may be administered in less than therapeutic doses, but the combination may still be therapeutically effective. Such combination use may limit or mitigate adverse effects in the patient because it may provide an opportunity for clinical use in situations where the therapeutic width of one or another component of the combination is narrow.

[0264] As an example of a combination with immunotherapy, a method is provided for treating a patient with cancer, comprising administering a therapeutically effective dose of a LILRB2 / 1 antibody product and one or more immune checkpoint inhibitors to the patient.

[0265] The term “immune checkpoint inhibitor” or “checkpoint inhibitor” generally refers to drugs that modulate immune checkpoint proteins (“checkpoint proteins”). Checkpoint inhibitors can achieve overall or partial reduction, inhibition, or interference with the activity of checkpoint proteins, or they can alter the binding of checkpoint proteins to their ligands by causing other changes to the structure of checkpoint proteins, and / or they can affect pathways related to checkpoint protein activity by acting, for example, as an antagonist to checkpoint proteins or their ligands. Immune checkpoint inhibitors may 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 called PD-1 antagonists and PD-L1 antagonists, respectively.

[0266] "PD-1 antagonist" means any chemical compound or biological molecule that inhibits 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 inhibits 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 therapeutic method, pharmaceutical product, or disclosed use in which a human individual is treated, a PD-1 antagonist inhibits the binding of human PD-L1 to human PD-1, preferably inhibiting the binding of both human PD-L1 and PD-L2 to human PD-1. The amino acid sequence of human PD-1 can be found in NCBI Locus No. NP005009. The amino acid sequences of human PD-L1 and PD-L2 can be found in NCBI Locus No. NP054862 and NP079515, respectively.

[0267] As disclosed herein, checkpoint proteins can interfere with T cell-mediated killing of cancer cells under certain circumstances and conditions. While checkpoint inhibitors can reverse the interference of checkpoint proteins, interference with checkpoint proteins is insufficient in certain types of cancer (e.g., certain solid tumors). This disclosure aims to mitigate macrophage-mediated T cell exhaustion and stimulate T cell effector function by combining the antibody products provided herein with other checkpoint inhibitors.

[0268] Checkpoint inhibitors can inhibit one or more checkpoint proteins. Non-exclusive examples of checkpoint proteins include PD-1, CD28, CTLA-4, ICOS, TMI IGD2, 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.

[0269] Checkpoint inhibitors can interact with ligands for 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, Examples include 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.).

[0270] Checkpoint inhibitors can be antagonists. For example, checkpoint inhibitors can antagonize checkpoint proteins. Checkpoint inhibitors can be antagonists to ligands of checkpoint proteins. Antagonists can be biological molecules, such as biopharmaceuticals. Checkpoint inhibitors can be antibodies or their antigen-binding moieties, such as monoclonal antibodies, humanized antibodies, fully human antibodies, fusion proteins, or combinations thereof. Checkpoint inhibitors can be small molecules. Checkpoint inhibitors can be rationally designed peptides. Checkpoint inhibitors can be cells or cell preparations (e.g., cells expressing the checkpoint inhibitor).

[0271] Checkpoint inhibitors can inhibit PD-1. Programmed cell death 1 (PD-1) is an important checkpoint receptor expressed by activated T cells and activated B cells that mediates immunosuppression. In particular, PD-1 limits T cell activity in peripheral tissues during the inflammatory response to infection. Furthermore, as a checkpoint protein, PD-1 inhibition can enhance T cell proliferation and cytokine production in response to antigen stimulation by specific antigen targets or allogeneic cells in mixed lymphocyte reactions.

[0272] While not intended to be theoretically bound, PD-1 inhibition, in combination with the LILRB2 / 1 antibody products disclosed herein, is thought to reduce macrophage-mediated T cell suppression / exhaustion, increase T cell proliferation and cytokine production, and improve immune cell effector function. PD-1 inhibition can be achieved by various mechanisms. For example, PD-1 inhibition can be achieved by inhibiting the binding of PD-1 to its ligand. PD-1 can be inhibited with checkpoint inhibitors, which are PD-1 antagonists. For example, PD-1 antagonists may be PD-1 antibodies (e.g., nivolumab, pembrolizumab, etc.). PD-1 antagonists can be small molecules [e.g., INCB-086550 (Incyte), or small molecules disclosed in, for example, Wu et al., Acta Pharmacol Sin. 2021 42:1-9, Jiao et al., Curr Pharm Des. 2018 24(41):4911-20, and Liu et al., Cancer Cell Int. 2021 21(1):239]. PD-1 antagonists can be or may contain reasonably designed peptides (e.g., APi2568). PD-1 antagonists can be or may contain cells or cell preparations (e.g., PD-1 conjugates, e.g., PD-1 antibodies, e.g., cells expressing HerinCAR-PD1).

[0273] Examples of PD-1 antibodies suitable for use in the method include, but are not limited to, nivolumab (ONO-4538, BMS-936558, MDX1106, Opdivo®; Bristol-Myers Squibb), pembrolizumab (MK-3475, Keytruda®; Merck), and semipirimab (e.g., semipirimab-rwlc (Libtayo)). TM ;Regeneron)), dostallimab (for example, dostallimab-gxly (Jemperli TM(GlaxoSmithKline), pimivalimab (IgG4) (JTX-4014; Jounce Therapeutics), spartalizumab (IgG4) (PDR001; Novartis), camrelizumab (SHR1210; Jiangsu HengRui Medicine), cintilimab (IBI308; Innovent and Eli Lilly), tisrelizumab (BGBA317; BeiGene), tripalimab (JS001; Shanghai Junshi) Examples include PD-1 binding domains (Bioscience), INCMGA00012 (MGA012; Incyte and MacroGenics), AMP-224 (PD-L2 / Ig fusion; AstraZeneca and GlaxoSmithKline), AMP-514 (IgG4κ) (MEDI0680; AstraZeneca), valstilimab (AGEN2034; Agenus), and / or any of these.

[0274] Another exemplary PD-1 antagonist is a rationally designed peptide, such as APi2568, which contains a B cell epitope (amino acids 92-110 derived from PD-1) linked to an indiscriminate T cell epitope (amino acid residues 288-302 derived from measles virus fusion protein) via a 4-amino acid linker, and, when combined with water for injection (WFI), forms formulation IMU-201, becoming PD1-Vaxx when emulsified with the excipient Montanide ISA 720 VG.

[0275] Another exemplary PD-1 antagonist is a cell that expresses a PD-1 antibody, such as a PD-1 antibody-expressing CAR-T cell (e.g., a HerinCAR-PD1 cell).

[0276] In some embodiments, the checkpoint inhibitor is a PD-L1 antagonist, such as a PD-L1 antibody. In some embodiments, the PD-L1 antibody is selected from the group consisting of avelumab, durvalumab, atezolizumab, emvafolimab, cosivelimab, LY3300054 CA-170, BMS-936559, and PD-L1 binding fragments or combinations thereof. In some embodiments, the PD-L1 antagonist includes a PD-L1 binding domain containing a CDR of an antibody selected from the group consisting of AUNP-12, BMS-986189, avelumab, durvalumab, atezolizumab, emvafolimab, cosivelimab (CK-301), LY3300054, CA-170, and BMS-936559, as well as their active fragments or combinations thereof.

[0277] Checkpoint inhibitors can inhibit cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or its ligand. CTLA-4 antibodies bind to CTLA-4, inhibiting its interaction with its ligand CD80 / CD86, which is then expressed on antigen-presenting cells. Therefore, CTLA-4 inhibitors that inhibit the interaction between CTLA-4 and its ligand can inhibit the negative downregulation of the immune response induced by the interaction of these molecules. Accordingly, checkpoint inhibitors can be CTLA-4 antagonists as described in U.S. Patents 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, among others. Furthermore, exemplary CTLA-4 antibodies include ipilimumab (10D1, MDX-D010, Yervoy) TMExamples include Bristol-Myers Squibb, tremelimumab (tisilimubab, CP-675, 206, Imjudo®; AstraZeneca), and quavonlimab (MK-1308; Merck). CTLA-4 antagonists may include the CTLA-4 binding domain or fragments thereof of any CTLA-4 antagonist. CTLA-4 antagonists may also include small molecules (see, for example, Wang et al., Biochim Biophys Acta Rev Cancer. 2019 1871(2):199-224).

[0278] Lymphocyte activation gene 3 (LAG-3, also known as CD223) is a CD4-related 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 may be LAG3 antagonists. LAG3 antagonists may be LAG-3 binding proteins (e.g., antibodies) or proteins that bind to LAG3 ligands. Non-specific examples of LAG-3 antibodies include LAG525 (IMP701, Novartis / Prima Biomed), MK-4280 (Merck Sharp & Dohme), REGN3767 (Regeneron Pharmaceuticals), relatrimab (BMS-986016, Bristol-Myers Squibb), and BI754111 (Boehringer Ingelheim).

[0279] T-cell immunoglobulin mucin 3 (TIM-3, also known as hepatitis A virus cell receptor (HAVCR2)) is a type I glycoprotein receptor that binds to galectin-9 (Gal-9), an S-type lectin. TIM-3 is a ligand widely expressed on lymphocytes, liver, small intestine, thymus, kidney, spleen, lung, muscle, reticulocytes, and brain tissue. Binding of Gal-9 by the TIM-3 receptor triggers downstream signaling, negatively regulating T-cell survival and function. Checkpoint inhibitors may be drugs that inhibit TIM-3. Checkpoint inhibitors may be TIM-3 antagonists, such as antibodies against TIM-3 antibodies or TIM-3 ligands. TIM-3 antagonists may contain the TIM-3 binding domain or fragments thereof of any TIM-3 antagonist. 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, for example, U.S. Patent Nos. 9,103,832, 8,552,156, 8,647,623, 8,841,418, U.S. Patent Publications 2016 / 0200815, 2015 / 0284468, 2014 / 0134639, and This information is disclosed in publications 2014 / 0044728, 2012 / 0189617, 2015 / 0086574, 2013 / 0022623, and PCT publications 2016 / 068802, 2016 / 068803, 2016 / 071448, 2011 / 155607, and 2013 / 006490.

[0280] T cell immunoglobulins and the ITIM domain (TIGIT) are inhibitory receptors 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. Checkpoint inhibitors can be TIGIT antagonists. TIGIT antagonists can bind to TIGIT or to TIGIT ligands. TIGIT antagonists can be TIGIT antibodies or antibodies against TIGIT ligands. TIGIT antagonists contain the TIGIT-binding domain or a fragment thereof of any TIGIT antagonist. Non-specific examples of TIGIT antagonists include tiragolumab (MTIG7192A, RG6058) (Genentech / Roche), AB154 (Arcus Bioscience), vivostrimab (MK-7684) (Merck), BMS-985207 (Bristol-Myers Squibb), ASP8374 (Astellas Pharma, Potenza Therapeutics), and ASP8374 (Astellas Pharma, Potenza Therapeutics).

[0281] An example of an anti-CD27 agonist is MK-5890 (Merck).

[0282] An example of an ICOS antibody is vopratelimab (JTX-2011; Jounce).

[0283] In some embodiments, the LILRB2 / 1 antibody product and the immune checkpoint inhibitor are co-formulated. In some embodiments, the LILRB2 / 1 antibody product and the immune checkpoint inhibitor are in separate formulations. In some embodiments, the LILRB2 / 1 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, with the dosage of each component controlled to provide a safe and effective treatment to the subject. In some embodiments, the LILRB2 / 1 antibody is administered with a co-formulation of pembrolizumab / quavonlimab (MK-1308A; Merck).

[0284] A method for treating cancer is also provided, comprising administering a therapeutically effective dose of the LILRB2 / 1 antibody product and colony-stimulating factor 1 (CSF1) antagonist provided herein. 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. CSF1 antagonists may be CSF1 antibodies or CSF1R inhibitors. Examples of such CSF1 antagonists include pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, emactozumab, AMG820, IMC-CS4, MCS110, PD-0360324, and cabilalizumab.

[0285] A method for treating cancer is also provided, comprising administering a therapeutically effective amount of the LILRB2 / 1 antibody product and agonist CD40 antibody provided herein.

[0286] A method for treating cancer is also provided, comprising administering a therapeutically effective amount of the LILRB2 / 1 antibody product and inhibitory CD47 antibody provided herein.

[0287] A method for treating cancer is also provided, comprising administering a therapeutically effective amount of the LILRB2 / 1 antibody product provided herein and an effective amount of a class IIa histone deacetylase (HDAC) inhibitor, such as TMP195.

[0288] A method for treating cancer is also provided, comprising administering a therapeutically effective amount of the LILRB2 / 1 antibody product and a TLR7 or TLR8 agonist provided herein. The Toll-like receptors TLR7 and TLR8 appear to be involved in the polarization of macrophages in the tumor microenvironment. Agonists of either or both of these receptors can promote the functional orientation of tumor-associated macrophages toward the M1-like phenotype. Reximod-loaded β-cyclodextrin nanoparticles have been reported to have an antitumor effect 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, for example, reximod (B848), motlimod (VTX-2337), and imiquimod.

[0289] Methods are also provided for treating disease conditions in subjects characterized by or mediated by LILRB2 expression by myeloid cells. Myeloid cells may be macrophages. Myeloid cells may be osteoclasts or osteoclast precursors. The method comprises administering a therapeutically effective dose of the LILRB2 / 1 antibody product disclosed herein to the subject.

[0290] Methods for treating or preventing bone metabolic disorders in the subject are also provided. Bone metabolic disorders may include osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or cancer metastasis to the bone, giant cell tumor, osteopenia, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, or osteogenesis imperfecta. Bone metabolic disorders may include osteoporosis. Osteoporosis may include postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of therapeutic drugs such as steroids or immunosuppressants, or osteoporosis associated with rheumatoid arthritis.

[0291] Other terms As used herein, the singular forms "a," "and," and "the" refer to multiple objects unless the context clearly indicates otherwise. Therefore, for example, a reference to "antibodies" refers to multiple antibodies.

[0292] As used herein, all numbers or numerical ranges include all integers within or encompassing such a range, and non-integer values ​​or integer values ​​within such a range, unless the context clearly indicates otherwise. Thus, for example, a reference to the range 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, references to the range of 1 to 5,000 times include 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x or 20x, as well as 1.1x, 1.2x, 1.3x, 1.4x or 1.5x, 2.1x, 2.2x, 2.3x, 2.4x or 2.5x, etc.

[0293] As used herein, the number "approximately" refers to a range that includes the number and extends from 10% below it to 10% above it. The range "approximately" refers to a range that extends from 10% below the lower limit of the range to 10% above the upper limit of the range.

[0294] As used herein, “intended,” “can,” “may be,” “possibly,” and “may be” all indicate what the inventors intend, which is functional and available as part of the subject matter provided. [Examples]

[0295] The following examples illustrate specific embodiments, but those skilled in the art will likely be able to conceive of variations and modifications. Therefore, only the limitations set forth in the claims should be imposed on the present invention.

[0296] Antibodies that bind to both LILRB2 and LILRB1 were discovered in rabbit B cells immunized with human LILRB2 protein. B cells from immunized rabbits were cultured at clonal density, and the IgG antibodies in the supernatant were evaluated for binding to human, as well as to LILRB1 and LILRB2 proteins, by enzyme-linked immunosorbent assay (ELISA).

[0297] Based on activity in a panel of functional and phenotypic assays using primary human macrophages and T cells, top LILRB2 / 1 clones were identified. Variable regions from positive hits were sequenced, cloned, and expressed as recombinant rabbit / human IgG1 and IgG4 Fc chimeras.

[0298] Selected clones were humanized using the in silico method.

[0299] Example 1: Immunization, cloning, and initial screening A dual LILRB2 / 1 antibody was discovered in 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: 43) (OncoResponse) using a standard immunization method. The rabbits were boosted on days 21, 42, and 73 after primary immunization, respectively. Pre-immunobleeding and test bleeding were evaluated for specific antibody titers by indirect ELISA. On day 83 after primary immunization, heparinized whole blood was collected for rabbit monoclonal antibody development. B cells from peripheral blood of both rabbits were collected after the final booster immunization, then isolated, purified, and cultured at clonal density using a proprietary method. Biopanning was performed using human LILRB2 ECD-human Fc fusion protein (SEQ ID NO: 44). B cell culture supernatants from 80 96-well plates were transferred to ELISA plates coated with human Fc-LILRB2 ECD fusion protein. Indirect ELISA was performed using secondary anti-rabbit IgG antibody (ImmunoPrecise, Antibodies Ltd.) for probing. Negative screening of positive candidates against unrelated human-Fc fusion proteins (ImmunoPrecise) was performed to recover antibodies specific to the target antigen. The B cell supernatants were also evaluated for their ability to block HLA-G-binding LILRB2-Fc (R&D Systems, No. 2078-T4) and LILRB1-Fc (R&D Systems, No. 2017-T2-050). The top 96 response wells were stored in standard RNA lysis buffer for antibody RNA isolation and recombinant plasmid DNA generation.

[0300] The top-level positive clone's rabbit antibody heavy and light chain (kappa) variable domains were cloned into separate mammalian expression vectors containing human IgG4 heavy chain and kappa constant domains. Recombinant monoclonal antibodies (rabbit-human chimeras) were generated using transfected CHO-K1 cells, purified using standard methods, and evaluated in several biochemical and cell-based functional assays.

[0301] Example 2: Antibody Humanization For humanization, we selected a rabbit / human chimeric clone, named B21A4, which is an IgG4 clone from among those identified in Example 1. Humanization was performed in silico using a proprietary methodology (fusion antibody, Belfast, Northern Ireland). This system generates a model of the parental variable domain to enable structure-induced humanization. The sequence was aligned to a panel of human germline sequences selected for preferred manufacturability characteristics, and non-human amino acids were converted using proprietary CDRx TM Humanization is performed using a humanization platform to graft the human sequence. The first humanization performed on the clone is EC 50 Since the criteria were met, only the first humanization process was performed.

[0302] Five humanized heavy chains and five light variable domain chains were generated for clone B21A4. These humanized mutants are generally referred to as B21H mutants in this specification.

[0303] Humanized amino acid sequences were submitted to GenScript. GenScript used its proprietary methodology to backtranslate each variable region sequence for mammalian cell expression and optimize the codons. Genes encoding the signal sequence + variable domain were synthesized in GenScript and cloned in-frame into the pTT5 vector with the heavy chain human constant IgG4 domain and the light chain human kappa constant domain. The resulting plasmid vectors, one for the light chain and one for the heavy chain, were transiently and co-transfected into HEK293-6E cells (National Research Council Canada), and the conditioned medium was collected after 7 days. Twenty-five IgG4 variants were expressed in mammalian cell cultures as combinatorial libraries of five light chains and five heavy chains. These IgG4 antibodies were named in the format "B21H4-XY," where "B21" refers to the parent clone, "H" signifies humanization, "4" signifies the IgG4 isotype, and the suffix -XY distinguishes between VH-VL combinations, with X and Y being 1-5 each, corresponding to the five humanized VH variants (VH1-VH5) and five humanized VL variants (VL1-VL5) shown in Table 3. Therefore, these IgG4 variant antibodies are named B21H4-11 to B21H4-55. In addition, a B21A4 chimera was included as a transfection control. The transfection supernatant concentrations of 25 humanized IgG4 variants and parent clone B21A4 chimeric antibodies were determined by IgG4-specific AlphaLISA® immunoassay (PerkinElmer, No. AL310C) (Table 4).

[0304] Next, these transfection supernatants were used to generate binding data for the B21H variant via ELISA using MSD (Meso Scale Diagnostics) instrumentation (Table 4). Success was defined as achieving a binding constant within twice that of the parent mAb, in which case EC 50The binding was determined by ELISA using a rabbit variable region fused to the human IgG4 constant region as the parent chimeric mAb. Based on the binding data obtained using transfection supernatant, 25 B21H IgG4 antibodies were selected down to 15 antibodies for further characterization. These 15 B21H IgG4 antibodies were expressed, purified by affinity chromatography, and assayed again for binding.

[0305] Next, all five humanized heavy chains were converted to IgG1 isotypes by using restriction enzyme cleavage sites common to vectors encoding either the human IgG4 or IgG1 constant region. These common restriction sites between the IgG4 and IgG1 vectors enable in-frame fusion of the variable and constant regions. Fifteen selected humanized IgG4 variants from the same set, as well as the original B21A4 chimera, were reexpressed as human IgG1 isotypes and tested for binding (Example 3 and Table 5). These IgG1 variants were named in the same format as the IgG4 variants described above, "B21H1-XY," where the prefix B21H1 signifies IgG1 and the suffix -XY is used to identify the same five heavy chain variants and five light chain variants. Thus, the 25 IgG1 variant antibodies are named B21H1-11 to B21H1-55.

[0306] Example 3: Antibody binding to LILRB1 and LILRB2 (human and cynomolgus monkey) by ELISA To confirm binding to human LILRB1 and human LILRB2, the transfection supernatant (obtained in Example 2) was evaluated for binding by ELISA. The conditioned media from these transient transfections were assayed for human IgG concentration using the AlphaLISA® immunoassay (PerkinElmer, No. AL310C) (Table 4). Plate-based MSD ELISA with immobilized human LILRB1-His or LILRB2-His was performed, and binding of the transfection supernatant of the humanized mAb mutant was detected via an anti-human IgG sulfo-tag labeled secondary antibody. IgG1 type from transfections of parental rabbit / human chimeric clones (named B21A1) was compared to the benchmark EC. 50 It served as a positive control. Antibody titer and EC 50 The values ​​are shown in Table 4.

[0307] Recombinant human and cynomolgus monkey LILRB1 protein (R&D Systems, No. 8989-T2-050; ACROBiosystems, No. CDJ-C52H3), and recombinant human and cynomolgus monkey LILRB2 protein (ACROBiosystems, No. LI2-H5220, No. LI2-C52H3) were diluted to 4 μg / mL in PBS and added at a rate of 10 μL / well to a 384-well multi-array high-binding plate (MSD, No. L21XA4), and incubated overnight at 4°C. The plates were washed several times using a microplate washer with washing buffer (0.05% Tween® 20 in PBS), and then blocked at room temperature (RT) for 1 hour with shaking at 700 rpm using 70 μL / well of 5% BSA in PBS. After blocking, 20 μL / well of anti-LILRB2 / LILRB1 antibody or isotype control was added to the plate, the plate was sealed, and incubated at RT for 2 hours with shaking at 700 rpm. After primary antibody binding, the plate was washed five times using a microplate washer with 70 μL / well of washing buffer (0.05% Tween® 20 in PBS). Secondary detection antibody (SULFO-TAG) TMLabeled goat anti-human IgG antibody (MSD, No. R32AJ-1) was diluted to 4 μg / mL in assay diluent (1% BSA in PBS), and 10 μL / well was added to the plate. The plate was incubated in a light-shielded RT for 1 hour with shaking at 700 rpm. After incubation, the plate was washed five times with washing buffer using a microplate washer. After removal of the final wash, 35 μL / well of MSD Gold Read Buffer B (MSD, No. R60AM-4) was added to all wells using a Multidrop combo washer / dispenser, and the plate was read using a MESO Sector 600MM instrument (MSD) within 5 minutes (min) of adding the Read Buffer. The signal was recorded as counts per minute (cpm). EC 50 The values ​​were calculated using GraphPad Prism based on the logarithmic concentration versus cpm value of the primary antibody.

[0308] [Table 4]

[0309] The binding of 15 purified humanized IgG1 antibodies to human and cynomolgus monkey LILRB1 and human LILRB2 is shown in Table 5 using the MSD ELISA method described above. All antibodies showed specific binding to human LILRB1 and LILRB2, as well as weaker binding to cynomolgus monkey LILRB1. Only minimal binding between the antibody and cynomolgus monkey LILRB2 was observed (data not shown). Table 5 shows the EC binding results of the MSD ELISA using purified antibodies. 50 The value is shown. NT = Not tested.

[0310] [Table 5]

[0311] Example 4: Affinity of LILRB2 / 1 antibody by biolayer interferometry (BLI) Affinity measurements of human LILRB1 and LILRB2 bound to immobilized humanized LILRB2 / 1 antibody mutants were determined by BLI using a 16-channel Octet® RH16 instrument with an anti-human IgG Fc capture (ACH2) biosensor (Sartorius) and compared to B21A1, the IgG1 form of the parental clone. Dynamic buffers were prepared using PBS, pH 7.4 (Bulldog Bio, No. 2N18501) containing 0.1% BSA (Fisher Scientific, No. BP9704-100), 0.05% Tween®-20 (Fisher Scientific, No. BP337-500), and 0.02% sodium azide (NaN3; RICCA Chemical, No. 7144.8-32). The dynamic buffers were filtered through a 0.2 μm filter unit (Thermo Scientific, No. 5690020) before use. All antibodies and analytes (LILRB2 His: Acro Biosystems, No. LI2-H5220; LILRB1 His: R&D Systems, No. 8989-T2-050) were prepared in dynamic buffer. The experiment was set up using a 96-well, black, flat-bottom microplate (Greiner Bio-One, No. 655209). The biosensor tip was hydrated in dynamic buffer for at least 10 minutes before the start of the assay. An initial baseline in dynamic buffer was run for 60 seconds. LILRB2 / 1 antibody was loaded onto the ACH2 biosensor at 4 μg / mL for 45 seconds. Following the 60-second baseline in dynamic buffer, the loaded biosensor was exposed to a range of analytes (LILRB1 or LILRB2) at concentrations (0–40 nM) for 300 seconds for the association step. During dissociation, the biosensor was immersed in dynamic buffer for 300 seconds. The regeneration step was performed between changes in each antibody / analyte by immersion in glycine, pH 1.5 buffer (Cytiva, No. BR100354), followed by a neutralization step to dynamic buffer for 5 seconds and in triple cycles. Background subtraction was used to correct for any sensor drift. All experiments were performed with shaking at 1,000 rpm.Background wavelength shift was measured from a reference biosensor loaded with antibody only. Octet® Analysis Studio 12.2 software was used, along with Savitzky-Golay filtering, to extract association and dissociation rates, and the data were fitted to a 1:1 binding model. K. D is, K off / K on The calculation was performed using the ratio. The dynamic constant range in the RH16 system is 1 mM to 1 pM. Any affinity higher than 1 pM is K D Shown as <1 pM. All experiments were performed in triplicate and as independent experiments. The humanized LILRB2 / 1 antibody tested was similar to the parent B21A1 in terms of K D It has a value, and in the LILRB1 protein, K D The ratio is 1.3-4.2 pM, and in the LILRB2 protein, K D It is <1 pM.

[0312] Example 5: Preparation of cells expressing human LILRB2 Cells stably expressing human LILRB2 were generated using human embryonic kidney cells (HEK293T / 17; ATCC, No. CRL-11268) and cultured in DMEM (Gibco, No. 11965-084) supplemented with 10% FBS (HyClone, No. SH30396.03; thermally inactivated before use) according to ATCC guidelines. Pre-packaged lentiviral particles containing a puromycin-selectable lentivector scaffold were purchased from G&P Biosciences and were either lacking the target gene (negative control, No. LTV0001), possessing human LILRB1 (No. LTV2991), or possessing human LILRB2 (No. LTV2992). HEK293T cells (5 × 10⁶) 4Human LILRB2 lentivirus particles were transfected with 10 multiples of infection (MOI) into 10 cells at 5% CO2 and 37°C for 24 hours in the presence of 8 μg / mL polyblen infection / transfection reagent (Millipore, No. TR-1003-G). The virus-containing medium was then removed, and the cells were restored to fresh medium for 2 days. Following this, they were selected in medium containing 0.75 μg / mL puromycin (Thermo Fisher, No. A1113803). LILRB2-expressing cell populations were isolated and cultured by selecting stable LILRB2 expression based on puromycin as a selection marker. LILRB2 expression in the puromycin-resistant cell population was analyzed using BD FACSymphony. TM Confirmation was performed by cytometry and flow cytometry using LILRB2-specific antibodies. In addition to LILRB2 transduction, the same protocol was used to transduce negative control lentiviral particles into cells to create stable puromycin-resistant control cell lines (containing a lentivector skeleton without gene insertions) for use in the assay.

[0313] Once target expression was confirmed, the cells were grown. A polyclonal pool of selected cells was built up at 4-5 million cells / mL / vial to create a master stock. Cell banking involved pelleting cells from the culture by centrifugation, removing the medium, resuspending the cells first in DMEM + 10% FBS at 4°C, and then adding an equal volume of 2× ATCC recommended freezing medium (DMEM + 10% FBS + 10% DMSO) so that the cells were at a density of 4-5 million cells / mL in 1× freezing medium (DMEM + 10% FBS + 5% DMSO). The cell suspension was then divided into smaller portions and stored in Mr. Frosty at 4°C. TM The cells were placed in an alcohol-based slow freezing system and immediately stored at -80°C. These frozen cell portions were stored at -80°C for one day and then transferred to a liquid nitrogen vapor tank.

[0314] For use in the assay, the master stock vial was thawed in a 37°C water bath for 1 minute, and the cells were pelleted by centrifugation. The cells were suspended in DMEM + 10% FBS, and the expression of the target protein was confirmed by flow cytometry. These cells were grown in culture to prepare 30 working stocks, and the expression of the target protein was confirmed by flow cytometry. These cells were grown in culture to prepare 30 working stocks.

[0315] Example 6: The bilayer LILRB2 / 1 antibody binds to cells expressing human LILRB1 and LILRB2. LILRB2 expression is primarily limited to bone marrow cells, while LILRB1 has broader expression in a subset of immune cells, including B cells, T cells, and NK cells. To confirm the binding of LILRB2 / 1 antibodies to cells expressing these receptors, flow cytometry binding assays were performed using HEK293 T cells modified to stably express human LILRB2 (Example 5), or the human B-cell lymphoma cell line 721.221, which naturally expresses human LILRB1. The LILRB1 binding of anti-LILRB2 / 1 antibodies was characterized using the B-cell lymphoma cell line 721.221 wild-type immortalized cancer cell line.

[0316] Frozen cells were removed from storage in gas-phase liquid nitrogen, thawed by gently swirling in a 37°C water bath, and then resuspended in DMEM medium containing 10% FBS. The cells were placed in a 15 mL conical tube, centrifuged at 300 × g for 5 minutes, and the supernatant was removed. The cells were then cultured in a T75 flask at a rate of 2 × 10⁶. 5 Cells were resuspended in DMEM + 10% FBS at a cell density of cells / mL, incubated at 5% CO2 and 37°C for 3 days, and then subcultured. The cells were then passed through to a cell density of 1 × 10⁶. 6Subculture was carried out when the cell density reached cells / mL. On the day of the assay, the cells were centrifuged and then the supernatant was removed. The cells were resuspended in blocking buffer B (FACS buffer containing 10% FBS and 0.5 mg / mL of human IgG1 (Athens Research, No. 16 - 16 - 090707 - 1M) + 0.05% NaN3, 5% (w / v)) in a 15 mL conical tube and then incubated at 4 °C for 30 minutes. 25 μL of the cells in blocking buffer B were transferred to a 384 - well plate at 2.5×10 4 cells / well, and 25 μL of the serially diluted AF647 - conjugated LILRB2 / 1 antibody or AF647 - conjugated IgG1 isotype control was added directly to each well at 2× the final assay concentration. The cells were incubated with the antibody at 4 °C for 1 hour. The cells were washed twice in FACS buffer (PBS containing 2 mM EDTA + 1% FBS + 0.05% NaN3), and then stained with Zombie Violet TM Fixable Viability Dye (BioLegend, No. 423114) for 10 minutes at room temperature, washed with FACS buffer, and resuspended in 75 μL of FACS buffer for acquisition on a BD FACSymphony TM or a BD FACSCanto TM II flow cytometer (BD Biosciences) as described in Example 6. The mean fluorescence intensity (MFI) binding of the LILRB2 / 1 antibody was gated on live cells using FlowJO software (10.5.3, FlowJO, LLC) and GraphPad Prism for EC 50 binding calculations (Table 6). The LILRB2 / 1 antibody bound to LILRB2 - expressing HEK293T cells in a dose - dependent manner and had an EC 50 value of 0.7 - 2.2 ng / mL. The LILRB2 / 1 antibody had an EC 50The antibody bound to 721.221 B-cell lymphoma cells at a value. The binding curves of the antibody to wild-type 721.221 cells (Figure 1A) and HEK293T-LILRB2 (Figure 1B) are shown. In Figure 1B, anti-LILRB1 antibodies (Comp1, VH SEQ ID NO: 43 and VL SEQ ID NO: 44) are included as negative controls, showing minimal binding to LILRB2-HEK293T cells, while anti-LILRB2 antibodies (Comp2, VH SEQ ID NO: 45 and VL SEQ ID NO: 46) are included as positive controls, showing the binding of the tested LILRB2 / 1 antibodies. NT = Not tested.

[0317] [Table 6]

[0318] Example 7: The LILRB2 / 1 antibody does not bind to other LILRB or LILRA family members. Cross-reactivity assays were performed to determine that the parental chimeric anti-LILRB2 / 1 antibody specifically binds to LILRB2 and LILRB1, but not to other LILRB and LILRA family members. Cross-reactivity was assessed by flow cytometry using antibodies that bound to transiently transfected HEK293-6E cells. Binding to HEK293-6E cells transiently transfected with human LILRB-1, -2, -3, -4, and 5 plasmids (Origene, No. RC219949, RC217935, RC211228, RC220932, RC206516) or cells transiently transfected with human LILRA-1, -2, -4, -5, and -6 plasmids (Origene, Nos. RC210808, RC205626, RC220452, RC212310, RC212965) was evaluated. AF647 mouse anti-human IgG-Fc secondary antibody (Jackson ImmunoResearch, No. 109-605-098) was used to detect bound human anti-LILRB2 / 1 antibodies on the cells. Specific binding to all tested targets was confirmed using mouse primary antibodies against LILRA and LILRB targets and corresponding isotype controls (R&D Systems). AF647 F(ab')2 fragment goat anti-mouse antibody (Jackson ImmunoResearch, No. 115-606-062) was used to detect bound positive control antibodies. Binding of parental B21A1 and humanized IgG1 mutants to other LILRA and LILRB family members is shown in Tables 7 and 8. No binding was observed to B21A1 or its humanized mutants with LILRB-3, -4, or -5 at a concentration of 1 μg / mL (Table 7). In addition, no binding was observed to LILRA-1, -2, -4, -5, or -6, confirming the specificity of B21A1 and its humanized mutants to LILRB1 and LILRB2 (Table 8).

[0319] [Table 7]

[0320] [Table 8]

[0321] Example 8: The LILRB2 / 1 antibody blocks LILRB2-Fc and LILRB1-Fc, which bind to HLA-G expressed in tumor cells. The binding of LILRB1 or LILRB2 on macrophages to HLA-G on cancer cells enhances the immunosuppressive function of myeloid cells. The ability of LILRB2 / 1 antibodies to block HLA-G binding in cancer cells is considered crucial for the clinical efficacy of anti-LILRB2 antibodies. LILRB2 / 1 antibodies were evaluated via flow cytometry to block the binding of recombinant LILRB1-Fc or LILRB2-Fc proteins to HLA-G expressed in 721.221B lymphoma cells.

[0322] Recombinant human LILRB1-Fc-Avitag protein (ACRO Biosystems, No. CDJ-H82F7) or LILRB2-Fc-Avitag protein (ACRO Biosystems, No. LI2-H82F5) was diluted to 7.5 μg / mL with FACS buffer and mixed with anti-LILRB2 / 1 antibodies at final concentrations of 40, 13, 4, or 1.5 μg / mL (for LILRB1-Fc protein) and 15, 7.5, 3.75, and 1.88 μg / mL (for LILRB2-Fc protein) in equal proportions by volume, and then incubated at 4°C for 1 hour. Next, B cells (721, 221) were washed with PBS and counted, and then 1.3 × 10⁶ cells were added to Fc blocks in FACS buffer (PBS containing 10% normal goat serum (Sigma, No. G6767), 2.5% FBS, 1% mouse anti-human CD32 (BD Biosciences, No. 555447), 2 mM EDTA, and 0.05% NaN3). 6 pieces / mL(50×10 3 The cells were resuspended at a cell density of (cells / well) and incubated at RT for 30 minutes. The cells were then washed with FACS buffer (100 μL / well), and the plate was pelleted by centrifugation at 450 × g for 5 minutes. The supernatant was removed, and a second wash was performed using FACS buffer at 250 μL per well, followed by pelleting again by centrifugation at 450 × g for 5 minutes.

[0323] In all blocking assays, 1:250 dilution of streptavidin-PE (BioLegend, No. 405204) (75 μL / well) was added, and the cell / antibody mixture was incubated in light-shielded RT for 30 minutes. The cells were then washed twice with PBS containing 1 mM EDTA at a volume of 200 μL / well, and then pelleted by centrifugation at 450 × g for 5 minutes. The cells were then placed in 50 μL of Zombie Violet cells. TM The cells were resuspended in viability dye (1:2000 dilution) and incubated in light-shielded RT for 10 minutes. A final wash was performed with 200 μL / well of FACS buffer, 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 6. The dual anti-LILRB2 / 1 clone blocked the binding of LILRB1-Fc and LILRB2-Fc to HLA-G expressing 721.221 cells in a dose-dependent manner, while the IgG1 isotype control did not affect the binding of LILRB2-Fc (Figure 2A) or LILRB1-Fc (Figure 2B) to 721.221 cells.

[0324] Example 9: Isolation and differentiation of primary human cells The evaluation of anti-LILRB2 / 1 antibodies in immunological assays requires the isolation of human T cells and monocytes, as well as the differentiation of monocytes into immunosuppressive macrophages. Various techniques are known in the art, such as 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 described techniques or another technique commonly used in the art. Briefly, human monocytes and T cells are isolated from white blood cells (WBC) according to standard techniques. (LeukoPak, No. 4510 - 01 Full LeukoPak, BloodWorks Northwest, Seattle, WA). Peripheral blood mononuclear cells (PBMC) 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). The supernatant is discarded, and the pellet is resuspended in 20 mL of EasySep TM buffer (STEMCELL Technologies, No. 20144) for counting PBMC and for further isolation of monocytes and T cells. Monocytes are isolated using the EasySep TM Human Monocyte Isolation Kit (STEMCELL Technologies, No. 19359) according to the manufacturer's instructions. Total CD8 + T cells are isolated using the EasySep TM Human CD8 + T Cell Isolation Kit (STEMCELL Technologies, No. 17953), and total NK cells are isolated using the EasySep TM Human NK Cell Isolation Kit (STEMCELL Technologies, No. 17955), and these are according to the manufacturer's instructions. These negative selection kits use antibodies to label unwanted cell types for removal, enabling the isolation of the desired target cells from untreated samples.

[0325] Example 10: Macrophage Generation Macrophages can be generated from PBMC-derived monocytes using commonly used techniques, as described below.

[0326] M0 macrophage generation: On day 0, monocytes (isolated from individual subjects as described in Example 9) were cultured in M0 culture medium (90% X-VIVO2). TM 15 + 10% FBS + 100 ng / mL human M-CSF (PeproTech, No. 300-25) containing 25-50 x 10 3 Cells were seeded at 100 μL / well in a 96-well culture plate (Thermo Fisher (Costar), No. 09-761-175). Cells were incubated at 37°C and 5% CO2 for 5-6 days to produce M0 macrophages.

[0327] Generation of immunosuppressive M2c macrophages: On day 5 of culture, M0 macrophages were polarized to M2c macrophages by gently aspirating the culture 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% CO2 for 2 days. On days 7-8 of culture, the M2c macrophages were ready for co-culture assay setup. The M2c macrophages were separated from the plates by incubation with Macrophage Detachment Solution DXF (PromoCell, No. C-41330) and washed with PBS before downstream assay.

[0328] Example 11: Generation of exhausted T cells Exhausted T cells are an indicator of the immunosuppressive tumor microenvironment and contribute to evasion of cancer immunity. To mimic exhausted T cells in TMEs, exhausted T cells with blast-like morphology were generated from human PBMCs by repeated (3×) phytohemagglutinin (PHA) stimulation. The cells were counted and 1×10⁶ cells were cultured 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)). 6 Cells were incubated at a concentration of cells / mL. Cells were divided into 1:2 or 1:3 groups every 3-4 days and cultured for a total of 10 days (2 divisions over 10 days; 3 total PHA stimulations). Fresh T cell blast culture medium was added to the cells at each division. Cells were harvested on day 10 and set up for co-culture assays or frozen for future use. The phenotype of exhausted T cells was confirmed by the expression of PD-1, TIM-3, and TIGIT, as well as the transcription factor EOMES (data not shown).

[0329] Example 12: LILRB2 / 1 antibody binds to monocytes, M0, and M2c macrophages. The ability of anti-LILRB2 / 1 antibodies to bind to LILRB1 and LILRB2-expressing myeloid cell subsets, including monocytes, M0, and M2c macrophages, was evaluated. M2c macrophages were used as a substitute for tumor-associated macrophages, which are suppressor macrophages commonly found in the tumor microenvironment. Frozen monocytes were removed from storage in gas-phase liquid nitrogen, thawed by gentle swirling in a 37°C water bath, and then incubated in assay medium (X-VIVO). TM The cells were resuspended in medium (15 + 10% FBS). The PBMCs were placed in a 15 mL conical tube, centrifuged at 300 × g for 5 minutes, and then the supernatant was removed. The cells were measured at 2.5 × 10⁶. 5The cells were resuspended in Assay Medium at a cell density of cells / mL. Cells were seeded in 96-well flat-bottom plates at 100 μL / well (25K cells / well). M0 and M2c macrophages were generated as described in Example 11 and harvested from the flask by incubation at RT for 15 minutes in Macrophage Detachment Solution DXF, and then removed from the flask into the assay medium. After centrifugation, the cells were resuspended in Blocking Buffer B (FACS buffer + 10% FBS + 0.5 mg / mL human IgG1 + 0.05% NaN3, 5% (w / v)) and incubated at 4°C for 30 minutes. Cells in 25 μL of Blocking Buffer B were divided into 2.5 × 10⁶ cells. 4 Cells were transferred to a 384-well plate at a concentration of cells / well, and 25 μL of titrated AF647 conjugate LILRB2 antibody or AF647 conjugate IgG1 isotype control was added directly to each well at the 2× final assay concentration. Cells were incubated with antibody at 4°C for 1 hour. Cells were washed twice with FACS buffer and then exposed to Zombie UV light. TM The samples were stained with a viability dye (BioLegend, No. 423107) (1:500 dilution) in the dark for 15 minutes using RT, washed with FACS buffer, and resuspended in 200 μL of FACS buffer for acquisition by flow cytometry as described in Example 6. GraphPad Prism was then subjected to EC2. 50 Used in the combined calculation, the mean EC for the two subjects tested. 50 This is shown (Table 9). B21A1 and the humanized LILRB2 / 1 mutant bind to human monocytes (Figure 3A), M0 macrophages (Figure 3B), and M2c macrophages (Figure 3C) in a dose-dependent manner. Representative targets for each cell type are shown in the dose-titer graph in Figure 3.

[0330] [Table 9]

[0331] Example 13: LILRB2 / 1 antibody binds to a subset of immune cells in human PBMCs. To further characterize the binding of LILRB2 / 1 antibodies to LILRB1-expressing immune cell subsets (i.e., B cells, T cells, and NK cells), binding assays were performed using primary human PBMCs. Single-cell suspensions of peripheral blood mononuclear cells (PBMCs) from healthy donors were seeded at 500,000 cells per well in 96-well plates (Thermo Fisher, No. 249946). PBMCs were blocked at 4°C for 45 minutes to 1 hour with 0.5 mg / mL of human IgG1 myeloma plasma (Athens Research, No. 16-16-090707-1M) in FACS buffer (PBS containing 2% FBS, 2 mM EDTA, and 0.05% NaN3). Immunotherapy phenotyping antibodies and the indicated AlexaFluor647-labeled test antibodies were then added to the cells, and the cells were incubated at 4°C for 45 minutes to 1 hour. The antibodies used for immunocytophenotyping included BUV496 anti-human CD14 (BD Biosciences, No. 741200), BV711 anti-human CD4 (BioLegend, No. 300558), BV786 anti-human CD16 (BD Biosciences, No. 563690), FITC anti-human CD56 (BioLegend, No. 318304), PE anti-human CD8 (BioLegend, No. 301008), PE-Cy7 anti-human CD19 (BioLegend, No. 302216), and APC / Fire750 anti-human CD3 (BioLegend, No. 300470).

[0332] Wash cells 1× with PBS and use Zombie Violet for viable cell identification. TM Cells were stained with a fixable viability dye using RT for 15 minutes. Cells were washed 1× with FACS buffer, fixed, and acquired using a FACS Symphony cytometer (BD Biosciences). Data analysis was performed using FlowJo software (v10.8.1). Gating was performed on the viable cell population, and classical monocytes (side-cutters (SSCs)) were analyzed. High CD3 - CD19 - CD14 + CD16 - ), intermediate monocytes (SSC)High CD3 - CD19 - CD14 + CD16 + ), non-classical monocyte (SSC) High CD3 - CD19 - CD14 - CD16 + ), B cells (SSC Low CD19 + ), CD4 T cells (SSC Low CD19 - CD3 + CD4 + CD8 - ), CD8 T cells (SSC Low CD19 - CD3 + CD4 - CD8 + ), CD56 Bright NK cells (SSCs) Low CD19 - CD3 - CD56 Bright CD16 - ), and CD56 Dim CD16 + NK cells (SSCs) Low CD19 - CD3 - CD56 Dim CD16 + ) was identified. The LILRB2 / 1 antibody targets classical, intermediate, and non-classical monocytes, as well as B cells, CD8 + T cells and CD56 dim CD16 + Binding to NK cells was observed (Figures 4A-D, 4F, 4H). Very little binding was observed to CD4 + Observed in T cells (Figure 4E), the target-dependent variable binding of CD56 bright CD16 - This was observed in NK cells (Figure 4G).

[0333] Example 14: LILRB2 / 1 antibody enhanced the IFN-γ response in LPS-stimulated PBMCs. To determine whether LILRB2 / 1 blockade enhances the innate immune response, subsequently increasing IFN-γ secretion and reducing IL-10 release, thereby decreasing immunosuppressive TME, we evaluated the effects of LILRB2 / 1 antibodies on IFN-γ and IL-10 secretion by lipopolysaccharide (LPS)-responsive PBMCs.

[0334] Frozen PBMCs were removed from storage in gas-phase liquid nitrogen, thawed by gently swirling in a 37°C water bath, and then resuspended in Assay Medium. The PBMCs were placed in a 15 mL conical tube, centrifuged at 300 × g for 5 minutes, and the supernatant was removed. Cells were divided into 3 × 10⁶ cells. 6 Cells were resuspended in Assay Medium at a concentration of cells / mL. Cells were seeded at 100 μL / well (300 K cells / well) in 96-well flat-bottom plates. This cell density is optimized for the mean level of IFN-γ response per subject. Fewer cells were seeded per well if the subject was known to elicit a higher cytokine response. Cells were allowed to stand at 37°C in 5% CO2 for 1 hour.

[0335] LILRB2 / 1 antibody X-VIVO TM The antibody was diluted to a 4x concentration (final concentration of 1 μg / mL) using 15, and 50 μL / well of the diluted antibody was added. 50 μL / well of X-VIVO was added to the wells where no antibody was added. TM 15 was added. The plate was incubated in 5% CO2 at 37°C for 2 hours before LPS stimulation. LPS (InvivoGen, No. Tlrl-pb5lps) was X-VIVO TM The solution was diluted to a 4× concentration (final concentration of 1 μg / mL in the assay) at 15°C, and 50 μL / well was added to the cell / antibody mixture; however, in control samples with known higher IFN-γ response, the final LPS concentration was 0.1 μg / mL. 50 μL / well of X-VIVO was added to the control well (without LPS). TM15 was added. Cells were incubated at 200 μL / well in 5% CO2 at 37°C for 24 hours. After 24 hours of incubation, the supernatant (150 μL) was collected and either frozen at -80°C or tested directly for IFN-γ secretion and IL-10 secretion by ELISA (R&D Systems). LILRB2 / 1 antibodies induced a pro-inflammatory native Th1-like phenotype, and PBMCs showed enhanced IFN-γ secretion and reduced IL-10 release when stimulated with LPS. All LILRB2 / 1 antibodies evaluated showed enhanced IFN-γ secretion compared to isotype controls, and variability was observed between donors. Data from four representative subjects regarding enhanced IFN-γ secretion are shown in Figures 5A-5D. Data from three representative subjects regarding reduced IL-10 release are shown in Figures 14A-14C.

[0336] LILRB2 / 1 antibody and Toll-like receptor 2 (TLR) ligand, for example, heat-sterilized Listeria monocytogenes (HKLM, InvivoGen, No. Tlrl-hklm, 2.5 × 10⁻¹⁴). 7 Combinations with cells / mL and Pam3CSK4 (InvivoGen, No. Tlrl-pms, 100 ng / mL) were also tested. LILRB2 / 1 antibody was tested with HKLM (2.5 × 10⁶). 7 PBMCs treated with (cells / mL) or Pam3CSK4 (100 ng / mL) enhanced IFN-γ secretion and reduced IL-10 release (data not shown).

[0337] Example 15: The LILRB2 / 1 antibody enhanced TNF-α secretion in CD40-activated macrophages. CD40L, expressed on T cells, is a primary molecule responsible for macrophage activation in TME through binding to CD40 expressed on macrophages via intercellular contact. The CD40 / CD40L interaction is important for activating macrophages to act as effector cells that mediate inflammation in T cell-mediated inflammatory processes. The inventors have developed an assay to evaluate inflammatory cytokine production by macrophages treated with LILRB2 / 1 antibody without the presence of T cells.

[0338] A modified HEK-293 cell line expressing CD40L (CrownBio, No. C2041) was used to mimic activation by activated T cells binding and subsequent stimulation. (Alternatively, a stable HEK-293 cell line expressing CD40L can be generated using conventional methods.) M0 macrophages were generated from monocytes (25K cells / well) as described in Example 11. The medium was removed from the wells and 100 μL / well of fresh Assay Medium was added. 50 μL / well of LILRB2 / 1 antibody at 5× concentrations (final concentrations of 5 and 10 μg / mL) was added and incubated on the macrophages at 5% CO2, 37°C for 2 hours. During the 2-hour incubation, CD40L-expressing HEK-293 cells were harvested from the flask and irradiated with 40 Gray. After pre-incubation with LILRB2 / 1 antibody, CD40L-expressing HEK-293 cells (5 × 10⁶) 3 The LILRB2 / 1 antibody was added at a volume of 100 μL / well in a 96-well plate, and the final well volume was adjusted to 250 μL / well. The mixture was incubated overnight at 37°C in 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 using homogeneous time-resolved fluorescence (HTRF) (CisBio, No.62HTNFAPET) according to the manufacturer's instructions. All LILRB2 / 1 antibodies tested showed equivalent enhancement of TNF-α secretion by CD40L-activated macrophages at both 5 μg / mL and 10 μg / mL concentrations, as shown by two representative subjects (Figures 6A-6B).

[0339] Example 16: LILRB2 / 1 antibody is M2c / CD8 + To mitigate M2c-mediated immunosuppression in T cell co-culture assays. Crosstalk between LILRB1- and LILRB2-expressing immunosuppressive myeloid cells and T cells is involved in T cell exhaustion and lack of antitumor immune response in TME. This crosstalk involves autologous monocyte-derived M2c macrophages and anti-CD3 stimulated CD8 +This can be modeled by in vitro co-culture with T cells. The reduction of immunosuppression can be evaluated by quantifying T cell proliferation, as well as by measuring IFN-γ secretion as a surrogate for T cell activation and antitumor activity. After polarizing M0 macrophages to M2c macrophages as described in Example 11, the supernatant was removed from the macrophages in the 96-well culture plate and replaced with 100 μL of Assay Medium containing OKT3 (BioLegend, No. 317326) at a final concentration of 0.25 μg / mL. LILRB2 / 1 antibody (dose-titer of 40-0.63 μg / mL) was added in a volume of 50 μL / well, and the plate was then incubated in 5% CO2 at 37°C for 1-2 hours. M2c macrophages were incubated with the antibody, and at the same time, autologous CD8 + T cells were isolated from PBMCs as described in Example 9. Isolated CD8 + T cells were diluted 1:2000 with 1×PBS in CellTrace TM The cells were labeled with violet (Thermo Fisher, No. C34557) and then incubated at 37°C for 20 minutes. CellTrace TM After incubation, preheated culture medium is added in a volume five times that of the labeled cells, and CellTrace TM Quench the pigment. Excessive CellTrace TM Wash with 5x volume of preheated Assay Medium and label CD8 + T cells were centrifuged at 300×g, then resuspended in Assay Medium, and then mixed with 1:1 M2c:CD8 in a volume of 100 μL. + 5 × 10⁶ T cell ratio 5 Cells were added to the M2c / antibody preparation at a concentration of cells / mL. The cells were then incubated in 5% CO2 at 37°C for 72 hours.

[0340] The supernatant containing T cells was transferred to a V-bottom 96-well plate, centrifuged to pelletize the T cells, and the culture supernatant was collected and frozen at -80°C for quantification of human IFN-γ secretion by MSD-ELISA (MSD, U-plex assay). The T cell pellet was stained with e780 viability dye at RT for 10 minutes in the dark, washed with 150 μL of FACS buffer, and then processed using BD FACSymphony. TM or FACSCanto TM For acquisition using a flow cytometer (BD Biosciences), the samples were resuspended in 100 μL of FACS buffer. Proliferative CD8 + The percentage and total number of T cells were analyzed using FlowJO software, and CellTrace was used. + This was reported as the total number of dividing cells.

[0341] The LILRB2 / 1 antibody was tested on three representative subjects using CD8 + In the M2c / T cell co-culture assay, M2c-macrophage-mediated immunosuppression was mitigated, as measured by restoring T cell proliferation and IFN-γ secretion (Figures 7A and 7B, respectively). In comparison, LILRB2 / 1 antibodies (Comp3, VH SEQ ID NO: 47 and VL SEQ ID NO: 48) failed to restore T cell proliferation and enhance IFN-γ secretion (Figures 7A and 7B, respectively).

[0342] Example 17: LILRB2 / 1 antibody rescues the IFN-γ response of exhausted T cells in co-culture with M2c macrophages. A prominent feature of an ineffective anti-cancer immune response is T cell exhaustion in the tumor microenvironment. Exhausted T cells are those with reduced cytokine expression and effector function. Reversing T cell exhaustion and restoring antitumor activity is a promising strategy for treating cancer. We evaluated the ability of a LILRB2 / 1 antibody to rescue the functional activity of exhausted T cells from LILRB2 / 1-mediated immunosuppression using an assay that measures macrophage-mediated immunosuppression with exhausted T cells and M2c cells in co-culture.

[0343] The ability of the LILRB2 / 1 antibody to rescue the functional activity of T cell blasts from M2c-mediated immunosuppression was measured using a co-culture assay with exhausted T cells and M2c macrophages. After polarizing 50,000 cells / well of M0 macrophages to M2c macrophages as described in Example 11, the supernatant was removed from the macrophages in a 96-well culture plate and replaced with Assay Medium containing the LILRB2 / 1 antibody or isotype control, and the cells were incubated at 5% CO2, 37°C for 2 hours. The OKT3 antibody was added to the wells at a final concentration of 0.25 μg / mL and incubated at 5% CO2, 37°C for 30 minutes. T cell blasts (Example 12) were added last to the M2c / LILRB2 / 1 antibody mixture + OKT3 in a 1:1 ratio and incubated at 5% CO2, 37°C for 72 hours. IFN-γ levels were quantified by ELISA from the supernatant collected 72 hours after OKT3 stimulation. B21A1 and humanized IgG1 mutants rescued the IFN-γ response of T cells exhausted from M2c macrophage-mediated immunosuppression in a dose-dependent manner, as shown in three representative subjects (Figure 8).

[0344] Example 18: B21A1 and humanized mutants enhance the cytotoxicity of NK cells. The ability of the LILRB2 / 1 antibody to enhance the cytotoxic capacity of NK cells was tested in primary human NK cells from healthy donors. Primary NK cells were isolated from frozen PBMCs using immunomagnetically negative selection as described in Example 9. The isolated NK cells were then subjected to 5 mL of X-VIVO2. TM The cells were resuspended in 15 medium + 10% FBS + 100 IU / mL (50 ng / mL) IL-2 (Peprotech, No. 200-02), then transferred to a T25 flask and incubated overnight at 5% CO2 and 37°C. Target cancer cells (721.221 wild-type or 721.221 expressing HLA-G) were harvested from the flask, resuspended in Assay Medium, and then counted using a 1:1 dilution with trypan blue (Thermo Fisher, No. 15250061). 721.221 WT or 721.221-HLA-G cells were traced using CellTrace as described in Example 17. TMLabeled with violet (Thermo Fisher, No. C34557). Excessive cell trace. TM The cells were washed with pre-warmed Assay Medium, and the labeled target tumor cells were resuspended in the assay medium. NK cells were seeded in 96-well plates at a final cell density of 50,000 cells / well in a volume of 50 μL / well. LILRB2 / 1 antibody was added to the wells containing NK cells at a concentration of 2× in 50 μL / well, and pre-incubated at 5% CO2, 37°C for 1 hour. 721.221 target cells were seeded in addition to the NK cells in a volume of 50 μL / well for a final cell density of 10,000 cells / well and a final NK cell:target cell ratio of 5:1. The NK:target cell mixture was incubated at 5% CO2, 37°C for 4 hours. After incubation, the cells were transferred to a V-bottom 96-well plate, centrifuged at 300×g, and the supernatant was removed. Cells were resuspended in a 1:2000 dilution of e780 viability stain (Thermo Fisher, No. 65-0865-18) in PBS and incubated in the dark at room temperature for 10 minutes. Following viability staining, 150 μL / well of FACS buffer was added to each well, and the dye was removed by centrifugation at 300 × g for 5 minutes. The supernatant was removed, and the cells were processed using BD FACSymphony. TM or FACSCanto TM For acquisition using a flow cytometer (BD Biosciences), cells were resuspended in 100 μL / well of FACS buffer. The percentage of dead target cells was analyzed using FlowJO software and reported as a percentage of cell death. An increase in NK cell-mediated target cell killing was observed in 721,221 HLA-G expressing (Figure 9B) and wild-type (HLA-G absent) (Figure 9A) cancer cells in the presence of LILRB2 / 1 antibody compared to isotype controls.

[0345] Example 19: LILRB2 / 1 antibody induces minimal cytokine release in whole blood from healthy subjects. Immunomodulatory therapeutic antibodies carry the risk of cytokine release syndrome, a rapid systemic inflammatory response characterized by the secretion of inflammatory cytokines by immune cells. Antibody target binding can induce cytokines by directly activating lymphocytes and myeloid cells, or by interacting with Fcγ receptors on myeloid cells and NK cells. Whole blood in vitro cytokine release assays are standard assays used to assess the risk of therapeutic antibody-mediated cytokine release syndrome. We evaluated whether treatment with a humanized LILRB2 variant triggers the release of inflammatory cytokines in whole blood from healthy test subjects.

[0346] LILRB2 / 1 antibody and control antibody were diluted to 10× final concentration in PBS in a dilution plate. The diluted LILRB2 / 1 antibody (25 μL / well) was transferred to a 96-well plate. Whole blood was purchased from healthy subjects (Bloodworks Northwest). Whole blood (225 μL / well) was added to the LILRB2 / 1 antibody without mixing to avoid cell lysis. The blood / antibody mixture was incubated at 5% CO2, 37°C for 24-48 hours. After incubation, the plate was centrifuged at 350×g for 5 minutes to pellet the cells. Plasma (65 μL) was collected from the surface of each well and immediately evaluated for IL-6, TNF-α, IFN-γ, and IL-1β cytokine secretion by MSD-ELISA according to the manufacturer's instructions (MSD). Whole blood cells from eight subjects were used to compare the release of IL-1β, IL-6, IFN-γ, and TNF-α in response to LILRB2 / 1 antibody treatment with cytokine induction induced by corresponding human IgG1 isotypes, untreated controls, and anti-CD52 (aremtuzumab, BOC Sciences, No. B0084-305393) positive control antibodies. Treatment with LILRB2 / 1 antibody did not trigger significant release of IFN-γ and IL-6 (Figures 10A-10H) or TNF-α and IL-1β (data not shown) compared to human IgG1 isotype control treatment. In contrast, anti-CD52 antibody significantly increased the release of IFN-γ and IL-6 at all evaluated dose levels compared to IgG1 isotype controls.

[0347] Example 18: The LILRB antibody binds to bone marrow cells, granulocytes, and lymphocytes in the whole blood of healthy subjects. LILRB2 is exclusively expressed in whole blood myeloid cells such as monocytes and neutrophils, while LILRB1 is found in whole blood myeloid cells and lymphocytes. Whole blood immunophenotyping assays were performed to determine whether LILRB2 / 1 antibodies bind to the relevant immune cell populations in whole blood. Whole blood was purchased from healthy subjects (Bloodworks Northwest). The tube containing heparin-containing blood was gently inverted to evenly distribute the plasma and cells, and then the blood was mixed with a blocking mix (10% FBS + 500 μg / mL human IgG1 myeloma plasma (Athens Research) + 0.05% NaN3, 5% (w / v)). The blood / blocking mix was then thoroughly mixed, pipetted into a 96-well deep plate, and incubated at 4°C for 1 hour. Cells were resuspended at a second time point, targeting 30 minutes, during incubation with the blocking mix. Antibody titration was performed in FACS buffer and added to the blood at final concentrations of 1 and 0.1 μg / mL. The blood mixture with the primary antibody was incubated at 4°C for 45 minutes in the dark. After incubation, the plate was RT for 15 minutes and incubated for 1 hour. Erythrocytes were refrozen in 1× RBC lysis buffer (BD). RBCs were lysed using a three-step RBC lysis procedure using Bio (No. 555899): adding RBC lysis buffer to the blood mixture, thoroughly mixing by pipetting up and down, and then incubating in light-shielded RT for 10 minutes. After incubation, the cell plate was sealed and centrifuged at 200×g for 5 minutes, and the supernatant was aspirated from the wells. RBC lysis was stopped by adding 1×PBS, and then centrifuged at 200×g for 5 minutes, and the supernatant was removed. The cells were then transferred to a 96-well V-bottom plate. The cells were then resuspended in blocking buffer (10% FBS + 1:10 diluted FcX block + FACS buffer) and incubated in RT for 15 minutes.Flow cytometry fluorophore conjugate antibody cocktail (BV421 anti-human CD3 (BioLegend, No. 300434), BV711 anti-human CD4, APC / Cy7 anti-human CD8 (BioLegend, No. 344714), PE anti-human CD11c (BioLegend, No. 337206), BUV496 anti-human CD14, BUV805 anti-human CD15 (BD Biosciences, No. 742057), BV786 anti-human CD16, BV605 anti-human CD19 (BioLegend, No. 302244), FITC anti-human CD56, and PE / Cy7 anti-human HLA-DR (BioLegend, No. 307616) (Table 10A) were added directly to wells on block buffer and incubated in light-shielded RT for 30 minutes. After incubation, FACS buffer was added to the wells for washing, and the plate was centrifuged at 350 × g for 5 minutes and subjected to FACSymphony. TM The cells were resuspended in 350 μL of FACS buffer for cytometry acquisition. Populations were identified according to the gating parameters listed in Table 10B. Representative data from three subjects are shown in Figures 11A–11C, where LILRB2 / 1 antibody was evaluated for binding to whole blood myeloid cell populations and lymphocyte cell populations at 1 μg / mL. LILRB2 / 1 antibody bound to myeloid cells, including classical, non-classical, and intermediate monocytes, myeloid dendritic cells (mDCs), and neutrophils. B21A1 and humanized variants also bound to human CD8 + It bound to T cells, B cells, and NK cells (Figures 11A-11C).

[0348] [Table 10A]

[0349] [Table 10B]

[0350] Example 21: Pharmacokinetic profiling of LILRB2 / 1 antibody 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 (hFcRn), the half-lives of monoclonal antibodies tested in mice do not correlate with those 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 lysosomes, preventing its degradation. FcRn-bound IgG antibodies are recycled to the extracellular surface, and at physiological pH, IgG dissociates from FcRn and returns to circulation, extending the half-life of these IgG monoclonal antibodies compared to IgG that does not bind to FcRn, otherwise being processed for degradation by lysosomes (Roopenian et al., Nat Rev Immunol. 2007 7:715-25, Roopenian et al., J Immunol. 2003 170:3528-33, Challa et al., Curr Top Microbiol Immunol. 2014 382:249-72). Pharmacokinetic studies of monoclonal antibodies in wild-type (WT) mice, humanized FcRn (hFcRn) mice, and non-human primates (NHPs) have shown that hFcRn mice predicted the antibody PK profile in a clinical setting better than WT mice (Avery LB et al., MAbs 2016).

[0351] Different FcRn transgenic mice have been created to knock out mouse FcRn and express human FcRn. Several groups have demonstrated that the pharmacokinetics (PKs) of human therapeutic antibodies in these humanized FcRn mice correlate well with human PKs and are comparable to those in non-human primates (Petkova et al., Int Immunol. 2006 18(12):1759-69, Tam et al., MAbs. 2013 5(3):397-405, Wang et al., Drug Metab Dispos 2011 39(9):1469-77, Roopenian et al., Methods Mol Biol. 2010 602:93-104, Avery et al., MAbs 2016 8(6):1064-78, Proetzel et al., Methods. 2014 65(1):148-53). For example, the Tg32 and Tg276 mouse strains are engineered in a C57BL / 6 background. These mice are immunoqualified and possess all mouse immune cells, and in these models only mouse FcRn is deleted, while human FcRn is expressed.

[0352] Homozygous male Tg32 FcRn mice were purchased at 6-8 weeks of age (Jackson Laboratory, No. 014565) and housed in micro-isolate cages under specific pathogen-free conditions at the Bloodworks Northwest animal facility. All procedures were performed according to the Bloodworks Northwest Institutional Animal Care and Use Committee (IACUC) Protocol No. 5390-01 facility guidelines. Mice were identified using ear tags. All mice were acclimatized for a minimum of 5 days before the start of antibody administration. Initial body weight of each mouse was recorded on the day of administration. Mice were assigned to three LILRB2 antibody treatment groups: B21A1 (IgG1 parental chimera), B21H1-25, and B21H1-55, with 3 mice per group in the B21A1 group and 5 mice per group in the B21H1-25 and B21H1-55 groups. Mice received a single intravenous (IV) bolus dose of 10 mg / kg of B21A1, B21H1-25, or B21H1-55 antibody. Blood (20–50 μL) was collected from the lateral saphenous vein without anesthesia, according to the approved IACUC protocol. The saphenous vein puncture method allowed for repeated sampling from the same animal until the limit of less than 1% of body weight per two weeks was reached. Blood was collected from each mouse at the following time points: 0.15, 1, 2, 5, 24, 48, 100, 168, 240, and 336 hours. Whole blood was allowed to coagulate at RT for at least 30 minutes. Blood clots were removed by centrifugation (2000 × g) at 4°C for 10 minutes. Serum was aliquoted into new microtubes and frozen at -80°C until analysis.

[0353] Human LILRB2 capture ELISA (following the method of Example 3) was performed, and antibody concentrations were determined for each group. Stocks of B21A1 and humanized mutants (B21H1-25 and B21H1-55) were diluted in assay buffer (1% BSA in PBS) to prepare a maximum standard concentration of 300 ng / mL. 15-point 2-fold serial dilutions of the standard material were diluted with 0.01 to 0.2% mouse serum. Serum samples from anti-LILRB2 / 1 treated mice were also diluted with assay buffer. Different dilutions (1:1,000 to 1:40,000) were tested depending on the timing of serum collection and antibody administration. Each standard dilution and mouse serum dilution were assayed in double wells.

[0354] Standard curves for B21A1, B21H1-25, and B21H1-55 were generated by plotting CPM versus antibody concentrations using Microsoft Excel and the XLFIT (IDBS) add-in. Antibody concentrations at each sample time point (0.15 hours to 336 hours) for each dilution were extrapolated from the standard curves using XLFIT. The optimal sample dilution(s) was selected so that the antibodies were adequately detected by their corresponding standard curves. The final concentrations of serum B21A1, B21H1-25, and B21H1-55 were adjusted and multiplied by the sample dilution factor.

[0355] The mean serum concentrations (μg / mL) of B21A1 chimeric IgG1 and humanized mutants B21H1-25 and B21H1-55 were calculated from 35 mice to generate composite PK profiles (Figures 12A-12C). Non-compartmental PK analysis after IV bolus injection was performed using the PK solver 2.0 add-in for Microsoft Excel (Table 11). Antibody serum exposure was graphed using GraphPad Prism for Windows (GraphPad Software). The LILRB2 / 1 antibodies B21A1, B21H1-25, and B21H1-55 showed half-lives of 7.6 days, 8.5 days, and 10.1 days, respectively, in humanized FcRn mice.

[0356] [Table 11]

[0357] Example 22: LILRB2 / 1 chimeric antibody inhibits tumor growth in humanized NSG-SGM3 mice carrying subcutaneous human SK-MEL-5 melanoma. The antitumor efficacy of B21A chimeric IgG4 (B21A4) 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 (strain No. 013062) expressing human IL-3, GM-CSF (CSF2), and SCF (KITLG) were tested by irradiating 3-week-old NSG-SGM3 mice with human UBC CD34. + Hematopoietic stem cells were reconstituted by intravenous injection. Human CD45 was used to evaluate humanization in peripheral blood. + Cell engraftment is monitored weekly by the Jackson Laboratory, and 25% of the cells are human CD45. + Only animals with minimal cell engraftment were received from Jackson Laboratory and enrolled in the study.

[0358] In vivo tumor xenograft model and LILRB2 / 1 antibody administration:

[0359] Female humanized NSG-SGM3 mice were housed in micro-isolate cages in a vivarium at Bloodworks Northwest under specific pathogen-free conditions. All procedures were performed according to the Bloodworks Northwest IACUC Protocol No. 5390-02 facility guidelines. Mice were identified using ear tags. All mice were acclimatized for a minimum of 5 days before the start of the study. Mice were placed in their right flanks in 100 μL of PBS containing 20% ​​Matrigel (R&D Systems, No. 3632-005-02), with 2 × 10⁶ units of PBS. 6 Individual SK-MEL-5 human melanoma cells (HLA class A*02:01, ATCC) were subcutaneously (SC) inoculated.

[0360] The tumor size was measured twice a week using digital calipers. The tumor volume was calculated: Tumor volume (mm²)3 )=(L×W 2 / 2) (where L is the maximum dimension and W is the minimum dimension). Approximately 50 mm 3 When the average tumor size is reached, the tumor size and human CD45 ++ Based on both cell engraftment, mice were randomized into groups, each containing 8-9 mice. Mice were administered 20 mg / kg of B21A4, LILRB2 antibody (Comp4, VH SEQ ID NO: 49 and VL SEQ ID NO: 50), or IgG4 isotype control intraperitoneally (IP) on the day of randomization (day 7), and a total of six doses were administered every 7 days (days 9, 16, 23, 30, and 37 after tumor inoculation) (arrows in Figure 13). Mice were sacrificed on day 41 for final tumor weight measurement.

[0361] The mean tumor growth inhibition (TGI) was calculated using the following formula:

[0362]

number

[0363] Statistical differences in tumor volume were examined using parametric repeated measures (RM) two-way ANOVA with Geisser-Greenhouse correction using GraphPad Prism software. The standard error of the mean was calculated daily for tumor volume. P values ​​were considered significant according to the following criteria: *P<0.05, **P<0.01, and ***P<0.001.

[0364] Mice treated with anti-LILRB2 / 1 B21A4 achieved 75% tumor growth inhibition (TGI) and 37.5% tumor regression by day 41 compared to the IgG4 control group, suggesting the tumor growth-delaying ability of B21A4 (Table 12 and Figure 13), and further suggesting that humanized variants of the antibody would have similar effects.

[0365] [Table 12]

[0366] Example 23: Phagocytic Assay LILRB2 and LILRB1 bind to classical (HLA-A and HLA-B) and non-classical (HLA-G, HLA-E, and HLA-F) MHC class I molecules. The expression of the common MHC class I component β2-microglobulin (β2M) by cancer cells directly protects them from phagocytosis by macrophages. This protection is mediated by MHC class I binding to LILRB1 or LILRB2 by providing a "don't eat me" signal, and interference with MHC class I binding to LILRB1 or LILRB2 has been shown to enhance phagocytosis in tumor cells (Barkal et al., Nat Immunol. 2018 Jan;19(1):76-84.doi:10.1038 / s41590-017-0004-z. Epub 2017 Nov 27. PMID:29180808;PMCID:PMC5832354, Mandel et al., J Immunother Cancer. 2022 Sep;10(9):e004859.doi:10.1136 / jitc-2022-004859.PMID:36096532;PMCID:PMC9472153, Tian et al. al., Cancer Immunol Res.2024 Feb 23.doi:10.1158 / 2326-6066.CIR-23-0568. Epub ahead of print. PMID:38393969).

[0367] The ability of LILRB2 / 1 antibodies to induce macrophage phagocytosis of tumor cells was investigated in vitro using 721.221 wild-type B-cell lymphoma tumor cells or 721.221 cells expressing HLA-G. Human monocytes were isolated from individual subjects (isolated as described in Example 9) and placed in a flat-bottom 96-well plate in macrophage medium (X-VIVO2 supplemented with 10% FBS and 100 ng / mL M-CSF as described in Example 9). TM15) The cells were seeded and incubated at 37°C in 5% CO2 for 5 days until the monocytes differentiated into M0 macrophages, and then polarized into M2c macrophages at 37°C for 2 days (as described in Example 10). On day 7, the M2c macrophages were ready for phagocytic assay setup. The medium was removed from the wells containing the M2c cells, and the wells were then washed 1× with PBS to remove non-adherent macrophages. LILRB2 / 1 antibody or isotype control was added to the macrophages at a 2× concentration (40 μg / mL) to a final concentration of 20 μg / mL in 50 μL / well, and incubated at 37°C in 5% CO2 for 1 hour.

[0368] During the 1-hour incubation of macrophages with the antibody, tumor target cells (721.221 wild-type or 721.221-HLA-G expressing) were treated with 1:2000 CellTrace diluted in 1×PBS. TM Cells were labeled with violet (CTV; Thermo Fisher, No. C34557) at 37°C for 20 minutes (as described in Example 16). After 20 minutes of incubation, excess CellTrace TM Wash the cells away from the target cells and then transfer them to the culture medium (X-VIVO TM The cells were resuspended in 15% FBS and then added to M2c macrophages with 100,000 target tumor cells / well in 50 μL / well to obtain a final 1:2 ratio of macrophages to tumor cells in 100 μL / well. Anti-CD47 phagocytosis-positive control antibody (maglorimab, Hu5F9, Selleckchem, No. A2036) was added to some wells. The target cells, macrophages, and antibody were incubated overnight at 37°C for 18 hours in 5% CO2 to enable phagocytosis.

[0369] The following day, the culture medium was discarded to remove non-phagocytic cells, the wells were washed with PBS (1×), and then the supernatant was discarded. 50 μL / well 1× TrypLE TMAdhering macrophages were detached from the wells by adding Express Enzyme solution (Thermo Fisher, No. 12604013), and incubated at 5% CO2 and 37°C. After 30 minutes of incubation, the cells were resuspended in 150 μL of warming medium, transferred to a V-bottom 96-well plate, centrifuged at 300 × g, and the supernatant was removed. The cells were then subjected to eFluorine in PBS. TM Cells were resuspended in a 1:2000 dilution of 780 (eF780) fixable viability stain (Thermo Fisher, No. 65-0865-18) and incubated in the dark at RT for 10 minutes. Following viability staining, 150 μL / well of FACS buffer was added to each well, and the stain was removed by centrifugation at 300 × g for 5 minutes. Cells were blocked in Fc blocks (2.5 μL / well) of 25 μL / well of FACS buffer and incubated at 4°C for 20 minutes, then 2.5 μL / well of PE conjugate anti-CD14 (BioLegend, No. 325606) was added to each well, and incubated at 4°C for 30 minutes. After staining, 150 μL / well of FACS buffer was added to each well, and the cells were centrifuged at 300 × g for 5 minutes. The supernatant was removed, and the cells were sterilized using BD FACSymphony. TM or FACSCanto TM For acquisition using a flow cytometer (BD Biosciences), cells were resuspended with 100 μL / well of FACS buffer. The phagocytic rate of tumor cells was determined using flow cytometry (eF780). - )CTV + CD14 + This was determined by reporting the percentage of cells. Increased M2c-mediated oncological phagocytosis was observed only in the presence of LILRB2 / 1 antibody or anti-CD47 phagocytosis control antibody against HLA-G expressing tumor cells, compared to isotype controls or culture media controls (Figure 14A). Phagocytosis in the presence of LILRB2 / 1 antibody was HLA-G dependent, as indicated by the lack of oncological phagocytosis against wild-type (HLA-G-less) 721,221 cancer cells.

[0370] 721.221 wild type and 721.221-HLA-G+ The ability of macrophages to induce phagocytosis of both target cells was confirmed by an anti-CD47-positive control antibody (Figures 14A and 14B, where *** represents p=0.001 and *** represents p=0.0001). Compared to isotype controls, the LILRB2 / 1 antibody of the present invention induced HLA-G-mediated phagocytosis of tumor cells by M2 macrophages (Figure 14A), but did not induce phagocytosis of 721.221 wild-type parental tumor cells (Figure 14B). In contrast, comparator LILRB2 / 1 antibodies (Comp3, VH SEQ ID NO: 47 and VL SEQ ID NO: 48) failed to induce macrophage phagocytosis of 721.221-HLA-G-expressing tumor cells or 721.221 parental cells.

[0371] All documents referenced in this application are incorporated herein by reference in their entirety.

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Claims

1. An antibody product comprising a variable region that specifically binds to human LILRB2 and LILRB1, wherein the variable region is a) When specified using the IMGT CDR definition, a first domain including CDR-H1 shown in SEQ ID NO: 16, CDR-H2 shown in SEQ ID NO: 17, and CDR-H3 shown in SEQ ID NO: 18, and a second domain including CDR-L1 shown in SEQ ID NO: 19, CDR-L2 having sequence TAS, and CDR-L3 shown in SEQ ID NO: 21, or b) An antibody product comprising a first domain containing CDR-H1 as shown in SEQ ID NO: 22, CDR-H2 as shown in SEQ ID NO: 23, and CDR-H3 as shown in SEQ ID NO: 24, and a second domain containing CDR-L1 as shown in SEQ ID NO: 25, CDR-L2 as shown in SEQ ID NO: 26, and CDR-L3 as shown in SEQ ID NO:

27.

2. The aforementioned variable region is a) An amino acid sequence that is at least 80% identical to SEQ ID NOs: 1, 6, 7, 8, 9, or 10, or b) The antibody product according to claim 1, comprising a heavy chain variable domain having an amino acid sequence shown in SEQ ID NOs: 1, 6, 7, 8, 9, or 10.

3. The aforementioned variable region is a) An amino acid sequence that is at least 80% identical to SEQ ID NOs: 2, 11, 12, 13, 14, or 15, or b) The antibody product according to claim 1, comprising a light chain variable domain having an amino acid sequence shown in SEQ ID NOs: 2, 11, 12, 13, 14, or 15.

4. The aforementioned variable region is a) Heavy chain variable domain containing Sequence ID 1, and light chain variable domain Sequence ID 2, b) A heavy chain variable domain containing SEQ ID NO. 6, and a light chain variable domain containing SEQ ID NO. 11, c) A heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 12, d) A heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 13, e) A heavy chain variable domain containing SEQ ID NO: 6, and a light chain variable domain containing SEQ ID NO: 14, f) A heavy chain variable domain containing SEQ ID NO. 6, and a light chain variable domain containing SEQ ID NO. 15, g) Heavy chain variable domain containing SEQ ID NO: 7, and light chain variable domain containing SEQ ID NO: 11 h) A heavy chain variable domain containing Sequence ID No. 7, and a light chain variable domain containing Sequence ID No. 12, i) A heavy chain variable domain containing Sequence ID No. 7, and a light chain variable domain containing Sequence ID No. 13, j) Heavy chain variable domain containing SEQ ID NO: 7, Light chain variable domain containing SEQ ID NO: 14, k) A heavy chain variable domain containing Sequence ID No. 7, and a light chain variable domain containing Sequence ID No. 15, l) A heavy chain variable domain containing SEQ ID NO: 8, and a light chain variable domain containing SEQ ID NO: 11, m) A heavy chain variable domain containing Sequence ID No. 8, and a light chain variable domain containing Sequence ID No. 12, n) A heavy chain variable domain containing SEQ ID NO: 8, and a light chain variable domain containing SEQ ID NO: 13, o) A heavy chain variable domain containing SEQ ID NO: 8, and a light chain variable domain containing SEQ ID NO: 14, p) A heavy chain variable domain containing Sequence ID No. 8, and a light chain variable domain containing Sequence ID No. 15, q) Heavy chain variable domain containing SEQ ID NO: 9, Light chain variable domain containing SEQ ID NO: 11, r) A heavy chain variable domain containing Sequence ID No. 9, and a light chain variable domain containing Sequence ID No. 12, s) Heavy chain variable domain containing Sequence ID No. 9, and light chain variable domain containing Sequence ID No. 13, t) Heavy chain variable domain containing Sequence ID No. 9, and light chain variable domain containing Sequence ID No. 14, u) A heavy chain variable domain containing Sequence ID No. 9, and a light chain variable domain containing Sequence ID No. 15, v) A heavy chain variable domain containing SEQ ID NO: 10, and a light chain variable domain containing SEQ ID NO: 11, w) A heavy chain variable domain containing sequence number 10, and a light chain variable domain containing sequence number 12, x) A heavy chain variable domain containing SEQ ID NO: 10, and a light chain variable domain containing SEQ ID NO: 13, y) A heavy chain variable domain containing SEQ ID NO: 10, and a light chain variable domain containing SEQ ID NO: 14, or z) The antibody product according to any one of claims 1 to 3, comprising a heavy chain variable domain containing SEQ ID NO: 10 and a light chain variable domain containing SEQ ID NO:

15.

5. An antibody product according to any one of claims 1 to 3, comprising a heavy chain containing a heavy chain variable domain (VH) and a human heavy chain constant domain (CH).

6. An antibody product according to any one of claims 1 to 3, comprising a light chain containing a variable light chain domain (VL) and a human constant light chain domain (CL).

7. An antibody product according to any one of claims 1 to 3, comprising a heavy chain containing a heavy chain variable domain (VH) and a human heavy chain constant domain (CH), and a light chain containing a light chain variable domain (VL) and a human light chain constant domain (CL).

8. The antibody product according to claim 5 or 7, wherein the heavy chain constant domain is an IgA, IgD, IgE, IgG, or IgM heavy chain constant domain.

9. The antibody product according to claim 8, wherein the heavy chain constant domain is an IgG1 constant domain, an IgG2 constant domain, or an IgG4 constant domain.

10. The antibody product according to claim 9, wherein the heavy chain constant domain is an IgG1 constant domain.

11. The heavy chain amino acid sequence is a) An amino acid sequence that is at least 80% identical to SEQ ID NO: 3, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, b) The antibody product according to claim 10, comprising the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO:

32.

12. The antibody product according to claim 9, wherein the heavy chain constant domain is an IgG4 constant domain.

13. The heavy chain amino acid sequence is a) An amino acid sequence that is at least 80% identical to SEQ ID NO: 4, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37, or b) The antibody product according to 12, comprising the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or 37.

14. An antibody product according to any one of claims 1 to 4, comprising a light chain including a light chain variable region (VL) and a human light chain constant region (CL).

15. The antibody product according to claim 14, wherein the human light chain constant region (CL) comprises a kappa domain or a fragment thereof.

16. The light chain amino acid sequence is a) An amino acid sequence that is at least 80% identical to SEQ ID NO: 5, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42, b) The antibody product according to 12, comprising the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO:

42.

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

15.

18. An antibody product that binds to human LILRB2, a) IgG 4 heavy chain having an amino acid sequence containing SEQ ID NO: 1, and kappa light chain having an amino acid sequence containing SEQ ID NO: 2 b) IgG 4 heavy chain having an amino acid sequence including SEQ ID NO: 6, and kappa light chain having an amino acid sequence including SEQ ID NO: 11, c) IgG 4 heavy chain having an amino acid sequence including SEQ ID NO: 6, and kappa light chain having an amino acid sequence including SEQ ID NO: 12, d) IgG 4 heavy chain having an amino acid sequence including SEQ ID NO: 6, and kappa light chain having an amino acid sequence including SEQ ID NO: 13, e) IgG 4 heavy chain having an amino acid sequence containing SEQ ID NO: 6, and kappa light chain having an amino acid sequence containing SEQ ID NO: 14, f) IgG quadrivalent heavy chain having an amino acid sequence containing SEQ ID NO. 6, and kappa light chain having an amino acid sequence containing SEQ ID NO. 15, g) IgG 4 heavy chain having an amino acid sequence containing SEQ ID NO: 7, and kappa light chain having an amino acid sequence containing SEQ ID NO: 11, h) An IgG quadrivalent heavy chain having an amino acid sequence containing SEQ ID NO: 7, and a kappa light chain having an amino acid sequence containing SEQ ID NO: 12, i) An IgG 4 heavy chain having an amino acid sequence containing SEQ ID NO: 7, and a kappa light chain having an amino acid sequence containing SEQ ID NO: 13, j) IgG 4 heavy chain having an amino acid sequence including SEQ ID NO: 7, and kappa light chain having an amino acid sequence including SEQ ID NO: 14, k) An IgG quadrivalent heavy chain having an amino acid sequence containing SEQ ID NO: 7, and a kappa light chain having an amino acid sequence containing SEQ ID NO: 15, l) IgG quadrivalent heavy chain having an amino acid sequence including SEQ ID NO: 8, and kappa light chain having an amino acid sequence including SEQ ID NO: 11, m) IgG quadrivalent heavy chain having an amino acid sequence containing SEQ ID NO: 8, and kappa light chain having an amino acid sequence containing SEQ ID NO: 12, n) An IgG 4 heavy chain having an amino acid sequence including SEQ ID NO: 8, and a kappa light chain having an amino acid sequence including SEQ ID NO: 13, or o) An antibody product comprising an IgG 4 heavy chain having an amino acid sequence including SEQ ID NO: 8, and a kappa light chain having an amino acid sequence including SEQ ID NO:

14.

19. The antibody product according to any one of claims 1 to 18, wherein the antibody product is a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single-chain antibody.

20. The antibody product according to any one of claims 1 to 18, wherein the antibody product is a monospecific, bispecific, tripspecific, or multispecific antibody.

21. An antibody product according to any one of claims 1 to 20, which specifically binds to human LILRB2 expressed by bone marrow cells or cancer cells.

22. The antibody product has a K content of less than 1 pM to approximately 1000 pM. Ｄ The antibody product according to claim 21, which specifically binds to human LILRB2.

23. An antibody product according to any one of claims 1 to 20, which specifically binds to human LILRB1 expressed by bone marrow cells, lymphocytes, or cancer cells.

24. The antibody product has a K content of approximately 0.1 pM to approximately 300 nM. Ｄ The antibody product according to claim 23, which specifically binds to human LILRB1.

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

26. The antibody product according to claim 25, wherein the antibody product binds to human immunosuppressive myeloid cells in the tumor microenvironment.

27. The antibody product according to claim 25 or 26, wherein the immunosuppressive myeloid cells are macrophages, myeloid dendritic cells, or myeloid-derived suppressor cells.

28. The antibody product according to claim 27, wherein the immunosuppressive myeloid cells are M2a, M2b, M2c, or M2d macrophages.

29. The antibody product contains 0.05 nM to 50 nM of K Ｄ The antibody product according to any one of claims 25 to 28, which specifically binds to human M2c macrophages.

30. The antibody product according to claim 29, wherein the binding of the antibody product to the immunosuppressive myeloid cells is not cytotoxic to the cells.

31. The antibody product according to any one of claims 1 to 20, wherein the antibody product binds to human lymphocytes.

32. The antibody product according to claim 31, wherein the antibody product binds to human lymphocytes in the tumor microenvironment.

33. The antibody product according to claim 31 or 32, wherein the lymphocytes are human B lymphocytes, human T lymphocytes, or NK cells.

34. The antibody product has a K content of 0.001 nM to 300 nM. Ｄ An antibody product according to any one of claims 31 to 33, which specifically binds to human lymphocytes.

35. The antibody product according to claim 34, wherein the binding of the antibody product to lymphocytes is not cytotoxic to the lymphocytes.

36. An antibody product according to any one of claims 1 to 20, which inhibits the immunosuppressive interaction between LILRB1 or LILRB2 expressed in bone marrow cells and HLA-G expressed in cells in the tumor microenvironment, such as tumor cells, cancer-associated fibroblasts, other immunosuppressive bone marrow cells, or lymphocytes.

37. An antibody product according to any one of claims 1 to 20, which inhibits the immunosuppressive interaction between LILRB1 expressed in lymphocytes and HLA-G expressed in tumor cells, cancer-associated fibroblasts, and immunosuppressive myeloid cells or lymphocytes.

38. An antibody product according to any one of claims 1 to 20, which inhibits the immunosuppressive interaction between LILRB1 or LILRB2 expressed in bone marrow cells and HLA class I expressed in the same bone marrow cells, or in tumor cells, cancer-associated fibroblasts, other immunosuppressive bone marrow cells, or lymphocytes.

39. An antibody product according to any one of claims 1 to 20, which inhibits the immunosuppressive interaction between LILRB1 expressed in lymphocytes and HLA-class I expressed in the same lymphocytes, or in tumor cells, cancer-associated fibroblasts, other immunosuppressive myeloid cells, or lymphocytes.

40. The antibody product according to any one of claims 1 to 20, which is bound by an Fc receptor expressed on immunosuppressive macrophages or other bone marrow cells or lymphocytes.

41. The antibody product according to claim 40, which is conjugated by CD16 (FcγRIIIIa), CD32 (FcγRII), or CD64 (FcγRI) expressed on immunosuppressive macrophages or other bone marrow cells or lymphocytes.

42. The antibody product according to claim 40 or 41, which binds to LILRB1 or LILRB2 expressed in the bone marrow cells and is bound by CD16 (FcγRIIIIa), CD32 (FcγRII), or CD64 (FcγRI) expressed in the same bone marrow cells.

43. The antibody product according to claim 40 or 41, which binds to LILRB1 expressed on the lymphocytes and is conjugated by CD16 (FcγRIIa), CD32 (FcγRII), or CD64 (FcγRI) expressed on the same lymphocytes.

44. The antibody product according to claim 40 or 41, which binds to LILRB1 or LILRB2 expressed in a first cell and is bound by CD16 (FcγRIIa), CD32 (FcγRII), or CD64 (FcγRI) expressed in a second cell.

45. A method for providing cancer immunotherapy to a subject in need thereof, wherein the cancer is associated with the presence of immunosuppressive macrophages, and the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody product according to any one of claims 1 to 44.

46. The method according to claim 45, comprising administering an amount of the pharmaceutical composition effective in inhibiting the immunosuppressive interaction between LILRB1 or LILRB2 expressed in bone marrow cells and HLA-G or HLA class I expressed in said bone marrow cells or in tumor cells, cancer-associated fibroblasts, other immunosuppressive bone marrow cells, or lymphocytes.

47. The method according to claim 45, comprising administering an amount of the pharmaceutical composition effective in inhibiting the immunosuppressive interaction between LILRB1 expressed in lymphocytes and HLA-G or HLA-class I expressed by the lymphocytes, or by tumor cells, cancer-associated fibroblasts, other immunosuppressive myeloid cells, or lymphocytes.

48. The antibody product binds to macrophages, and the binding of the antibody product to macrophages has the following effects: a) CD4 ＋ T cells, CD8 ＋ Promotes the activation of T cells, NK cells, or any combination thereof. b) CD4 ＋ T cells, CD8 ＋ To promote the proliferation of T cells, NK cells, or any combination thereof. c) To prevent the polarization of macrophages into immunosuppressive macrophages, and The method of claim 45, which results in at least one of the following: d) enhancing the congenital antitumor response.

49. CD4 ＋ T cells, CD8 ＋ The method according to claim 48, wherein said activation of CD4 T cells, CD8 T cells, NK cells, or any combination thereof is measured as an enhanced level of IFN-γ, TNF-α, or perforin, or any combination thereof.

50. The method according to claim 48, wherein the binding of the antibody product to the macrophage is not cytotoxic to the macrophage.

51. The binding of the antibody product to macrophages is a) Internalization of the antibody product by the macrophages, b) Secretion of TNFα, IL-6, perforin, or any combination thereof c) Inhibition of IL-10 secretion from macrophages, d) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof, e) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof, f) The method according to claim 45, which brings about enhanced tumor cell killing in the tumor microenvironment.

52. The method according to claim 51, wherein the combination results in two or more of (a) to (f), three or more of (a) to (f), four or more of (a) to (e), five of (f) beyond (a), or all of (a) to (e).

53. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to macrophages increases immunostimulatory activity in the tumor microenvironment.

54. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to the macrophage reduces the immunosuppressive activity of the macrophage.

55. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to the macrophage reduces the tumor-promoting activity of the macrophage.

56. The binding of the antibody product to macrophages is CD4 ＋ T cell activation, CD4 ＋ T cell proliferation, or CD4 ＋ The method according to any one of claims 48 to 52, which promotes both T cell activation and proliferation.

57. The binding of the antibody product to macrophages is CD8 ＋ T cell activation, CD8 ＋ T cell proliferation, or CD8 ＋ The method according to any one of claims 48 to 52, which promotes both T cell activation and proliferation.

58. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to macrophages promotes cytotoxic lymphocyte-mediated killing of cancer cells.

59. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to macrophages promotes NK cell-mediated tumor cell killing.

60. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to macrophages reduces the suppression of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment.

61. The method according to any one of claims 48 to 52, wherein the binding of the antibody product to macrophages promotes macrophage-mediated phagocytosis of tumor cells expressing HLA-G.

62. The antibody product binds to lymphocytes, and the binding of the antibody product to lymphocytes is a) Promote NK cell-mediated killing of tumor cells, CD8 ＋ T cells enhance IFN-γ secretion and its cytotoxic activity. b) NK cells and CD8 ＋ Activate T cells, or c) The method according to claim 43, which results in blocking LILRB1-mediated suppression of lymphocytes.

63. The method according to claim 45, wherein the cancer is a sarcoma, carcinoma, or a cancer of blood origin.

64. The method according to claim 63, wherein the cancer is glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, pancreatic adenocarcinoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell carcinoma, gastric cancer, Merkel cell carcinoma, dendritic sarcoma, non-small cell lung cancer, papillary thyroid cancer, cutaneous squamous cell carcinoma, or ovarian cancer.

65. The method according to claim 63, wherein the cancer is a B-cell malignant tumor selected from B-cell lymphoma (diffuse large B-cell lymphoma, small lymphocytic lymphoma or chronic lymphocytic leukemia, non-Hodgkin lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, etc.), B-cell leukemia, acute myeloid leukemia, and multiple myeloma.

66. The method according to claim 63, wherein the cancer cells express LILRB2 or LILRB1.

67. The method according to claim 63, wherein the cancer cells overexpress LILRB2 or LILRB1.

68. The method according to any one of claims 45 to 67, further comprising administering an effective amount of the anti-cancer treatment product to the subject.

69. The method according to claim 68, wherein the anti-cancer treatment product comprises an immune checkpoint inhibitor.

70. The method according to claim 69, comprising administering to the subject an effective amount of an anti-cancer treatment product containing an immune checkpoint inhibitor in an amount effective to reduce T-cell immunosuppression.

71. The method according to claim 69 or 70, wherein the immune checkpoint inhibitor is a PD-1 antagonist, a PD-L1 antagonist, or a CLTA-4 antagonist.

72. The method according to claim 71, wherein the immunosuppression of the T cells includes immunosuppression mediated through the interaction between the T cells and myeloid cells expressing PD-L1.

73. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain variable domain described in claim 2.

74. An isolated nucleic acid comprising a nucleotide sequence encoding the light chain variable domain described in claim 3.

75. An expression vector comprising the nucleic acid described in claim 73.

76. An expression vector comprising the nucleic acid described in claim 74.

77. An expression vector comprising the nucleic acid described in claims 75 and 76.

78. A host cell comprising the expression vector according to any one of claims 75 to 77.

79. A method for producing a protein containing an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain, a) Growing the host cells according to claim 78 under conditions such that the host cells express the protein containing the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain, b) A method comprising purifying the protein containing the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain.

80. A method for producing an antibody product that binds to human LILRB2, a) Growing host cells containing the expression vector described in claim 77 under conditions such that the host cells express a protein containing the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain, thereby producing the antibody product, b) A method comprising purifying the antibody product.

81. A composition comprising an antibody product according to any one of claims 1 to 44 and an excipient.

82. A product comprising the composition according to claim 81 and a container.

83. Use of an antibody product according to any one of claims 1 to 42 or a composition according to claim 81 for the manufacture of a pharmaceutical for the treatment of cancer in a subject requiring cancer treatment.

84. A pharmaceutical composition comprising an antibody product according to any one of claims 1 to 44 and a pharmaceutically acceptable excipient.

85. An antibody product according to any one of claims 1 to 44, or a pharmaceutical composition according to claim 84, for use in treating a subject having cancer expressing LILRB2.

86. Use of an antibody product according to any one of claims 1 to 44 in the manufacture of a pharmaceutical product for treating a subject having cancer expressing LILRB2.

87. A method for detecting LILRB2 in a sample, tissue, or cell using an antibody product according to any one of claims 1 to 44, comprising contacting the sample, tissue, or cell with the antibody product and detecting the antibody product.

88. A method for reducing the biological activity of LILRB1 or LILRB2 in a subject requiring such reduction, the method comprising administering a therapeutically effective amount of the antibody product described in any one of claims 1 to 44 or the pharmaceutical composition described in claim 84.

89. The method according to claim 88, wherein the antibody product mediates the depletion of at least one cancer cell expressing LILRB2.

90. A method for promoting an immune response in a subject requiring such promotion, the method comprising administering a therapeutically effective amount of an antibody product according to any one of claims 1 to 44 or a pharmaceutical composition according to claim 84.

91. A method for providing cancer immunotherapy to a subject in need of such therapy, wherein the cancer cells express LILRB2, and the method comprises administering to the subject a therapeutically effective amount of an antibody product according to any one of claims 1 to 44.

92. The method according to 91, comprising administering an amount of the antibody product effective in mediating the death of the cancer cells via antibody-dependent cytotoxicity.

93. The method according to claim 91, comprising administering an amount of the antibody product effective in reducing the LILRB1- or LILRB2-mediated suppression of T cells in the subject.

94. The method according to claim 91, further comprising administering to the subject a PD-1 antagonist or a PD-L1 antagonist in an amount sufficient to reduce PD-1 / PD-L1 system-mediated immunosuppression of T cells in the subject, or a CTLA-4 antagonist in an amount sufficient to reduce CTLA-4 system-mediated immunosuppression of T cells in the subject.

95. The method according to claim 94, wherein the PD-1 antagonist comprises a PD-1 antibody product.

96. The method according to claim 94, wherein the PD-L1 antagonist comprises a PD-L1 antibody product.

97. The method according to claim 94, wherein the CTLA-4 antagonist comprises a CTLA-4 antibody product.

98. The antibody product according to any one of claims 1 to 44, wherein the antibody product comprises a detectably labeled or conjugated toxin, drug, receptor, enzyme, or receptor ligand.

99. The antibody product described in claim 98 comprises a therapeutic portion or a cytotoxic portion.

100. A pharmaceutical composition comprising an antibody product according to any one of claims 1 to 44 and a physiologically acceptable carrier or excipient, wherein the antibody product reduces or prevents the binding of LILRB1 or LILRB2 to its ligand, and / or reduces or prevents LILRB1- or LILRB2-mediated signaling.

101. The pharmaceutical composition according to claim 100, 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.

102. The pharmaceutical composition according to claim 100 or 101, wherein the ligand is expressed on the surface of bone marrow cells, lymphocytes, or tumor cells.

103. A method for treating cancer in a subject requiring treatment, comprising administering an effective amount of a pharmaceutical composition according to any one of claims 100 to 102 to the subject.

104. The method according to claim 103, wherein the subject is a cancer comprising cells that express or overexpress a ligand for LILRB2.

105. The method according to claim 103 or 104, wherein the antibody product or its antigen-binding fragment increases the immune response, delays or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages and alters their activity, reduces tumor-associated macrophage-mediated immunosuppression, reduces or reverses T cell suppression, or a combination thereof.

106. The method according to any one of claims 103 to 105, wherein the cancer or tumor comprises macrophages expressing LILRB2.

107. The method according to any one of claims 103 to 106, further comprising administering a therapeutic dose of a second therapeutic product to the subject.

108. The method according to claim 107, wherein the second therapeutic product comprises an immune checkpoint inhibitor.

109. The method according to claim 108, wherein the second therapeutic product comprises a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

110. a) A heavy chain containing SEQ ID NO: 1, and a light chain containing SEQ ID NO: 2 b) A heavy chain containing SEQ ID NO: 6, and a light chain containing SEQ ID NO: 11, c) A heavy chain containing SEQ ID NO: 6, and a light chain containing SEQ ID NO: 12, d) A heavy chain containing SEQ ID NO: 6, and a light chain containing SEQ ID NO: 13, e) A heavy chain containing SEQ ID NO: 6, and a light chain containing SEQ ID NO: 14, f) A heavy chain containing Sequence ID No. 6, and a light chain containing Sequence ID No. 15, g) Heavy chain containing SEQ ID NO: 7, and light chain variable containing SEQ ID NO: 11 h) A heavy chain containing SEQ ID NO: 7, and a light chain containing SEQ ID NO: 12, i) A heavy chain containing SEQ ID NO: 7, and a light chain containing SEQ ID NO: 13, j) Heavy chain containing SEQ ID NO: 7, Light chain containing SEQ ID NO: 14, k) A heavy chain containing Sequence ID No. 7, and a light chain containing Sequence ID No. 15, l) A heavy chain containing SEQ ID NO: 8, and a light chain containing SEQ ID NO: 11, m) Heavy chain containing SEQ ID NO: 8, and light chain containing SEQ ID NO: 12, n) A heavy chain containing SEQ ID NO: 8, and a light chain containing SEQ ID NO: 13, o) A heavy chain containing SEQ ID NO: 8, and a light chain containing SEQ ID NO: 14, p) Heavy chain containing Sequence ID No. 8, and light chain containing Sequence ID No. 15, q) Heavy chain containing SEQ ID NO: 9, Light chain containing SEQ ID NO: 11, r) A heavy chain containing SEQ ID NO: 9, and a light chain containing SEQ ID NO: 12, s) Heavy chain containing SEQ ID NO: 9, and light chain containing SEQ ID NO: 13, t) Heavy chain containing SEQ ID NO: 9, and light chain containing SEQ ID NO: 14, u) A heavy chain containing Sequence ID No. 9, and a light chain containing Sequence ID No. 15, v) A heavy chain containing SEQ ID NO: 10, and a light chain containing SEQ ID NO: 11, w) Heavy chain containing SEQ ID NO: 10, and light chain containing SEQ ID NO: 12, x) A heavy chain containing SEQ ID NO: 10, and a light chain containing SEQ ID NO: 13, y) A heavy chain containing SEQ ID NO: 10, and a light chain containing SEQ ID NO: 14, or z) The antibody product according to claim 1, comprising a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO:

15.

111. a) A heavy chain containing SEQ ID NO: 1, and a light chain containing SEQ ID NO: 2 b) A heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33, and a light chain containing SEQ ID NO: 38, c) A heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33, and a light chain containing SEQ ID NO: 39, d) A heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33, and a light chain containing SEQ ID NO: 40, e) A heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33, and a light chain containing SEQ ID NO: 41, f) A heavy chain containing SEQ ID NO: 28 or SEQ ID NO: 33, and a light chain containing SEQ ID NO: 42, g) A heavy chain containing SEQ ID NO: 29 or SEQ ID NO: 34, and a light chain containing SEQ ID NO: 38, h) A heavy chain containing SEQ ID NO: 29 or SEQ ID NO: 34, and a light chain containing SEQ ID NO: 39, i) A heavy chain containing SEQ ID NO: 29 or SEQ ID NO: 34, and a light chain containing SEQ ID NO: 40, j) A heavy chain containing SEQ ID NO: 29 or SEQ ID NO: 34, and a light chain containing SEQ ID NO: 41, k) A heavy chain containing SEQ ID NO: 29 or SEQ ID NO: 34, and a light chain containing SEQ ID NO: 42, l) A heavy chain containing SEQ ID NO: 30 or SEQ ID NO: 35, and a light chain containing SEQ ID NO: 38, m) A heavy chain containing SEQ ID NO: 30 or SEQ ID NO: 35, and a light chain containing SEQ ID NO: 39, n) A heavy chain containing SEQ ID NO: 30 or SEQ ID NO: 35, and a light chain containing SEQ ID NO: 40, o) A heavy chain containing SEQ ID NO: 30 or SEQ ID NO: 35, and a light chain containing SEQ ID NO: 41, p) A heavy chain containing SEQ ID NO: 30 or SEQ ID NO: 35, and a light chain containing SEQ ID NO: 42, q) A heavy chain containing SEQ ID NO: 31 or SEQ ID NO: 36, and a light chain containing SEQ ID NO: 38, r) A heavy chain containing SEQ ID NO: 31 or SEQ ID NO: 36, and a light chain containing SEQ ID NO: 39, s) A heavy chain containing SEQ ID NO: 31 or SEQ ID NO: 36, and a light chain containing SEQ ID NO: 40, t) A heavy chain containing SEQ ID NO: 31 or SEQ ID NO: 36, and a light chain containing SEQ ID NO: 41, u) A heavy chain containing SEQ ID NO: 31 or SEQ ID NO: 36, and a light chain containing SEQ ID NO: 42, v) A heavy chain containing SEQ ID NO: 32 or SEQ ID NO: 37, and a light chain containing SEQ ID NO: 38, w) A heavy chain containing SEQ ID NO: 32 or SEQ ID NO: 37, and a light chain containing SEQ ID NO: 39, x) A heavy chain containing SEQ ID NO: 32 or SEQ ID NO: 37, and a light chain containing SEQ ID NO: 40, y) A heavy chain containing SEQ ID NO: 32 or SEQ ID NO: 37, and a light chain containing SEQ ID NO: 41, or z) The antibody product according to claim 1, comprising a heavy chain containing SEQ ID NO: 32 or SEQ ID NO: 37, and a light chain containing SEQ ID NO: 42.

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