Compositions and methods for modulating macrophage activity

Abstract

An antigen-binding protein capable of specifically binding to human LILRB1 and human LILRB2, which does not block the interaction between human LILRB1 and HLA-G tetramers and / or does not block the interaction between human LILRB2 and HLA-G tetramers, and which is capable of reprogramming macrophages.

Description

[Technical Field]

[0001] Technical Field The present invention relates to antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, that are capable of specifically binding to both human LILRB1 and human LILRB2, respectively, and their use to modify the behavior of macrophages, in particular tumor-associated macrophages (TAMs), for the treatment of diseases such as cancer and immunosuppressive conditions. [Background technology]

[0002] Background to the Invention Tumors develop as ecosystems consisting of tumor cells, stromal cells, and infiltrating immune cells. Tumor formation is determined by the intrinsic properties of cancer cells and their interactions with components of the tumor microenvironment (TME). The poor prognostic outcome of neoplastic lesions is determined not only by the type of mutations that have already occurred, but also by the tumor stromal composition; cytotoxic lymphocytes (e.g., CD8 + The recruitment and activation of tumor-associated macrophages (T cells) can suppress lethal tumor development, which is facilitated by the infiltration of tumor-associated macrophages (TAMs).TAMs are key components of the tumor ecosystem and correlate with clinical stage, poor overall survival, and shortened recurrence-free survival in a variety of cancers.

[0003] Tumor-associated macrophages are the most abundant immune cells in the TME. During the initial stages of tumor development, macrophages can either directly promote antitumor responses by killing tumor cells or indirectly recruit and activate other immune cells. When genetic changes occur within tumors, T helper 2 (TH2) cells begin to dominate the TME, and TAMs begin to exhibit an immunosuppressive and tumor-promoting phenotype that promotes tumor progression, metastasis, and resistance to therapy. Therefore, targeting TAMs has emerged as a strategy for cancer treatment. TAM targeting strategies focus on macrophage depletion and inhibition of their recruitment to the TME. However, although clinical trials, along with combination therapies, are still ongoing, these strategies have shown limited therapeutic efficacy. The fact that macrophages have the potential for antitumor activity has led the field of TAM targeting to develop TAM-reprogramming strategies to support this antitumor immune response. Macrophages, when appropriately activated, can directly kill cancer cells and thus potentially mount a robust antitumor response; they can do so by presenting tumor antigens and by activating cytotoxic CD8 + They can support adaptive immune responses by producing chemokines and cytokines that recruit and activate T cells and NK cells. Thus, when these immune responses dominate in the tumor microenvironment, malignant tumor development is suppressed. However, in many cases, the tumor microenvironment shifts macrophage function from pro-inflammatory (i.e., tumoricidal) to trophic, resembling the function of macrophages in developing tissues. TAMs bind to CD8 upon binding of immune checkpoint receptors, programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). + They express immunosuppressive receptors, such as programmed death-ligand 1 (PD-L1), which limit T cell activity. As a result, these tumor-educated macrophages promote rather than suppress malignant tumorigenesis.

[0004] Leukocyte Ig-like receptor subfamily B (LILRB) is a group of type I transmembrane glycoproteins with extracellular Ig-like domains and cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITAMs). This group of ITIM-containing receptors includes LILRB1 (also known as CD85J, LIR1, and ILT2), LILRB2 (also known as CD85D, LIR2, and ILT4), LILRB3 (also known as CD85A, LIR3, and ILT5), LILRB4 (also known as CD85K, LIR5, and ILT3), and LILRB5 (also known as CD85C and LIR8). The biological functions and clinical significance of many of these LILRBs (ILTs) are still under investigation. There is also the LILRB subfamily A (LILRA), a group of type I transmembrane glycoproteins with extracellular Ig-like domains and cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The LILRA family includes six members: LILRA1 (also known as CD85I, LIR6), LILRA2 (also known as CD85H, LIR7, ILT1), LILRA3 (also known as CD85E, LIR4, ILT6, monocyte inhibitory receptor HM43 / 31), LILRA4 (also known as CD85G, ILT7), LILRA5 (also known as CD85F, LIR9, ILT11), and LILRA6 (also known as ILT8).

[0005] LILR receptors have two to four extracellular Ig-like domains, which can be inhibitory (LILRB) or activating (LILRA). With the exception of LILRA3, which is expressed only in a soluble form, LILRs are expressed as membrane-bound receptors. Inhibitory receptors (LILRB1-B5) have long cytoplasmic tails within ITIM motifs. Activating receptors LILRA1-A6, with the exception of A3, have short cytoplasmic tails and couple to ITAM-bearing Fc receptors. Individual LILR receptors are classified as group 1 (LILRB1, LILRB2, and LILRA1-3) or group 2 (LILRB3-5 and LILRA4-6) members based on the conservation of LILRB1 residues, which can recognize human leukocyte antigen (HLA) class I molecules. Expression of individual LILRs has been identified in immune cells such as neutrophils, eosinophils, macrophages, dendritic cells, NK cells, B cells, T cells, and osteoclasts, as well as non-immune cells such as endothelial cells and neurons. Human LILRB and mouse PIR-B can regulate the function of ITAM-bearing receptors such as FcR, B cell receptor (BCR), and T cell receptor (TCR). LILRs also regulate Toll-like receptor (TLR) signaling and function. LILRs can exert immunomodulatory effects on a wide range of immune cells and can regulate a wide range of immune functions, including immune cell function, cytokine release, antibody production, and antigen presentation.

[0006] A subset of LILRs recognizes MHC class I (also known as HLA class I in humans). LILR family members can have both activating and inhibitory functions. The inhibitory receptors LILRB1 and LILRB2 exhibit broad specificity for classical and non-classical MHC alleles. LILRB1 exhibits preferential binding to β2-microglobulin-associated complexes. Unlike LILRB1, LILRB2 binding to HLA ligands does not require β2-microglobulin. The activating receptor LILRA1 and the soluble protein LILRA3 prefer β2-microglobulin-independent free heavy chains of MHC class I and especially HLA-C alleles.

[0007] In humans, the leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1, CD85J, LIR1, ILT2) protein is encoded by the LILRB1 gene, found in a gene cluster in chromosome region 19ql3.4. This receptor is expressed on immune cells, binds to MHC class I molecules on antigen-presenting cells, and transmits negative signals that inhibit the stimulation of immune responses. LILRB1 has also been reported to be expressed in human gastric cancer cells and may promote tumor growth. It is thought to help regulate inflammatory responses and cytotoxicity, focusing the immune response and limiting self-reactivity. Multiple transcript variants encoding different isoforms have been found for this gene.

[0008] The human leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2, CD85D, LIR2, ILT4) protein is encoded by the LILRB2 gene, found in the gene cluster in the chromatid region 19ql3.4. This receptor is expressed on immune cells, binds to MHC class I molecules on antigen-presenting cells, and transmits a negative signal that inhibits stimulation of the immune response. This is thought to help control inflammatory responses and cytotoxicity, focusing the immune response and limiting autoreactivity. This receptor is also expressed in human non-small cell lung cancer cells. Multiple transcript variants encoding different isoforms have been found for this gene.

[0009] Leukocyte immunoglobulin-like receptor subfamily A member 3 (LILRA3, CD85E, LIR4, ILT6, monocyte inhibitory receptor HM43 / 31), also known as CD85 antigen-like family member E (CD85e), immunoglobulin-like transcript 6 (ILT-6), and leukocyte immunoglobulin-like receptor 4 (LIR-4) protein, is encoded in humans by the LILRA3 gene, which is located within the leukocyte receptor complex on chromosome 19q13.4. Unlike most of its family members, LILRA3 lacks a transmembrane domain. The function of LILRA3 is currently unknown; however, it is highly homologous to other LILRA genes and can bind to human leukocyte antigen (HLA) class I. Thus, when secreted, LILRA3 can impair the interaction of membrane-bound LILRAs (such as LILRB1, an inhibitory receptor expressed on effector and memory CD8 T cells) with their HLA ligands, thereby modulating immune responses and affecting disease susceptibility. Like its close relative, LILRA1, LILRA3 binds to both normal and "unfolded" free heavy chains of HLA class I and prefers the free heavy chains of HLA-C alleles. LILRA3 also binds to both classical HLA-A and non-classical HLA-G1, but with lower affinity than either LILRB1 or LILRB2. The role of LILRA3 in cancer is less well understood. However, mutations in LILRA3 have been reported in humans and are associated with immune disorders. For example, a homozygous 6-7-kb deletion of LILRA3, which reduces LILRA3 mRNA and protein expression, is associated with Sjögren's syndrome, multiple sclerosis, and rheumatoid arthritis. Therefore, it is suggested that LILRA3 contributes to suppression of inflammation and immunity.

[0010] WO2020023268 (Amgen) describes a combination therapy comprising administering a first antibody or antigen-binding fragment thereof that binds to PD-1, PD-L1, or PD-L2; and a second antibody or antigen-binding fragment thereof that binds to LILRB1, LILRB2, or HLA-G.

[0011] Disclosed anti-LILRB1 antibodies include antibody clones MAB20171 and MAB20172 (R&D Systems), anti-LILRB1 clone 3D3-1D12 (Sigma-Aldrich), anti-LILRB1 clone GH1 / 75 (Novus Biologicals), and a LILRB4 antibody that also cross-reacts with LILRB1, as described in U.S. Patent Application No. 2018 / 0086829 (WO 2016144728, University of Texas, see below). These antibodies are derived from non-human species.

[0012] Disclosed anti-LILRB2 antibodies include clone MAB2078 (R&D Systems), anti-LILRB2 clone 1D4 (Sigma-Aldrich), and an anti-LILRB4 antibody that also cross-reacts with LILRB1, as described in U.S. Patent Application No. 2018 / 0086829 (WO 2016144728, University of Texas, see below). These antibodies are derived from non-human species.

[0013] WO2020023268 also describes the generation of anti-LILRB1 antibodies by immunization of XENOMOUSE® transgenic mice. Hybridoma supernatants that bind to human LILRB1 but not to human LILRA1 and human LILRA2 were selected, and the sequences of three representative anti-LILRB1 antibodies, 3C1, 30A10, and 19D6, are disclosed. The LILRB1-binding domains were determined for antibodies 3C1 (LILRB1 domain 4), 19D6 (LILRB1 domain 4), and 30A10 (LILRB1 domain 3).

[0014] [Table 1]

[0015] [Table 2]

[0016] WO 2020136145 (Innate Pharma) describes LILRB1 antibodies that bind to the D1 or D4 region of LILRB1. Many of the anti-LILRB1 antibodies are said to bind to LILRA3 in addition to LILRB1, either alone (i.e., LILRB1 and LILRA3 cross-reactivity) or with additional binding to LILRB2 or LILRB3. Antibodies 1C11, 1D6, 9G1, 19F10a, 27G10, and commercially available antibodies 586326 and 292305 bound to LILRB1 and LILRA3. Antibody 586326 (mouse IgG2b, Bio-Techne #MAB30851), a mouse monoclonal IgG2b antibody, is the only antibody reported in WO2020136145 to bind to LILRB2 in addition to LILRB1 and LILRA3; however, no experimental data is shown to support this assertion, which contradicts the technical information for this commercially available antibody, which discloses that the antibody was raised against recombinant human LILRA1 / CD85i / LIR-6 Pro17-Asn461 from the mouse myeloid cell line NS0, which states that "in direct ELISA, 100-400% cross-reactivity with recombinant human (rh) ILT2 is observed, and no cross-reactivity with rhILT3, 4, 5, 6, rhLIR-7 or -8 is observed." Thus, according to the supplier, antibody 586326 exhibits 100-400% cross-reactivity with recombinant human LILRB1 and LILRA1, and no cross-reactivity is observed with recombinant human LILRB2, LILRB3, LILRB4, LILRA3, LILRA4 or LILRA6.

[0017] The commercially available antibody 292305 bound to LILRB3 in addition to LILRB1 and LILRA3. The commercially available antibody 292319 bound to LILRA2 in addition to LILRB1. A subset of antibodies, exemplified by 3H5, 12D12, 26D8, 18E1, 27C10, and 27H5, bound only to LILRB1 and not to other LILR family member proteins.

[0018] [Table 3]

[0019] [Table 4]

[0020] Antibodies 3H5, 12D12, 26D8, 18E1, 27C10, 27H5, 1C11, 1D6, 9G1, 19F10a and 27G10 all blocked LILRB1 binding to HLA-G and HLA-A2. Antibodies 12D12, 2A8A, 2A8B, 2A9, 2B11, 2C4, 2C8, 2D8, 2E2B, 2E2C, 2E8, 2E11, 2G5, 2H2A, 2H2B, 2H12, 1A9, 1A10B, 1A10C, 1A10D, 1E4B, 1E4C, 3A7A, 3A7B, 3A8, 3B5, 3E5, 3E7A, 3E7B, 3E9A, 3E9B, 3F5, 4A8, 4C11B, 4E3A, 4E3B, 4H3, 5C5, 5D9, 6C6, 10H1, 48F12, 15D7, and 2C3 all blocked LILRB1 (ILT2) binding to HLA-G and HLA-A2.

[0021] Antibodies 3H5, 12D12, and 27H5 bound to an epitope in domain D1 of LILRB1. Antibodies 26D8, 18E1, and 27C10 all bound to the D4 domain of LILRB1. Antibodies 12D12, 2H2B, 48F12, 1E4C, 1A9, 3F5, and 3A7A bound to an epitope in domain D1 of LILRB1. Antibodies 26D8 and 18E1 lost binding after amino acid substitutions F299I, Y300R, D301A, W328G, Q378A, K381N or substitutions W328G, Q330H, R347A, T349A, Y350S, and Y355A. 26D8 also lost binding to mutant LILRB1 with the amino acid substitutions D341A, D342S, W344L, R345A, and R347A, while antibody 18E1 showed reduced (but not complete) binding to the same mutants. 27C10 also lost binding to the same mutants, but not to other mutants. These amino acid residues, together with the loss of binding to human LILRA3 polypeptide, suggest that these may identify an epitope that characterizes anti-LILRB1 antibodies that promote cytotoxicity in primary NK cells.

[0022] The disclosed LILRB1 antibodies were characterized for their ability to block interactions between HLA-G or HLA-A2 expressed on the surface of cell lines, and recombinant LILRB1 proteins were evaluated by flow cytometry. This allowed the identification of a panel of anti-LILRB1 antibodies that were highly effective in blocking LILRB1 interaction with its HLA class I ligand, HLA-G. Antibodies 3H5, 12D12, 26D8, 18E1, 27C10, 27H5, 1C11, 1D6, 9G1, 19F10a, and 27G10 all blocked LILRB1 binding to HLA-G and HLA-A2. Such blocking antibodies are suggested to be useful in treating a wide variety of cancers characterized by tumor cells expressing HLA-G (and / or other LILRB1 ligands, such as HLA-A2) or HLA-E in addition to HLA-G. Therefore, neutralization of LILRB1 binding to HLA is considered a desirable antibody property.

[0023] LILRA3 naturally exists as a soluble protein and binds to HLA class I molecules, suggesting that LILRA3 may thereby compete with LILRB1 for HLA class I molecule binding and act as an inhibitor of LILRB1 signaling; consequently, it would be desirable to identify antibodies that bind to LILRB1 but not to LILRA3.

[0024] GHI / 75 is a murine monoclonal LILRB1 antibody that has been shown to enhance macrophage phagocytic activity by promoting anti-CD47 blockade-mediated phagocytosis of cancer cells, but has not been shown to have any effect on its own (see Barkal et al., "Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy", Nat. Immunol. Jan;19(1):76-84).

[0025] WO2021028921 (Biond Biologics) describes antibodies 19E3, 15G8, and 17F2 that bind to LILRB1. Cross-reactivity to LILRA3 and LILRA1 was examined using a binding ELISA, and none of the antibodies cross-reacted with human LILRA3 or LILRA1. 15G8 binds to an epitope between the D1 and D2 domains, which is thought to be the interaction region of LILRB1 that binds to beta-2-microglobulin (B2M) when complexed with HLA.

[0026] The antibodies were selected according to their favorable binding, cross-reactivity profiles, and functional activity in various assays examined. Each LILRB1 antibody was shown to block LILRB1-biotin binding to cells expressing HLA-G, and the 15G8 antibody blocked LILRB1-MHC-I interaction. Functional blocking was examined in human Jurkat cells (T cells) by co-culturing LILRB1-expressing Jurkat cells with or without MHC-I-expressing A375 cancer cells. It was shown that MHC-I from cancer cells strongly inhibited the secretion of the pro-inflammatory cytokine IL-2, and the 15G8 antibody, which blocks LILRB1 / MHC-I interaction, increased IL-2 secretion in a dose-dependent manner. The inhibitory effect of LILRB1 was enhanced by transfecting A375 cancer cells with HLA-G, rendering them MHC-I and HLA-G positive.

[0027] We also show that blocking LILRB1 antibodies 19E3, 15G8, and 17F2 can promote the phagocytosis of HLA-G-positive A375 cells by macrophages, and that LILRB1-blocking antibody 15G8 can promote the phagocytosis of various MHC-I-positive cancer cell lines. The presence of LILRB1-blocking antibodies during macrophage differentiation from monocytes was shown to increase the expression of HLA-DR and CD80, markers of the inflammatory macrophage phenotype.

[0028] [Table 5]

[0029] WO 2022034524 (Biond Biologics) describes an antibody that binds to an epitope within the ILT2 (LILRB1) interdomain between the D1 and D2 domains, the interaction domain between ILT2 and beta-2-microglobulin (B2M), directly blocking the interaction between LILRB1 and its HLA-G ligand. Anti-ILT2 antibodies used as monotherapy have been shown to promote phagocytosis of cancer cells.

[0030] WO 2022026360 (University of Texas) describes antibodies that bind to LILRB1 at D1-D2, particularly at epitopes located within the linker region located between the D1 and D2 domains of human LILRB1, and block the interaction of LILRB1 with HLA-G. Antibodies that bind to LILRB1 or to LILRB1 and LILRA1 but do not bind to other LILRB or LILRA family members are disclosed.

[0031] WO 2022025585 (LG Chem) describes antibodies specific for LILRB1. Antibodies 10, 11, and 13 increase cell death of HLA-G-overexpressing HEK293 cells by natural killer cells KHYG-1 compared to a human IgG4 isotype control, indicating that these antibodies enhance NK cell cytotoxicity.

[0032] WO 2021222544 (NGM) describes antibodies that bind to human LILRB1, human LILRB2, and both human LILRB1 and human LILRB2, including 73D1 and Hz73Dl.vl, anti-LILRB1 / anti-LILRB2 dual antagonist monoclonal antibodies. In addition to binding to LILRB1 and LILRB2, the anti-LILRB1 / LILRB2 antibodies exhibit cross-reactivity with LILRA1, but not with LILRB3, LILRB4, LILRB5, LILRA2, LILRA4, LILRA5, and LILRA6. As part of the characterization process, representative antibodies were evaluated for their ability to inhibit or block the interaction of LILRB1 or LILRB2 with their natural ligands in competition experiments using a Biacore system. Natural ligands for LILRB1 and LILRB2 include, but are not limited to, HLA class I molecules, including HLA-A, HLA-B, HLA-C, HLA-E, and HLA-G. The anti-LILRB1 and anti-LILRB1 / LILRB2 antibodies described therein inhibited the interaction between LILRB1 and its ligands. Furthermore, the anti-LILRB2 and anti-LILRB1 / LILRB2 antibodies described therein inhibited the interaction between LILRB2 and its ligands. The anti-LILRB1 / LILRB2 antibodies can bind to both targets, i.e., LILRB1 and LILRB2, and are biologically functional in preventing the interaction of both targets with their ligands.

[0033] To further characterize the effects of anti-LILRB1, anti-LILRB2, and anti-LILRB1 / LILRB2 antibodies on macrophage function, we performed phagocytosis assays. Anti-LILRB1 / LILRB2 antibodies (e.g., Hz73Dl.vl) and anti-LILRB1 antibodies (e.g., 27F9) enhanced macrophage phagocytic activity toward Raji tumor cells opsonized with anti-CD47 antibodies. Anti-LILRB2 antibodies (e.g., 48A5) had no effect on macrophage phagocytosis. Antibody 24E7, an anti-i-LILRB1 antibody that does not disrupt MHC-I interactions, failed to induce macrophage phagocytosis. These data suggest that anti-LILRB1 and anti-LILRB1 / LILRB2 antibodies that enhance macrophage phagocytosis do so by disrupting macrophage LILRB1 interactions with MHC-I on tumor cells, thereby inhibiting LILRB1-induced suppression of macrophages and thus increasing macrophage phagocytosis of tumors. Anti-LILRB1 antibodies that cannot block the interaction with MHC-I, such as 24E7, do not induce macrophage phagocytosis.

[0034] Anti-LILRB1 / LILRB2 antibodies and anti-LILRB1 and anti-LILRB2 antibodies were evaluated for their ability to induce pro-inflammatory cytokine release from PBMCs after LPS stimulation. LILRB2 and LILRB1 / 2 antibodies (but not LILRB1-selective antibodies) were able to induce increased release of pro-inflammatory TNF-alpha and GM-SCF after LPS stimulation. These data indicate that LILRB2 can suppress pro-inflammatory cytokine release from PBMCs after LPS stimulation. LILRB1 / 2 and LILRB2 antibodies (but not LILRB1-selective antibodies) were able to reduce the immunosuppressive activity of MDSCs in an MLR assay. The MLR assay is used to determine allogeneic T cell activation. Macrophages are traditionally characterized as either pro-inflammatory (M1) or immunosuppressive (M2) based on the surface-expressed markers CD80, CD86 (M1), CD163, CD204, and CD206 (M2). Hz73Dl.vl (a dual LILRB1 / LILRB2 antibody) induced a decrease in M2-like macrophage phenotype markers CD163, CD204, and CD206, as well as additional M2-like markers CD14 and CD209, consistent with the M2-to-M1-like polarization of differentiating monocytes. The anti-LILRB2-specific antibody 48A5, but not the anti-LILRB1-specific antibody 27F9, induced a shift in M1- and M2-like marker profile comparable to the dual LILRB1 / LILRB2 antibody, suggesting that LILRB2 interactions contribute to M2-to-M1-like polarization.

[0035] These data indicate that anti-LILRB1 / LILRB2 antibodies enhance macrophage phagocytosis in the presence of CD47 antibodies via LILRB1 and induce a more pro-inflammatory M1-like phenotype during macrophage differentiation via LILRB2, mediated by inhibition of LILRB1 or LILRB2 interaction with MHC-1.

[0036] [Table 6]

[0037] WO 2018187518 (Merck, Agenus) disclosed an anti-LILRB2 (anti-ILT4) antibody, 1E1, that binds to a nonlinear conformational epitope that overlaps with the HLA-G-bound epitope. Epitope characterization was provided only for 1E1. Other anti-LILRB2 antibodies, 1G2, 2A6, 2D5, 3E6, 3G7, 2C1, and 5A6, were disclosed with specific characteristics, such as the ability to bind to cynomolgus monkey ILT4 (LILRB2), the ability to block HLA-G Fc ligand binding to ILT4, and the rescue of spontaneous IL2 suppression and HLA-G-dependent suppression.

[0038] WO 2019126514 (Jounce) discloses anti-LILRB2-specific antibodies, but none of the disclosed antibodies bind to LILRB1, LILRB4, LILRB5, LILRA3, or LILRA6. WO 2019126514 also discloses anti-LILRB2 antibodies capable of blocking the interaction between HLA-G / A and LILRB2. A positive correlation between M1-promoting activity (as measured by an increase in TNF-alpha) and the ability of anti-LILRB2 mAbs to block HLA-G / A:LILRB2 interaction was reported. A chimeric (hIgG4) anti-LILRB2 antibody was selected based on its specificity for cell-expressed hLILRB2 over 10 other human LIL family members, its ability to block ligand interactions for cell-expressed LILRB2, and its ability to convert M2-like macrophages into M1-like macrophages with an inflammatory activation state in a primary human macrophage assay. The selected LILRB2-specific ligand-blocking antibodies were further screened for binding to non-human primate (NHP) monocytes. JTX-8064 (Jounce) is a humanized IgG4 monoclonal antagonist antibody that selectively binds to LILRB2, thereby preventing LILRB2 from binding to its ligands, classical and non-classical MHCI molecules. By blocking the ability of LILRB2 to bind to HLA-A / B and / or HLA-G, markers of immune tolerance in cancer cells, JTX-8064 was shown to enhance pro-inflammatory cytokine production in macrophages. Antagonism of LILRB2 has been reported to result in the repolarization of human macrophages from an M2 (suppressive) to an M1 (pro-inflammatory) phenotype and promote antitumor immunity in mouse models.

[0039] WO2016144728A2 (University of Texas) identifies a group of antibodies that bind to LILRB2, 3, and 4. Figure 19 of that application shows the cross-reactivity of antibodies to LILRB1-5 and the absence of antibodies that bind to LILRB1.

[0040] WO2022087188 (ImmuneOnc) describes the antibody B2-19 and its variants that specifically bind to LILRB2 and block the interaction of LILRB2 with multiple ligands involved in cancer-associated immunosuppression, including HLA-G, ANGPTL, SEMA4A, and CD1d.

[0041] WO2022079045 describes antagonist antibodies that bind to human and macaque LILRB1 and / or LILRB2. All antibodies (B.1.2.1 and B.1.2.2) did not bind to human LILRA2, LILRA4, LILRA5, LILRB3, LILRB4, or LILRB5, or to macaque LILRA1.1, LILRA1.2, LILRA2.1, LILRA2.2, LILRA4, LILRB3, or LILRB4.

[0042] WO2019144052 (Adanate) discusses antibodies that bind to various LILRBs and LILRs, but does not provide antibody sequences; antibodies 5G11.H6, 9C9.E6, 9C9.D3, 5G11.G8 and 16D11.D10 are said to bind to LILRB1, LILRB2, LILRB3, LILRB5, LILRA1, LILRA3 and LILRA5, but not LILRB4, LILRA2 and LILRA4, and are HLA-G blocking antibodies.

[0043] Through cis- or trans-interactions with human leukocyte antigen (HLA)-G, the two most commonly expressed inhibitory LILRs, LILRB1 and LILRB2 (also known as LILRB1 / 2, CD85j / d, and ILT2 / 4), are involved in immune tolerance during pregnancy and transplantation, autoimmune disease, and tumor-mediated immune evasion. LILRB1 / 2 contains four extracellular Ig-like domains, D1, D2, D3, and D4. D1D2 are thought to be involved in binding to HLA class I (HLA-I), whereas the role of D3D4 is unclear. Crystallography of the four-domain LILRB1 and HLA-G1 complex structures supports a model in which D1D2 contributes to HLA binding, while D3D4 acts as a scaffold (Wang et al. (2020) Cell Mol Immunol. 2020 Sep;17(9):966-975. doi:10.1038 / s41423-019-0258-5. Epub 2019 Jul 4).

[0044] LILRB1 and LILRB2, particularly the D1D2 domain involved in ligand binding, are attractive targets for anticancer approaches when immune regulatory processes are disrupted to evade antitumor immunity by inducing macrophage phagocytosis through blockade of LILRB1 interactions and inducing pro-inflammatory macrophage reprogramming through blockade of LILRB2 ligand interactions with MHC-1 and HLA-G. However, preclinical efficacy in cancer models following treatment with LILRB1 or LILRB2 antibodies has been mixed to date, with reports of partial growth inhibition or tumor regression observed in only a subset of animals. Therefore, further approaches to target the LILR family need to be developed. Summary of the Invention [Means for solving the problem]

[0045] Statement of the Invention The present invention provides the following: 1. An antigen-binding protein capable of specifically binding to human LILRB1 and human LILRB2, which does not block the interaction between human LILRB1 and HLA-G tetramers and / or does not block the interaction between human LILRB2 and HLA-G tetramers, and is capable of reprogramming macrophages. 2. The antigen-binding protein of clause 1, which is capable of reprogramming fully differentiated macrophages towards an anti-tumor (pro-inflammatory) phenotype. 3. The antigen binding protein of clause 1 or clause 2, wherein reprogramming is indicated / detected by induction of a marker of macrophage reprogramming. 4. The antigen binding protein of any one of clauses 1 to 3, wherein reprogramming is indicated / detected by release of pro-inflammatory cytokines from macrophages following exposure of macrophages to the antigen binding protein and stimulation selected from LPS stimulation, R848, IL1 beta, HMGB1 peptide, c-di-AMP and poly(I:C). 5. The antigen binding protein of any of clauses 1 to 4, wherein reprogramming is indicated / detected by release of the pro-inflammatory cytokines TNF-alpha and / or GM-CSF from macrophages following exposure to the antigen binding protein and LPS stimulation. 6. The following: (a) stimulating the production of GM-CSF and / or TNF-alpha upon LPS stimulation of iPS-derived macrophages and / or primary monocyte-derived macrophages; (b) stimulating the production of GM-CSF and / or TNF-alpha upon LPS stimulation of iPS-derived macrophages expressing LILRB1; (c) stimulating the production of GM-CSF and / or TNF-alpha upon LPS stimulation of primary monocyte-derived macrophages expressing LILRB1 and LILRB2; (d) stimulating the production of GM-CSF and / or TNF-alpha upon LPS stimulation of human macrophages expressing LILRB1 and LILRB2; and (e) Stimulating the production of GM-CSF and / or TNF-alpha upon LPS stimulation of LILRB1-expressing human macrophages. 6. The antigen-binding protein of any of clauses 1 to 5, having one or more properties selected from: 7. An antigen-binding protein comprising: (a) Induce phagocytosis, (b) induce phagocytosis in the absence of a second signal; (c) inducing phagocytosis in the absence of a second antibody (e.g., an anti-CD47 antibody or an anti-EGFR antibody); (d) induces phagocytosis in the absence of a second opsonizing antibody (e.g., an anti-CD47 antibody or an anti-EGFR antibody); (e) inducing phagocytosis of cancer cells in the absence of a second opsonizing antibody (e.g., a tumor-binding antibody); and (f) inducing phagocytosis of MHC class I-positive and / or MHC class I-negative cancer cells; 7. The antigen-binding protein of any of clauses 1 to 6, having one or more properties selected from the ability to 8. An antigen-binding protein comprising: (a) Human LILRB1, human LILRB2, and human LILRA3; (b) human LILRB1, human LILRB2, human LILRA3, and human LILRA1; (c) human LILRB1, human LILRB2, human LILRB3, human LILRA3, human LILRA4 and human LILRA6; and / or (d) human LILRB1, human LILRB2, human LILRB3, human LILRA1, human LILRA3, human LILRA4, and human LILRA6 8. The antigen-binding protein of any of clauses 1 to 7, which is capable of specifically binding to 9. The antigen-binding protein of any of clauses 1 to 8, which does not bind to human LILRB4, human LILRB5, human LILRA2 or human LILRA5. 10. The antigen-binding protein of any of clauses 1-9, wherein binding is assessed by flow cytometry or ELISA. 11. The antigen binding protein of any of clauses 1 to 10, which is capable of specifically binding to a homologue of the LILRB1 / 2 ectodomain of rhesus monkey (SEQ ID NO: 43) and / or cynomolgus monkey (SEQ ID NO: 44). 12. An antigen-binding protein comprising: (a) Human LILRB1, human LILRB2, and human LILRA3; (b) human LILRB1, human LILRB2, human LILRA3, and human LILRA1; (c) human LILRB1, human LILRB2, human LILRB3, human LILRA3, human LILRA4 and human LILRA6; and / or (d) human LILRB1, human LILRB2, human LILRB3, human LILRA1, human LILRA3, human LILRA4, and human LILRA6 12. The antigen-binding protein of any one of clauses 1 to 11, which binds to an epitope common to 13. The epitope is (a) the sequence of human LILRB1 AEFPMGPVTSAHAGT ​​(SEQ ID NO: 78); (b) the sequence AEFPMGPVTSAHAGT ​​(SEQ ID NO: 78) and the sequence LTHPSDPLEL (SEQ ID NO: 79) of human LILRB1 formed by; Epitopes are mapped using hydrogen-deuterium exchange (HDX) mass spectrometry; 13. The antigen-binding protein of any one of clauses 1 to 12. 14. The epitope is (a) the sequence FVLYKDGERDF (SEQ ID NO: 80) in human LILRB1, the sequence GYDRFVLYKEGERD (SEQ ID NO: 81) in human LILRB2, and the sequence YDRFVLYKEWGRD (SEQ ID NO: 82) in human LILRA3; (b) the sequence SSEWSAPSDPLD (SEQ ID NO: 83) in LILRB1, the sequence ECSAPSDPLDI (SEQ ID NO: 84) in LILRB2, and the sequence SEWSAPSDPLD (SEQ ID NO: 85) in LILRA3; (c) the sequence LQCVSDVGYD (SEQ ID NO: 86) in LILRB2 and the sequence FQCGSDAGYDRF (SEQ ID NO: 87) in LILRA3; (d) the sequence FLLTKEGAADDPW (SEQ ID NO: 88) in LILRB1 and the sequence AADAPLRLRSIHEY (SEQ ID NO: 89) in LILRB2; (e) the sequence RSYGGQYR (SEQ ID NO: 90) in LILRB1 and the sequence PVSRSYGGQYRC (SEQ ID NO: 91) in LILRB2; (f) the sequence LDILIAGQFYD (SEQ ID NO: 92) in LILRB1, the sequence APSDPLDILI (SEQ ID NO: 93) in LILRB2, and the sequence PSDPLDILI (SEQ ID NO: 94) in LILRA3 formed by; The epitope is mapped using binding to a peptide microarray. 14. The antigen-binding protein of any one of clauses 1 to 13. 15. The antigen-binding protein of any one of clauses 1 to 14 which is an antibody or antigen-binding fragment thereof. 16. The antigen-binding protein of any one of clauses 1 to 15, which is a human antibody or antigen-binding fragment thereof. 17. The antigen-binding protein of any one of clauses 1 to 16, which is a monoclonal antibody, such as a human monoclonal antibody. 18. The antigen-binding protein according to any one of clauses 1 to 17, comprising an Fc, such as a human IgG1 Fc or a human IgG4 Fc. 19. An antigen-binding protein comprising: (a) Antibody 1 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; (b) Antibody 2 of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; (c) antibody 3 of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22; (d) Antibody 4 of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30; and (e) Antibodies 5 of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 comprising six CDRs (HCDR1, HCRD2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively) of an antibody selected from: The sequences are defined using the Kabat nomenclature. 19. The antigen-binding protein of any one of clauses 1 to 18. 20. An antigen-binding protein comprising: (a) Antibody 1 of SEQ ID NO: 7 and SEQ ID NO: 8; (b) antibody 2 of SEQ ID NO: 15 and SEQ ID NO: 16; (c) antibody 3 of SEQ ID NO: 23 and SEQ ID NO: 24; (d) antibody 4 of SEQ ID NO: 31 and SEQ ID NO: 32; and (e) Antibody 5 of SEQ ID NO: 39 and SEQ ID NO: 40 each comprising a VH and a VL of an antibody selected from: 20. The antigen binding protein of any one of clauses 1 to 19, wherein the sequence is defined using the Kabat nomenclature system. 21. An antigen-binding protein, e.g., a human antibody or antigen-binding fragment thereof, that is capable of competing with an antigen-binding protein, such as the antibody or antigen-binding fragment thereof, of any one of clauses 1 to 20 for binding to human LILRB1, human LILRB2 and / or human LILRA3. 22. The antigen-binding protein, such as a human antibody or antigen-binding fragment thereof, according to clause 21, wherein competition for binding is assessed using a competition assay selected from a cell-based binding assay, a cell-free binding assay, an immunoassay, ELISA, HTRF, flow cytometry, a fluorescence microvolume assay technology (FMAT) assay, Mirrorball, a high-content imaging-based fluorescence immunoassay, a radioligand binding assay, biolayer interferometry (BLI), surface plasmon resonance (SPR), and a thermal shift assay. 23. A composition comprising an antigen-binding protein according to any one of clauses 1 to 22 and a diluent. 24. (a) For use as a medicine; (b) for use as a medicament for the treatment of cancer; (c) for use in the treatment of cancer; (d) For use in the manufacture of a medicament for the treatment of cancer (Optionally, the cancer of (b), (c), or (d) is (i) Acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), brain low-grade glioma (LGG), invasive breast cancer (BRCA), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney clear cell carcinoma (KIRC), kidney papillary renal cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) , pancreatic adenocarcinoma (PAAD), sarcoma (SARC), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), endometrial carcinoma (UCEC), uveal melanoma (UVM), colorectal cancer, prostate cancer, childhood cancer, lymphoma and leukemia, such as DLBCL, NHL, multiple myeloma, Hodgkin's lymphoma, etc.; (ii) cancer that is positive for LILRB1 or LILRB2 or both LILRB1 and LILRB2; (iii) cancers positive for immunosuppressive macrophages (as measured by CD163 or CD68 positivity) and / or tumor-infiltrating T cells; (iv) cancers with elevated or decreased classical or non-classical MHC class I expression; (v) cancer positive for one or more of LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 (selected from); (e) For use as a drug for the treatment of immunosuppressive diseases; (f) for use in the treatment of immunosuppressive diseases; or (g) For use in the manufacture of a medicament for the treatment of an immunosuppressive disease, 24. The antigen-binding protein of any one of clauses 1 to 22 or the composition of clause 23. 25. A method of treating cancer or treating an immunosuppressive disease comprising administering to a subject an antigen binding protein according to any one of clauses 1 to 22 or a composition according to clause 23. 26. An isolated recombinant DNA or RNA sequence comprising a sequence encoding an antigen binding protein according to any one of clauses 1 to 22. 27. The isolated recombinant DNA sequence of clause 26, which is a vector, optionally wherein the vector is an expression vector. 28. The isolated recombinant DNA sequence of clause 26 or 27 encoding the antigen binding protein of any one of clauses 1 to 22 under the control of a promoter. 29. A host cell comprising a DNA or RNA sequence according to any one of clauses 26 to 28, optionally capable of expressing an antigen binding protein according to any one of clauses 1 to 22. 30. A method of producing an isolated antigen binding protein according to any one of clauses 1 to 22, the method comprising culturing a host cell according to clause 29 under conditions suitable for expression of the isolated antibody or antigen binding fragment thereof. 31. A method for identifying an antigen-binding protein according to any one of clauses 1 to 22, comprising: (a) (i) human LILRB1, LILRB2 and / or LILRA3; (ii) human LILRB1, LILRB2, and LILRA3 proteins; (iii) human LILRB1, LILRB2, LILRA1, and LILRA3 proteins; (iv) human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4 and LILRA6 proteins; and / or (v) human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 proteins providing one or more antigen binding proteins capable of binding to the (b), (c), and (d): (b) assessing the ability of one or more antigen binding proteins to modulate one or more biological activities / phenotypes of human macrophages, such as promoting phagocytosis and / or pro-inflammatory cytokine release (such as TNF-alpha or GM-SCF), expression of macrophage activation markers (such as HLA-DR and / or CD80), or down-regulation of CD163 (a marker of macrophage activation to the M1 phenotype); (c) evaluating the ability of one or more antigen binding proteins to block the binding of LILRB1 and / or LILRB2 to cells expressing a ligand for LILRB1 and / or LILRB2, e.g., HLA-G, and selecting one or more antibodies that bind to LILRB1, LILRB2, and LILRA3 but do not block the binding of LILRB1 and / or LILRB2 to target cells expressing a ligand for LILRB1 and / or LILRB2, e.g., HLA-G; (d) evaluating the ability of another antigen binding protein to block the binding of a ligand (e.g., HLA-G) to cells expressing LILRB1 and / or LILRB2, and selecting one or more antibodies that do not block the binding of a ligand for LILRB1 and / or LILRB2, e.g., HLA-G, to cells expressing LILRB1 and / or LILRB2. conducting one or more assessments selected from: (e) selecting one or more antigen binding proteins capable of specifically binding to human LILRB1 and human LILRB2, wherein the antigen binding proteins do not block the interaction of human LILRB1 with HLA-G tetramers and are capable of reprogramming macrophages; and optionally, (f) formulating the one or more antigen binding proteins with one or more excipients into a composition; A method comprising: 32. A method for identifying an antibody or antigen-binding fragment thereof according to any one of clauses 1 to 22, comprising: (a) (i) human LILRB1, LILRB2 and / or LILRA3 proteins; (ii) human LILRB1, LILRB2, and LILRA3 proteins; (iii) human LILRB1, LILRB2, LILRA1, and LILRA3 proteins; (iv) human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4 and LILRA6 proteins; and / or (v) human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 proteins providing one or more antibodies or antigen-binding fragments thereof capable of binding to (b), (c) and (d): (b) evaluating the ability of another antibody or antigen-binding fragment thereof to block the binding of LILRB1 and / or LILRB2 to cells expressing a ligand for LILRB1 and / or LILRB2, e.g., HLA-G, and selecting one or more antibodies that bind to LILRB1, LILRB2, and LILRA3 but do not block the binding of LILRB1 and / or LILRB2 to target cells expressing a ligand for LILRB1 and / or LILRB2, e.g., HLA-G; (c) evaluating the ability of another antibody or antigen-binding fragment thereof to block the binding of a ligand (e.g., HLA-G) to cells expressing LILRB1 and / or LILRB2, and selecting one or more antibodies that do not block the binding of a ligand of LILRB1 and / or LILRB2, e.g., HLA-G, to cells expressing LILRB1 and / or LILRB2; and (d) assessing the ability of one or more antibodies or antigen-binding fragments thereof to modulate one or more biological activities / phenotypes of human macrophages, such as to enhance phagocytosis and / or pro-inflammatory cytokine release (such as TNF-alpha or GM-SCF) or expression of macrophage activation markers (such as HLA-DR and / or CD80). conducting one or more assessments selected from: A method comprising:

[0046] The present invention provides antigen-binding proteins, such as antibodies or antigen-binding proteins, such as human monoclonal antibodies, that specifically bind to human LILRB1 and human LILRB2, respectively. Antibodies of the present invention also bind to human LILRA3 (e.g., antibodies 1, 2, 3, 4, and 5), and in some embodiments, antibodies of the present invention bind to LILRA3 and human LILRA1 (e.g., antibody 3). In some embodiments, antibodies of the present invention bind to human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4, and LILRA6. In some embodiments, antibodies of the present invention bind to human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6. In preferred embodiments, antibodies 1 to 5 of the present invention do not bind to human LILRB4, human LILRB5, human LILRA2, or human LILRA5.

[0047] The antibodies of the present invention are "non-blocking" in that they do not disrupt the interaction of LILRB1 and / or LILRB2 with HLA-G.

[0048] "Not blocking," "non-blocking activity," or "non-blocking" or "not blocking" means that in the assays described herein, the assay signal is greater than 10% of the signal observed for the isotype control. The isotype control is 100% signal, blocking is less than 10% of the signal observed for the isotype control, and non-blocking is greater than 10% of the signal observed for the isotype control. The percent of blocking can be determined by normalizing to a negative control IgG. The percentage of blocking can be calculated at various concentrations of the test antibody. The antibodies of the present invention block the binding of HLA-G tetramers to LILRB1; blocking can be detected and / or quantified by any suitable means known in the art or described herein. For example, blocking of HLA-G tetramer-LILRB1 interaction can be detected and / or quantified using a tetramer blocking assay as described herein. The ability of an antibody to block receptor binding to its ligand can be assessed using HEK293 cells overexpressing the human LIRB1 receptor by incubating the cells with human HLA-G PE-labeled tetramer in the absence or presence of 500 nM test antibody; cells that bind to HLA-G can be quantified by flow cytometry.

[0049] The antibodies of the present invention can induce the reprogramming of macrophages into an anti-tumor, pro-inflammatory phenotype, such as fully differentiated macrophages. Macrophages with an anti-tumor phenotype secrete high levels of pro-inflammatory cytokines, such as GM-CSF, TNFα, IL1, IL6, and IL12. Macrophages reprogrammed to become anti-tumor macrophages have increased secretion of one or more pro-inflammatory cytokines (e.g., GM-CSF, TNFα, IL1, IL6, and / or IL12) compared to the macrophages before reprogramming. Surface markers of the anti-tumor phenotype include CD80 high and CD86 high, CD206 low, CD209 low, and CD163 low. Fully differentiated macrophages are macrophages with adhesive properties and express differentiation markers such as CD163, CD206, and / or 25F9. Human macrophages include human iPS-derived macrophages, human monocyte-derived macrophages, human tumor-derived macrophages, and human ascites-derived macrophages, human mono / macrophage cell lines (THP-1, mono / mac, U937, etc.), and TAMs.

[0050] In some embodiments, antibodies of the invention (e.g., antibodies 1, 3, and 5) are capable of inducing phagocytosis of cancer cells, including immortalized and / or transformed cell lines, cell lines containing oncogenes that cause uncontrolled growth, and those with oncogenes that cause uncontrolled growth, cells isolated from human tumors, cells isolated from human ascites, and / or cells isolated from patient-derived tumor xenograft models.

[0051] The antibodies of the present invention can also induce macrophage phagocytosis, regardless of the HLA status of tumor cells and in the presence or absence of LILRB1 / 2 ligand expression on tumor cells. Therefore, physical interaction between tumor cells expressing the ligand and LILRB1 and / or LILRB2 tumor-associated macrophages (TAMs) is not essential for the antibodies of the present invention to reprogram TAMs. Furthermore, antibodies 1, 3, and 5 of the present invention, which can induce phagocytosis, can do so in the absence of a second signal or antibody, such as an anti-CD47 antibody or an anti-EGFR antibody. The antibodies of the present invention induce the release of pro-inflammatory cytokines (such as TNF-alpha or GM-SCF) and / or the expression of macrophage activation markers (such as HLA-DR and / or CD80) from fully differentiated macrophages, indicating macrophage "reprogramming" to an anti-tumor (pro-inflammatory) phenotype. The antibodies of the present invention do not bind to ITAM domain-containing LILRA2.

[0052] LILR family members have overlapping and distinct patterns of ligand binding and expression and can be either immunostimulatory or immunosuppressive. Therefore, to maximize antitumor immunity, tailored approaches are needed to minimize or avoid immunostimulatory LILR signaling (LILRA1, 2, 4, 5, 6) while inhibiting immunosuppressive signaling (LILRB1, 2). To overcome compensatory resistance mediated by targeting redundancy, it may be advantageous to target both LILRB1 and LILRB2 receptors, which share a common ligand and expression pattern. LILRB1 and LILRB2 are highly homologous, bind to a common HLA, are both expressed on myeloid cells, including macrophages, and both contain intracellular immunosuppressive ITIM domains. Therefore, without wishing to be bound by theory, it may be advantageous to target both LILRB1 and LILRB2 while avoiding binding to the ITAM-domain containing LILRA2 and LILRA5 receptors.

[0053] LILRA3 is considered an "off-target," i.e., an undesired target, and the art has reported that LILRB1 binding but not LILRA3 binding is associated with NK cell cytotoxicity, and selectivity for LILRB1 over LILRA3 has been reported as a positive characteristic in selecting preferred antibodies. Furthermore, it has been suggested that LILRA3, as a soluble factor, may compete with LILRB1 and LILRB2 for ligand binding, thereby acting as a natural competitor for LILRB1 and LILRB2 ligand binding.

[0054] However, we hypothesize that LILRA3 binding may be advantageous based on the clinical manifestations of inflammatory conditions associated with genetic loss-of-function mutations in LILRA3 and the potential immunosuppressive role of LILRA3 in humans, which involves reduced affinity of LILRA3 for HLA-G and HLA-A compared with LILRB1 and LILRB2. We further investigated LILRA3 expression in other tumor types using the TCGA database, which identified overexpression of LILRA3 in multiple cancer types (Figure 1).

[0055] Bioinformatics analysis of single-cell RNA-sequencing datasets across multiple cancer types comparing LILRB1 and LILRB2 expression specifically in melanoma cancer patients also revealed mixed expression of LILRB1 and LILRB2 in the tumor environment, identifying LILRB1-positive, LILRB2-positive, and both LILRB1- and LILRB2-positive macrophages (Figures 2 and 3).

[0056] The antibodies of the present invention are capable of binding to LILRB1 and LILRB2. The antibodies of the present invention are capable of binding to LILRB1, LILRB2, and LILRA3. The antibodies of the present invention are capable of binding to LILRB1, LILRB2, LILRA3, and LILRA1. The antibodies of the present invention do not specifically bind to LILRA2.

[0057] LILRB1 and LILRB2 bind to MHC class I, and altered MHC class I expression is a known tumor immune evasion mechanism, reducing tumor antigen presentation and subsequent T cell activation. For example, downregulation of classical MHC class I (e.g., HLA-A) is observed in approximately one in three melanoma patients and has been associated with innate and acquired resistance to T cell checkpoint therapy. Many cancer types also upregulate non-classical MHC class I, such as HLA-G. Tumors are also highly heterogeneous, and immune infiltrates, including macrophages, NK cells, and T cells, are not uniformly distributed within the tumor microenvironment and do not always come into direct contact with ligand-expressing tumor cells. However, successful activation of these immune cell types results in the release of cytokines and chemokines, contributing to antitumor immunity within the tumor. LILRB1 expression in the tumor microenvironment has also been associated with poor clinical responses to immunotherapy, even in the absence of HLA-G. Therefore, it would be highly advantageous if LILRB1 / 2 antibodies could activate the anti-tumor properties of macrophages (such as GM-SCF release and phagocytosis) independently of ligand interaction. Despite this, to date, the ability of antibodies to not only bind to LILRB1 and / or LILRB2 but also block ligand interaction has been an important part of antibody selection during drug discovery, with binding and blocking of MHC class I and HLA-G interactions considered key properties related to the effective induction of macrophage reprogramming and phagocytosis. Consistent with this strategy, non-ligand-blocking LILRB1 antibodies have been used as controls in antibody characterization due to their lack of functional effects, as described above. Taking these implications into account, drug discovery strategies aimed at identifying functionally active, non-ligand-blocking (hereinafter "non-blocking") LILRB1 and LILRB2 antibodies are unlikely to be successful. However, without wishing to be bound by theory, the inventors hypothesize that non-ligand blocking antibodies, which can exert immune activation independent of the ligand expression status of the tumor or the proximity of receptor-expressing immune cells to ligand-expressing tumor cells, may improve a patient's therapeutic response and eligibility for treatment.

[0058] In summary, we conducted an antibody strategy against the extracellular domain of LILRB1, which is highly conserved with LILRB2 and shares high sequence homology and structural integrity with the HLA-G binding region of LILRA3. Blocking and non-blocking antibodies were evaluated in macrophage functional assays to identify and compare blocking and non-blocking antibodies for ligand binding. Although binding to LILRA3 is generally considered unfavorable due to its natural inhibitory effect on LILRB1 and LILRB2 through competition for MHC-I binding, we investigated LILRA3 binders and non-binders. Phagocytosis and reprogramming assays were performed using induced pluripotent stem cell-derived macrophages (iPSC-DMs) and macrophages (iPSC-DMs) expressing both LILRB1 and LILRB2.

[0059] Thirty-nine antibodies were tested for their ability to reprogram fully differentiated macrophages and induce phagocytosis. Of these 39 antibodies, only five were identified as being able to induce significant reprogramming of fully differentiated macrophages. Surprisingly, all five antibodies did not block HLA-G binding to LILRB1 or LILRB2, and they cross-competed with each other for LILRB1 binding in epitope binning and cross-competition ELISA experiments, whereas they did not compete with the other 34 antibodies or with reference antibody 2. These five antibodies also bound to LILRA3 but not to LILRA2. Antibodies 1 to 5 were found to bind to human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6, but not to LILRB4, LILRB5, LILRA2, or LILRA5. Furthermore, these antibodies were able to induce macrophage phagocytosis of cancer cells positive and negative for MHC-I (including HLA-G) expression; thus, operating in a ligand-independent manner, they were able to induce the reprogramming of fully differentiated (mature) macrophages, as measured by cytokine release in both iPSC-DMs and MDMs, to achieve pro-inflammatory reprogramming of fully differentiated (mature) macrophages.

[0060] The antibodies of the invention demonstrate ligand-independent activity and can induce reprogramming and phagocytosis regardless of the MHC-I status of the tumor or the proximity of macrophages to tumor cells, which may lead to improved therapeutic responses and patient benefit.

[0061] Detailed Description of the Invention The present invention relates to antigen-binding proteins and antigen-binding fragments thereof, such as antibodies and antigen-binding fragments thereof, in particular human antibodies and antigen-binding fragments thereof that can specifically bind to human LILRB1, human LILRB2, human LILRB3, human LILRA1, human LILRA3, human LILRA4, and human LILRA6.

[0062] The antibodies of the present invention (e.g., antibodies 1, 2, 3, 4, and 5) can specifically bind to an epitope common to human LILRB1, human LILRB2, and human LILRA3. The antibodies of the present invention (e.g., antibodies 1, 2, 3, 4, and 5) do not specifically bind to human LILRA2. In some embodiments, the antibodies of the present invention (e.g., antibody 3) can specifically bind to an epitope common to human LILRB1, human LILRB2, human LILRA3, and human LILRA1. Antibodies 1 to 5 of the present invention selectively bind to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6, but do not bind to the highly homologous LILRB4, LILRB5, and LILRA2.

[0063] In some embodiments, antibodies of the present invention (e.g., antibodies 1, 2, 3, 4, and 5) are capable of specifically binding to an epitope present in the human LILRB1 ectodomain (SEQ ID NO: 41), which includes domains D1, D2, D3, and D4, but do not bind to the D1-D2 fragment of LILRB1 (SEQ ID NO: 42). The D1-D2 region is defined as amino acids 24-223 of human LILRB1, human LILRB2, and human LILRA3; the D3-D4 region is defined as amino acids 224-458 of human LILRB1 and LILRB2 and amino acids 224-439 of LILRA3 (Figure 19). In some embodiments, antibodies of the present invention (e.g., antibodies 1, 2, 3, 4, and 5) are capable of specifically binding to an epitope formed by the amino acid sequence in the human LILRB1 ectodomains D3 and D4.

[0064] The antibodies of the present invention are cross-reactive in that they bind to homologues of the LILRB1 / 2 ectodomain from rhesus monkeys (SEQ ID NO: 43) and cynomolgus monkeys (SEQ ID NO: 44).

[0065] The antibodies of the present invention bind to the major allelic variants of the human LILRB1 ectodomain with similar binding efficiencies, but "binding" indicates that the signal obtained for the LILRB1 variant was at least three times higher than that observed for the control protein.

[0066] The antibodies of the present invention are capable of specifically binding to human LILRB1 and / or human LILRB2 expressed in fully differentiated (mature) human macrophages, such as TAMs, and are capable of modulating one or more biological activities / phenotypes of human macrophages selected from the following: (a) promote the maintenance of an anti-tumor phenotype as assessed by increased release of TNFα and GM-CSF and / or improved cancer cell phagocytosis upon LPS stimulation; (b) promoting a pro-inflammatory phenotype as assessed by increased release of TNFα and GM-CSF upon LPS stimulation; (c) alleviating immunosuppression through the production of pro-inflammatory cytokines, as assessed by increased release of TNFα and GM-CSF upon LPS stimulation; (d) promoting tumor-specific phagocytosis through an HLA-G-independent mechanism, as assessed by enhancing human macrophage phagocytosis of HLA-G-negative or -positive cancer cells; (e) promoting tumor-specific phagocytosis through an MHC class I-independent mechanism, as assessed by enhanced human macrophage phagocytosis of MHC class I-negative or -positive cancer cells; (f) promotes a pro-inflammatory macrophage phenotype, as assessed by increased release of TNFα and GM-CSF upon LPS stimulation from human macrophages, but not from unstimulated peripheral blood mononuclear cells;

[0067] The antibodies or antigen-binding fragments thereof of the present invention may be produced by recombinant means.

[0068] A "recombinant antibody" is an antibody produced by recombinantly modified host cells. The antibodies or antigen-binding fragments thereof according to the invention are optionally isolated or purified.

[0069] The terms "antibody" or "antibody molecule" describe an immunoglobulin, whether natural or partly or wholly synthetically produced. The antigen-binding proteins of the present invention may be antibodies, preferably monoclonal antibodies, which may be human or non-human, chimeric or humanized.

[0070] The antibody molecule is preferably a monoclonal antibody, preferably a human monoclonal antibody. Examples of antibodies are immunoglobulin isotypes, such as immunoglobulin G, and their isotype subclasses, such as IgG1, IgG2, IgG3, and IgG4, and fragments thereof. The four human subclasses (IgG1, IgG2, IgG3, and IgG4) each contain a different heavy chain; however, they are highly homologous and differ primarily in the hinge region, resulting in different degrees of activation of the host immune system. IgG1 and IgG4 contain two interchain disulfide bonds in the hinge region, IgG2 has four, and IgG3 has 11 interchain disulfide bonds.

[0071] The terms "antibody" and "antibody molecule" as used herein include antibody fragments, such as Fab and scFv fragments, which contain an antigen-binding site based on the CDRs for an epitope of a target antigen.

[0072] Antibodies of the present invention may be monovalent or bivalent and may or may not contain an Fc. Bivalent antibodies of the present invention may be monoparatopic, having two identical paratopes for epitope binding, or biparatopic, having two different paratopes for epitope binding. Bivalent antibodies of the present invention may be monospecific antibodies that bind to one epitope, or bispecific antibodies that bind to two different epitopes. Bivalent antibodies of the present invention may be bispecific antibodies that bind to two different epitopes, each from a different target antigen. Bivalent antibodies of the present invention may be bispecific biparatopic antibodies that bind to two distinct (non-overlapping) epitopes on the same target antigen. For optimal macrophage reprogramming activity, particularly under immunosuppressive M2 conditions, antibodies of the present invention are preferably provided in a bivalent monoparatopic format and preferably contain an Fc for association with Fc receptors on the macrophage membrane.

[0073] Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv), and domain antibodies (sdAb). Unless otherwise required by context, the terms "antigen-binding protein," "antibody," or "antibody molecule," as used herein, are thus equivalent to "antibody or antigen-binding fragment thereof."

[0074] Antibodies are immunoglobulins with the same basic structure: two heavy chains and two light chains forming two Fab arms containing identical domains, the Fc domain giving the antibody a classic "Y" shape, connected by a flexible hinge region to the stem. The Fab domain consists of two variable and two constant domains, with the variable heavy (VH) and constant heavy 1 (CH1) domains on the heavy chain and the variable light (VL) and constant light (CL) domains on the light chain. The two variable domains (VH and VL) form a variable fragment (Fv), which confers antigen specificity based on the antibody's CDRs, and the constant domains (CH1 and VL) act as a structural framework. Each variable domain contains three hypervariable loops known as complementarity-determining regions (CDRs). In each of the VH and VL, the three CDRs (CDR1, CDR2, and CDR3) are flanked by four less variable framework (FR) regions (FR1, FW2, FW3, and FW4), resulting in the structure FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. The CDRs provide specific antigen recognition sites on the surface of the antibody.

[0075] Both Kabat and ImMunoGeneTics (IMGT) numbering nomenclature may be used herein. Generally, unless otherwise indicated (explicitly or by context), amino acid residues are numbered herein according to the Kabat numbering scheme (Kabat et al., 1991, J Immunol 147(5):1709-19). For examples where the IMGT numbering scheme is used, amino acid residues are numbered herein according to the ImMunoGeneTics (IMGT) numbering scheme described in Lefranc et al., 2005, Dev Comp Immunol 29(3):185-203.

[0076] Techniques for producing and isolating exogenous, eg, human, antibodies and fragments thereof in transgenic non-human mammals, such as mice and rats, are well known in the art.

[0077] Using monoclonal and other antibodies and recombinant DNA technology, it is possible to generate other antibodies or chimeric molecules that largely retain the specificity of the original antibody. Such techniques may involve transferring CDRs into a different immunoglobulin framework or grafting variable regions onto a different immunoglobulin constant region. Transferring the CDRs of one immunoglobulin into another is described, for example, in EP-A-184187, GB-A-2188638A, or EP-A-239400. Alternatively, hybridomas or other cells that produce antibody molecules may be subject to genetic mutation or other changes that may or may not alter the binding specificity of the antibody produced.

[0078] Antibody humanization involves the transfer or "grafting" of key non-human amino acids onto a human antibody framework. Primarily, this involves the grafting of amino acids in the complementarity-determining regions (CDRs), but potentially also other framework amino acids important for the VH:VL interface and for CDR orientation. Humanization seeks to retain the original binding activity of the non-human parent antibody while introducing human content to reduce the risk of immunogenicity. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences; optionally, additional framework region modifications may be made within the human framework sequences. The term "antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences and optimized (e.g., by affinity maturation) by modifying or otherwise altering amino acid residues in one or more of the CDRs and / or in one or more framework sequences to, for example, adjust or improve the antibody's biological properties, e.g., to improve affinity or adjust the on-rate and / or off-rate for binding of the antibody to its target epitope. The term "humanized antibody" includes antibodies that have been optimized (e.g., by affinity maturation); thus, an antibody of the present invention may be humanized or humanized and optimized, e.g., humanized and affinity matured.

[0079] Because antibodies can be modified in many ways, the term "antigen-binding protein" or "antibody" should be interpreted to encompass antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide containing an immunoglobulin-binding domain, aptamer, affimer, or bicyclic peptide, whether natural or wholly or partially synthetic. Thus, chimeric molecules comprising an immunoglobulin-binding domain fused to another polypeptide or equivalent are included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

[0080] An example of an antibody fragment containing both CDR sequences and a CH3 domain is a minibody, which comprises an scFv linked to a CH3 domain (Hu et al. (1996) Cancer Res 56(13):3055-61).

[0081] Domain (single-domain) antibodies are peptides, usually about 110 amino acids long, containing one variable domain (VH) of a heavy-chain antibody or of an IgG. Single-domain antibodies (sdAbs), such as nanobodies, are antibody fragments consisting of one monomeric variable antibody domain. Like whole antibodies (containing two heavy chains and two light chains), they are antigen-binding proteins capable of selectively binding to a specific antigen. Domain antibodies have a molecular weight of only 12-15 kDa and are therefore significantly smaller than antibodies composed of two heavy protein chains and two light chains (150-160 kDa). Domain antibodies are also smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable domains, one from a light chain and one from a heavy chain). Single-domain antibodies are engineered from heavy-chain antibodies found in camelids; these are called VHH fragments. Cartilaginous fish also possess heavy chain antibodies (IgNAR, "immunoglobulin new antigen receptor") from which single domain antibodies, called VNAR fragments, can be derived. Domain (single domain) antibodies can be VH or VL. Domain antibodies can be VH or VL of human or murine origin. While most single domain antibodies are heavy chain variable domains, light chain single domain antibodies (VL) have also been shown to specifically bind target epitopes. Protein scaffolds have a relatively defined three-dimensional structure and generally contain one or more regions amenable to specific or random amino acid sequence variation to generate antigen-binding regions within the scaffold that can bind antigen.

[0082] Binding in this context may refer to specific binding. The term "specific" may refer to a situation in which an antibody molecule does not exhibit any significant binding to molecules other than its specific binding partner. The term "specific" may also be applicable when an antibody molecule is specific for a particular epitope as described herein that is carried by many antigens, in which case the antibody molecule will be able to bind to a variety of antigens that carry the epitope.

[0083] The antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, of the present invention bind to epitopes present in human LILRB1, human LILRB2, and human LILRA3. The antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, bind to epitopes present in human LILRB1, human LILRB2, and human LILRA3, but do not bind to LILRB1 ectodomain-truncated protein fragments that contain only the D1 and D2 domains but not the D3 and D4 domains. The antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, bind to an epitope common to human LILRB1, human LILRB2, and human LILRA3. In some embodiments, the antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, bind to an epitope common to human LILRB1, human LILRB2, human LILRA3, and human LILRA1. In some embodiments, the antibodies of the present invention bind to an epitope common to LILRB1, LILRB2, LILRB3, LILRA3, LILRA4, and LILRA6. In some embodiments, antibodies of the present invention bind to an epitope common to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6. In some embodiments, antibodies of the present invention bind to an epitope common to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6. Antibodies 1 to 5 of the present invention selectively bind to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6, but do not bind to the highly homologous LILRB4, LILRB5, LILRA2, and LILRA5. Antibodies of the present invention are not antibodies listed in any one of Tables 1 to 4 or otherwise described in the prior art in the background to the present invention.

[0084] PEPperPRINT CRO used their proprietary PEPperCHIP® linear and "conformational" peptide microarrays to map putative epitopes on the D3-D4 region of the human LILRB1, LILRB2, and LILRA3 molecules for antibodies 1-5 (Figure 24). Strong binding to the peptide microarray was observed only for antibody 2, suggesting that it binds to a relatively unstructured epitope. The two putative epitopes present in all three target proteins corresponded to the peptides: epitope 1 (sequence FVLYKDGERDF (SEQ ID NO: 80) in LILRB1, sequence GYDRFVLYKEGERD (SEQ ID NO: 81) in LILRB2, and sequence YDRFVLYKEWGRD (SEQ ID NO: 82) in LILRA3) and epitope 2 (sequence SSEWSAPSDPLD (SEQ ID NO: 83) in LILRB1, sequence ECSAPSDPLDI (SEQ ID NO: 84) in LILRB2, and sequence SEWSAPSDPLD (SEQ ID NO: 85) in LILRA3). Binding was also observed to additional putative epitopes: epitope 3 (sequence LQCVSDVGYD (SEQ ID NO: 86) in LILRB2 and sequence FQCGSDAGYDRF (SEQ ID NO: 87) in LILRA3) and epitope 4 (sequence FLLTKEGAADDPW (SEQ ID NO: 88) in LILRB1 and sequence AADAPLRLRSIHEY (SEQ ID NO: 89) in LILRB2). Binding to similar peptides was observed for Antibody 1, although the signal was weaker. Antibody 4 was found to bind to two putative epitopes; epitope 5 (sequence RSYGGQYR (SEQ ID NO: 90) in LILRB1 and sequence PVSRSYGGQYRC (SEQ ID NO: 91) in LILRB2). Weak binding to the peptide array was observed for antibody 5, suggesting one putative epitope; epitope 6 (sequence LDILIAGQFYD (SEQ ID NO: 92) in LILRB1, sequence APSDPLDILI (SEQ ID NO: 93) in LILRB2, and sequence PSDPLDILI (SEQ ID NO: 94) in LILRA3). No significant binding to the peptide array was observed for antibody 3.

[0085] Amino acids may be referred to by their one-letter or three-letter codes, or by their full names. The one-letter and three-letter codes and full names for each of the 20 common amino acids are shown below.

[0086] [Table 7]

[0087] In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention may comprise a set of six CDRs of an antibody: (a) Clone 1 (HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3), LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5), and LCDR3 (SEQ ID NO: 6)); (b) Clone 2 (HCDR1 (SEQ ID NO: 9), HCDR2 (SEQ ID NO: 10), HCDR3 (SEQ ID NO: 11), LCDR1 (SEQ ID NO: 12), LCDR2 (SEQ ID NO: 13) and LCDR3 (SEQ ID NO: 14)), (c) Clone 3 (HCDR1 (SEQ ID NO: 17), HCDR2 (SEQ ID NO: 18), HCDR3 (SEQ ID NO: 19), LCDR1 (SEQ ID NO: 20), LCDR2 (SEQ ID NO: 21) and LCDR3 (SEQ ID NO: 22)), (d) Clone 4 (HCDR1 (SEQ ID NO: 25), HCDR2 (SEQ ID NO: 26), HCDR3 (SEQ ID NO: 27), LCDR1 (SEQ ID NO: 28), LCDR2 (SEQ ID NO: 29) and LCDR3 (SEQ ID NO: 30)) or (e) Clone 5 (HCDR1 (SEQ ID NO: 33), HCDR2 (SEQ ID NO: 34), HCDR3 (SEQ ID NO: 35), LCDR1 (SEQ ID NO: 36), LCDR2 (SEQ ID NO: 37) and LCDR3 (SEQ ID NO: 38)) (as defined by Kabat nomenclature).

[0088] The antibody or antigen-binding fragment thereof of the invention may comprise the VH and VL sequences of the antibody: (a) Clone 1 (SEQ ID NO: 7 and SEQ ID NO: 8), (b) Clone 2 (SEQ ID NO: 15 and SEQ ID NO: 16), (c) Clone 3 (SEQ ID NO: 23 and SEQ ID NO: 24), (d) Clone 4 (SEQ ID NO: 31 and SEQ ID NO: 32) or (e) Clone 5 (SEQ ID NO: 39 and SEQ ID NO: 40).

[0089] The antibodies or antigen-binding fragments thereof of the invention may include one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, additional amino acid modifications in the VH and / or VL sequences, provided that the functional properties of the antibody are retained.

[0090] The modification may be an amino acid substitution, deletion or insertion. Preferably, the modification is a substitution.

[0091] In preferred embodiments where one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example, according to the following table: In some embodiments, amino acids in the same category in the middle column are substituted for each other, i.e., a nonpolar amino acid is substituted with another nonpolar amino acid, In some embodiments, amino acids in the same row in the right-hand column are substituted for each other.

[0092] [Table 8]

[0093] In some embodiments, substitutions may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., binding affinity) of an antibody molecule comprising the substitution, when compared to a comparable unsubstituted antibody molecule.

[0094] In a preferred embodiment, an antibody or antigen-binding fragment thereof of the present invention may comprise a VH and / or VL domain sequence that has one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues) compared to the VH and / or VL sequences of the present invention described herein, preferably no more than 20 alterations, no more than 15 alterations, no more than 10 alterations, no more than 5 alterations, no more than 4 alterations, no more than 3 alterations, no more than 2 alterations or no more than 1 alteration.

[0095] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a VH domain amino acid sequence comprising the set of three HCDRs of the antibody: (a) Clone 1 HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), and HCDR3 (SEQ ID NO: 3); and the VH domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of Clone 1 set forth in SEQ ID NO: 7; (b) Clone 2 HCDR1 (SEQ ID NO: 9), HCDR2 (SEQ ID NO: 10), and HCDR3 (SEQ ID NO: 11); and the VH domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of Clone 2 set forth in SEQ ID NO: 15; (c) Clone 3 HCDR1 (SEQ ID NO: 17), HCDR2 (SEQ ID NO: 18), and HCDR3 (SEQ ID NO: 19); and the VH domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of Clone 3 set forth in SEQ ID NO: 23; (d) clone 4 HCDR1 (SEQ ID NO:25), HCDR2 (SEQ ID NO:26), and HCDR3 (SEQ ID NO:27); and the VH domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of clone 4 set forth in SEQ ID NO:31; or (e) Clone 5 HCDR1 (SEQ ID NO:33), HCDR2 (SEQ ID NO:34), and HCDR3 (SEQ ID NO:35) and the VH domain have an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of Clone 5 set forth in SEQ ID NO:39; The sequences are defined by the Kabat nomenclature.

[0096] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises the antibody VH domain sequence: (a) a VH domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (b) a VH domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (c) a VH domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (d) a VH domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; or (e) a VH domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and the sequences are defined by the Kabat nomenclature.

[0097] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a VL domain amino acid sequence comprising the set of three LCDRs of the antibody: (a) clone 1 LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5), and LCDR3 (SEQ ID NO: 6); and the VL domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of clone 1 set forth in SEQ ID NO: 8; (b) clone 2 LCDR1 (SEQ ID NO:12), LCDR2 (SEQ ID NO:13), and LCDR3 (SEQ ID NO:14); and the VL domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of clone 2 set forth in SEQ ID NO:16; (c) Clone 3 LCDR1 (SEQ ID NO:20), LCDR2 (SEQ ID NO:21), and LCDR3 (SEQ ID NO:22); and the VL domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of Clone 3 set forth in SEQ ID NO:24; (d) clone 4 LCDR1 (SEQ ID NO:28), LCDR2 (SEQ ID NO:29), and LCDR3 (SEQ ID NO:30); and the VL domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of clone 4 set forth in SEQ ID NO:32; or (e) clone 5 LCDR1 (SEQ ID NO:36), LCDR2 (SEQ ID NO:37), and LCDR3 (SEQ ID NO:38); and the VL domain has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of clone 5 set forth in SEQ ID NO:40; The sequences are defined by the Kabat nomenclature.

[0098] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises the antibody VL domain sequence: (a) a VL domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (b) a VL domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (c) a VL domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (d) a VL domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; or (e) a VL domain having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; These sequences are defined by the Kabat nomenclature.

[0099] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention is an antibody (a) Clone 1 HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3), LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5), and LCDR3 (SEQ ID NO: 6); (b) clone 2 HCDR1 (SEQ ID NO: 9), HCDR2 (SEQ ID NO: 10), HCDR3 (SEQ ID NO: 11), LCDR1 (SEQ ID NO: 12), LCDR2 (SEQ ID NO: 13) and LCDR3 (SEQ ID NO: 14); (c) Clone 3 HCDR1 (SEQ ID NO: 17), HCDR2 (SEQ ID NO: 18), HCDR3 (SEQ ID NO: 19), LCDR1 (SEQ ID NO: 20), LCDR2 (SEQ ID NO: 21) and LCDR3 (SEQ ID NO: 22); (d) clone 4 HCDR1 (SEQ ID NO: 25), HCDR2 (SEQ ID NO: 26), HCDR3 (SEQ ID NO: 27), LCDR1 (SEQ ID NO: 28), LCDR2 (SEQ ID NO: 29) and LCDR3 (SEQ ID NO: 30); or (e) Clone 5 HCDR1 (SEQ ID NO: 33), HCDR2 (SEQ ID NO: 34), HCDR3 (SEQ ID NO: 35), LCDR1 (SEQ ID NO: 36), LCDR2 (SEQ ID NO: 37) and LCDR3 (SEQ ID NO: 38); The amino acid sequences of the VH and VL domains contain a set of six HCDR LCDRs, and the sequences are defined by the Kabat nomenclature.

[0100] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is an antibody: (a) Clone 1 set forth in SEQ ID NOs: 7 and 8; (b) Clone 2 set forth in SEQ ID NOs: 15 and 16; (c) Clone 3 set forth in SEQ ID NOs: 23 and 24; (d) Clone 4 set forth in SEQ ID NOs: 31 and 30; or (e) Clone 5 set forth in SEQ ID NOs: 39 and 40; and VH and VL domain sequences, which sequences are defined by the Kabat nomenclature.

[0101] The terms "antibody clone," "clone," and "antibody" (eg, clone 1 or antibody 1) are used interchangeably herein to refer to antibodies 1-5 of the present invention.

[0102] Sequence identity is generally determined with reference to the algorithm GAP (Wisconsin GCG Package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used, with a gap creation penalty of 12 and a gap extension penalty of 4. Although the use of GAP may be preferred, other algorithms may be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147:195-197), or the TBLASTN program of Altschul et al. (1990), supra, typically using default parameters. In particular, the psi-Blast algorithm may be used (Nucl. Acids Res. (1997) 25 3389-3402). Sequence identity may be determined using the Bioedit, ClustalW algorithm.

[0103] The antibody may comprise a CH2 domain. The CH2 domain is preferably located N-terminal to the CH3 domain, as in human IgG molecules. The antibody CH2 domain is preferably a human IgG1, IgG2, IgG3, or IgG4 CH2 domain, more preferably a human IgG1 CH2 domain. Sequences of human IgG domains are known in the art.

[0104] The antibody may comprise an immunoglobulin hinge region or portion thereof at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate to form a dimer. Preferably, the hinge region or portion thereof is a human IgG1, IgG2, IgG3, or IgG4 hinge region or portion thereof. More preferably, the hinge region or portion thereof is an IgG1 hinge region or portion thereof.

[0105] The sequence of the CH3 domain is not particularly limited. Preferably, the CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3, or IgG4 CH3 domain, most preferably a human IgG1 CH3 domain.

[0106] The antibodies of the present invention may comprise a human IgG1, IgG2, IgG3, or IgG4 constant region or a modified version thereof. The sequences of human IgG1, IgG2, IgG3, or IgG4 CH3 domains are known in the art. The antibodies of the present invention may comprise a human IgG constant region, for example, a human IgG1 constant region.

[0107] The antibodies of the present invention may comprise a human IgG Fc with effector function. The antibodies of the present invention may comprise an Fc with effector function, an Fc with enhanced effector function, an Fc with reduced effector function, or an Fc without effector function.

[0108] Fc receptors (FcRs) are key immunoregulatory receptors that link antibody-mediated (humoral) immune responses with cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM), and FcδR (IgD). Three classes of receptors for human IgG found on leukocytes are CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRIs are classified as high-affinity receptors (KD in the nanomolar range), whereas FcγRII and FcγRIII have low- to intermediate-affinity receptors (KD in the micromolar range).

[0109] In antibody-dependent cellular cytotoxicity (ADCC), FcγRs on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) bind the Fc region of IgG, which itself binds to target cells. Upon binding, signaling pathways are initiated, resulting in the secretion of various substances, such as lytic enzymes, perforin, granzymes, and tumor necrosis factor, that mediate target cell destruction. The level of ADCC effector function varies for IgG subtypes. While this depends on the allotype and the specific FcγR, briefly, ADCC effector function is high for human IgG1 and IgG3 and low for IgG2 and IgG4. See below for IgG subtype variations in effector function, listed in descending order of potency.

[0110] [Table 9]

[0111] FcγRs bind IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has led to attempts to modify it to tailor IgG effector function.

[0112] Antibody potency can be improved by enhancing their ability to mediate cellular cytotoxic functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). Numerous mutations within the Fc domain have been identified that enhance Fc receptor binding, either directly or indirectly, and significantly enhance cytotoxicity: mutations S239D / A330L / I332E ("3M"), F243L, or G236A. Alternatively, enhanced effector function can be achieved by modifying the glycosylation of the Fc domain; FcγR interacts with carbohydrates on the CH2 domain, and the glycan composition has a substantial effect on effector function activity. Defucosylated (non-fucosylated) antibodies exhibit significantly enhanced ADCC activity through increased binding to FcγRIIIa.

[0113] Although activation of ADCC and CDC may be desirable for some therapeutic antibodies, in some embodiments, antibodies that do not activate effector function are preferred.

[0114] Due to their lack of effector functions, IgG4 antibodies are the preferred IgG subclass for receptor blockade without cell depletion. However, IgG4 molecules can exchange half molecules in a dynamic process called Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. The S228P mutation has been shown to prevent this recombination process, allowing the design of IgG4 antibodies with a reduced propensity for Fab-arm exchange.

[0115] Fc engineering approaches have been used to determine the key interaction sites for the IgG1 Fc domain with Fcγ receptors and C1q, and then mutate these positions to reduce or eliminate binding. Through alanine scanning, the binding site of C1q to the region covering the hinge and upper CH2 of the Fc domain was identified. The CH2 domain of the antibody or fragment of the present invention may contain one or more mutations to reduce or eliminate binding of the CH2 domain to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII, etc., and / or complement. The CH2 domain of a human IgG domain typically binds to Fcγ receptors and complement; reduced binding to Fcγ receptors is expected to reduce antibody-dependent cell-mediated cytotoxicity (ADCC), and reduced binding to complement is expected to reduce the complement-dependent cytotoxicity (CDC) activity of the antibody molecule. Mutations to reduce or eliminate binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art. Antibody molecules of the present invention may comprise an Fc with the modifications K322A / L234A / L235A or L234F / L235E / P331S ("TM"), which almost completely eliminate FcγR and C1q binding. Antibody molecules of the present invention may comprise a CH2 domain, wherein the CH2 domain contains alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 according to the IMGT numbering) ("LALA mutation"). Furthermore, complement activation and ADCC can be reduced by, for example, mutating Pro329 (positions according to the EU numbering) to either P329A or P329G. Antibody molecules of the invention may comprise a CH2 domain comprising alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 according to the IMGT numbering) and an alanine (LALA-PA) or glycine (LALA-PG) at EU position 329 (position 114 according to the IMGT numbering). Additionally or alternatively, antibody molecules of the invention may comprise an alanine, glutamine, or glycine at EU position 297 (position 84.4 according to the IMGT numbering).

[0116] Modification of glycosylation at asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction, can result in loss of FcR binding; loss of FcR binding has been observed with N297 point mutation. The antibody molecules of the present invention may comprise an Fc with an N297A, N297G, or N297Q mutation. Antibody molecules of the present invention with a deglycosylated Fc domain can be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor, or after expression of the Fc domain in bacteria.

[0117] IgG naturally persists in serum for extended periods due to FcRn-mediated recycling, with a typical half-life of approximately 21 days. Altering the pH-dependent interaction of the Fc domain with FcRn to improve affinity at pH 6.0 while maintaining minimal binding at pH 7.4 can extend half-life. The T250Q / M428L variant conferred an approximately two-fold extension of IgG half-life (assessed in rhesus monkeys), while the M252Y / S254T / T256E variant ("YTE") provided an approximately four-fold increase in IgG half-life (assessed in cynomolgus monkeys). Prolonging half-life may offer the potential for reduced dosing frequency while maintaining or improving efficacy.

[0118] Immunoglobulins are known to have modular structures comprising separate domains that can be combined in many different ways to generate multispecific, e.g., bispecific, trispecific, or tetraspecific, antibody formats. Exemplary multispecific antibody formats are described, for example, in Spiess et al. (2015) Mol Immunol 67:95-106 and Kontermann (2012) Mabs 4(2):182-97. The antibodies of the present invention can be used in such multispecific formats.

[0119] The present invention provides antibodies or antigen-binding fragments thereof, such as human antibodies or antigen-binding fragments thereof, that can compete with the antibodies of the present invention described herein (e.g., comprising the HCDR and LCDR of clone 1, 2, 3, 4 or 5 and / or the set of VH and VL amino acid sequences of clone 1, 2, 3, 4 or 5 as defined by the Kabat nomenclature) for binding to an epitope of human LILRB1, human LILRB2 and / or human LILRA3.

[0120] Competitive assays include cell-based and cell-free binding assays, including immunoassays such as ELISA, HTRF, flow cytometry, fluorescence microvolume assay technology (FMAT) assays, Mirrorball, high-content imaging-based fluorescent immunoassays, radioligand binding assays, biolayer interferometry (BLI), surface plasmon resonance (SPR), and thermal shift assays.

[0121] An antibody that binds to the same epitope as a reference antibody or an epitope that overlaps with a reference antibody refers to an antibody that blocks binding of the reference antibody to its binding partner (e.g., antigen or "target") by 50% or more in a competition assay, and / or conversely, the reference antibody blocks binding of the antibody to its binding partner by 50% or more in a competition assay. Such antibodies are said to compete for binding to the epitope of interest.

[0122] The antigen-binding protein, such as an antibody or antigen-binding fragment thereof, of the present invention can be conjugated to a detectable label (e.g., a radioisotope) or to a biologically active molecule. In this case, the antigen-binding protein, such as an antibody or antigen-binding fragment thereof, can be referred to as a conjugate. Such conjugates can be applied in the treatment and / or diagnosis of diseases as described herein.

[0123] The antigen-binding proteins (including conjugates) of the present invention may be useful for detecting (e.g., in vitro detection of) epitopes bound by antibodies of the present invention (epitopes present on human LILRB1, LILRB2 and LILRA3, preferably epitopes present on human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4 and LILRA6). Accordingly, the present invention relates to the use of the antigen-binding proteins of the present invention for detecting the presence of epitopes bound by antibodies of the present invention in a sample. The antigen-binding proteins may be conjugated to a detectable label, as described elsewhere herein.

[0124] In a preferred embodiment, the present invention relates to an in vitro method for detecting an epitope of the invention in a sample, the method comprising incubating an antigen binding protein of the invention with a sample of interest and determining binding of the antigen binding protein to an epitope of the invention present in the sample, wherein binding of the antigen binding protein indicates the presence of the epitope of the invention in the sample. Methods for detecting binding of an antigen binding protein to its target antigen are known in the art and include ELISA, ICC, IHC, immunofluorescence, Western blot, IP, SPR and flow cytometry.

[0125] The sample of interest may be a sample obtained from an individual. The individual may be a human. Samples include, but are not limited to, tissues, such as tumor tissue, tumor lysate, primary or cultured cells or cell lines, cell supernatants, cell lysates, cerebrospinal fluid (CSF), platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, saliva, sputum, tears, sweat, mucus, and tissue culture fluid, tissue extracts, such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0126] After incubation, antigen-binding proteins, such as antibodies to antigens, are detected using an appropriate detection system. Detection methods can be direct or indirect and can produce fluorescent or chromogenic signals. Direct detection involves the use of a primary antibody directly conjugated to a label. Indirect detection methods use a labeled secondary antibody directed against the primary antigen-binding protein, e.g., antibody, host species. Indirect methods can include an amplification step to enhance signal intensity. Labels commonly used for visualization (i.e., detection) of antigen-binding protein-antigen (e.g., antibody-epitope) interactions include fluorophores and enzymes that convert a soluble substrate into an insoluble chromogenic end product.

[0127] The term "detecting" is used broadly herein to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of a target molecule in a sample as well as determining whether the target molecule is present in a sample at detectable levels. Detection can be direct or indirect.

[0128] Suitable detectable labels that can be conjugated to antigen-binding proteins such as antibodies are known in the art and include radioisotopes such as iodine-125, iodine-131, yttrium-90, indium-111, and technetium-99; fluorescent dyes such as fluorescein, rhodamine, phycoerythrin, Texas Red, and cyanine dye derivatives such as Cy7, Alexa 750, and Alexa Fluor 647; chromogenic dyes such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; iodine or laser dyes with spectroscopically isolated absorption or emission characteristics; electro-chemiluminescent labels that can be detected via electrical stimulation in an appropriate chemical environment, such as sulfo-TAG; and chemical moieties that can be detected via binding to a specific cognate detectable moiety, such as labeled avidin or streptavidin, such as biotin.

[0129] The antigen-binding proteins, such as antibodies or fragments thereof, of the present invention can be conjugated to the detectable label by any suitable covalent or non-covalent bond, such as a disulfide or peptide bond. Suitable peptide linkers are known in the art and can be 5-25, 5-20, 5-15, 10-25, 10-20, or 10-15 amino acids in length.

[0130] The present invention also provides a nucleic acid or set of nucleic acids encoding an antibody or antigen-binding fragment of the present invention, as well as a vector comprising such a nucleic acid or set of nucleic acids. When a nucleic acid encodes the VH and VL domains or the heavy and light chains of an antibody molecule of the present invention, the two domains or chains can be encoded in the same or separate nucleic acid molecules.

[0131] Isolated nucleic acid molecules can be used to express antibody molecules of the present invention. The nucleic acid is generally provided in the form of a recombinant vector for expression. Accordingly, another aspect of the present invention provides a vector comprising such a nucleic acid. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive expression of the nucleic acid in a host cell. The vector can be a plasmid, virus, such as a phage or phagemid, as appropriate.

[0132] A nucleic acid molecule or vector as described herein can be introduced into a host cell. Techniques for introducing a nucleic acid or vector into a host cell are well established in the art, and any suitable technique can be used. A range of host cells suitable for the production of recombinant antibody molecules are known in the art and include bacterial, yeast, insect, or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NS0, or HEK cells, e.g., HEK293 cells.

[0133] Recombinant host cells comprising the nucleic acid or vector of the invention are also provided. Such recombinant host cells can be used to produce the antigen binding proteins (e.g., antibodies) of the invention. Accordingly, there is also provided a method of producing the antigen binding proteins, e.g., antibodies, of the invention, the method comprising culturing recombinant host cells under conditions suitable for the production of the antigen binding protein, e.g., antibody. The method may further comprise the step of isolating and / or purifying the antigen binding protein, e.g., antibody.

[0134] Thus, the present invention provides methods for producing an antigen binding protein, e.g., an antibody, of the invention, comprising expressing a nucleic acid encoding the antigen binding protein, e.g., an antibody, in a host cell and, optionally, isolating and / or purifying the antigen binding protein so produced. Methods for culturing host cells are well known in the art, and techniques for purification of recombinant antigen binding proteins, e.g., antibodies, are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography, e.g., using Protein A or Protein L. In some embodiments, purification may be carried out using an affinity tag on the antigen binding protein, e.g., the antibody. The method may also include formulating the antigen binding protein, e.g., the antibody, into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described below.

[0135] The antigen binding proteins, e.g., antibodies, of the present invention are expected to find application in therapeutic applications, particularly in humans, for example in the treatment of cancers including, but not limited to: (i) Acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), brain low-grade glioma (LGG), invasive breast cancer (BRCA), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney clear cell carcinoma (KIRC), kidney papillary renal cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) , pancreatic adenocarcinoma (PAAD), sarcoma (SARC), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), endometrial carcinoma (UCEC), uveal melanoma (UVM), colorectal cancer, prostate cancer, childhood cancer, lymphoma and leukemia, such as DLBCL, NHL, multiple myeloma, Hodgkin's lymphoma, etc.; (ii) cancer that is positive for LILRB1 or LILRB2 or both LILRB1 and LILRB2; (iii) cancers positive for immunosuppressive macrophages (as measured by CD163 or CD68 positivity) and / or tumor-infiltrating T cells; (iv) Cancers with elevated or decreased expression of classical or non-classical MHC class I; (vi) Cancer that is positive for one or more of LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6.

[0136] Compositions, such as pharmaceutical compositions, comprising an antigen binding protein, such as an antibody, according to the invention, and an excipient, such as a pharmaceutically acceptable excipient, are also provided.

[0137] The present invention further provides an antigen-binding protein, e.g., an antibody, of the present invention for use in a method of treatment. A method of treating a patient is also provided, which method comprises administering to the patient a therapeutically effective amount of an antigen-binding protein, e.g., an antibody, according to the present invention. Further provided is the use of an antigen-binding protein, e.g., an antibody, according to the present invention for use in the manufacture of a medicament. The patient, as referred to herein, is preferably a human patient.

[0138] The present invention also provides an antigen binding protein, eg, an antibody, of the present invention for use in a method of treating cancer in a patient.

[0139] There is also provided a method of treating cancer, such as breast cancer, in a patient, comprising administering to the patient a therapeutically effective amount of an antigen binding protein, e.g., an antibody, according to the invention.

[0140] There is further provided the use of an antigen binding protein, such as an antibody, according to the present invention for use in the manufacture of a medicament for the treatment of cancer, including but not limited to: (i) acute myeloid leukemia (LAML), bladder urothelial carcinoma (BLCA), brain low-grade glioma (LGG), invasive breast cancer (BRCA), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney clear cell carcinoma (KIRC), kidney papillary renal cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) , pancreatic adenocarcinoma (PAAD), sarcoma (SARC), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), endometrial carcinoma (UCEC), uveal melanoma (UVM), colorectal cancer, prostate cancer, childhood cancer, lymphoma and leukemia, such as DLBCL, NHL, multiple myeloma, Hodgkin's lymphoma, etc.; (ii) cancer that is positive for LILRB1 or LILRB2 or both LILRB1 and LILRB2; (iii) cancers positive for immunosuppressive macrophages (as measured by CD163 or CD68 positivity) and / or tumor-infiltrating T cells; (iv) cancers with elevated or decreased classical or non-classical MHC class I expression; (v) Cancer that is positive for one or more of LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6.

[0141] Treatment may further comprise administering to the patient a second therapy, which may be administered to the patient simultaneously, separately or sequentially with the antigen binding protein, e.g., antibody, of the invention.

[0142] In another aspect, the present invention relates to an antigen binding protein, e.g. an antibody, of the invention for use in: a) treating cancer; b) slowing the progression of cancer; c) prolonging the survival of patients suffering from cancer; d) reducing tumor immune evasion; e. Reducing cancer metastasis; f) reducing resistance to second treatments; g) improving response rates or overall survival after standard care treatment; h) reducing tumor volume prior to surgical resection; and / or i) Reducing tumor recurrence after surgery or neoadjuvant therapy.

[0143] Thus, antigen binding proteins, e.g., antibodies, as described herein may be for use in therapeutic applications, particularly for the treatment of cancers, including but not limited to: (i) Acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), brain low-grade glioma (LGG), invasive breast cancer (BRCA), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney clear cell carcinoma (KIRC), kidney papillary renal cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) , pancreatic adenocarcinoma (PAAD), sarcoma (SARC), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), endometrial carcinoma (UCEC), uveal melanoma (UVM), colorectal cancer, prostate cancer, childhood cancer, lymphoma and leukemia, such as DLBCL, NHL, multiple myeloma, Hodgkin's lymphoma, etc.; (ii) cancer that is positive for LILRB1 or LILRB2 or both LILRB1 and LILRB2; (iii) cancers positive for immunosuppressive macrophages (as measured by CD163 or CD68 positivity) and / or tumor-infiltrating T cells; (iv) cancers with elevated or decreased classical or non-classical MHC class I expression; (v) Cancer that is positive for one or more of LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6.

[0144] Antigen binding proteins, such as antibodies, as described herein may be used in methods of treatment of the human or animal body.

[0145] Related aspects of the present invention provide: (i) an antigen-binding protein, e.g., an antibody, as described herein for use as a medicament; (ii) an antigen binding protein, e.g., an antibody, as described herein, for use in a method of treating a disease or disorder; (iii) use of an antigen binding protein, e.g., an antibody, described herein in the manufacture of a medicament for use in the treatment of a disease or disorder; and (iv) A method of treating a disease or disorder in an individual comprising administering to the individual a therapeutically effective amount of an antigen binding protein, e.g., an antibody, as described herein.

[0146] The individual may be a patient, preferably a human patient.

[0147] Treatment can be any treatment or therapy that achieves some desired therapeutic effect, such as inhibiting or slowing the progression of the condition, including slowing the rate of progression, halting the rate of progression, alleviating the condition, curing or relieving (whether partial or complete) the condition, preventing, delaying, ameliorating or arresting one or more symptoms and / or signs of the condition, or prolonging the survival of an individual or patient beyond that expected without treatment.

[0148] Treatment as a preventative measure (i.e., prevention) is also included. For example, an individual who is susceptible to or at risk of developing cancer, e.g., breast cancer, may be treated as described herein. Such treatment may prevent or delay the onset or recurrence of disease in the individual. Methods of treatment as described may include administering at least one additional treatment to the individual in addition to the antigen binding protein, e.g., antibody. Thus, the antigen binding proteins, e.g., antibodies, described herein may be administered to the individual alone or in combination with one or more other treatments. When an antigen binding protein, e.g., antibody, is administered to an individual in combination with another treatment, the additional treatment may be administered to the individual simultaneously with, sequentially with, or separately from the administration of the antigen binding protein, e.g., antibody. When the additional treatment is administered simultaneously with the antigen binding protein, e.g., antibody, the antigen binding protein, e.g., antibody, and the additional treatment may be administered to the individual as a combined formulation. For example, the additional treatment may be a known treatment or therapeutic agent for the disease to be treated.

[0149] While an antigen-binding protein, e.g., an antibody, can be administered alone, an antigen-binding protein, e.g., an antibody, is usually administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antigen-binding protein, e.g., an antibody. Accordingly, another aspect of the present invention provides a pharmaceutical composition comprising an antigen-binding protein, e.g., an antibody, as described herein. Methods are also provided that comprise formulating an antigen-binding protein, e.g., an antibody, into a pharmaceutical composition.

[0150] Pharmaceutical compositions may include, in addition to an antigen-binding protein, e.g., an antibody, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) within the scope of sound medical judgment, and are free of excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection, or any other suitable route, as discussed below.

[0151] For parenteral administration, e.g., by injection, e.g., subcutaneous or intravenous administration, of a pharmaceutical composition comprising the antigen-binding protein, e.g., an antibody, can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. One skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Buffers such as phosphate, citric acid, and other organic acid buffers; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; and the like. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be used as needed, including amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0152] In some embodiments, the antigen binding protein, e.g., antibody, may be provided in lyophilized form for reconstitution prior to administration, e.g., a lyophilized antigen binding protein, e.g., antibody, may be reconstituted in sterile water or saline prior to administration to an individual.

[0153] The amount administered may be a therapeutically effective amount, which is sufficient to show benefit to the individual. The actual amount administered and the rate and time course of administration will depend on the nature and severity of what is being treated, the particular individual being treated, the individual's clinical condition, the cause of the disorder, the site of delivery of the composition, the type of antigen-binding protein, e.g., an antibody, the method of administration, the administration schedule, and other factors known to medical professionals. Determination of treatment prescriptions, e.g., dosage, etc., is within the responsibility of general practitioners and other medical professionals and may depend on the severity of the symptoms and / or the progression of the disease being treated. Appropriate dosages of antigen-binding proteins, e.g., antibodies, are well known in the art. A therapeutically effective amount or appropriate dosage of an antigen-binding protein, e.g., an antibody, can be determined by comparing in vitro activity and in vivo activity in animal models. Methods for the estimation of effective dosages in mice and other test animals relative to humans are known. The exact dosage will depend on many factors, including the size and location of the area to be treated and the appropriate properties of the antigen-binding protein, e.g., an antibody.

[0154] Typical antibody doses range from 100 μg to 1 g for systemic administration and 1 μg to 1 mg for local administration. An initial higher loading dose may be administered, followed by one or more lower doses. This is the dose for a single treatment of an adult individual; it may be adjusted proportionally for children and infants, and for other antibody formulations depending on molecular weight.

[0155] Treatments may be repeated at daily, twice-weekly, weekly, or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may depend on the pharmacological and bioinformatic properties of the antibody composition, the route of administration, and the nature of the condition being treated.

[0156] Treatment can be cyclic, with the period between administrations being about 2 weeks or more, e.g., about 3 weeks or more, about 4 weeks or more, about monthly or more, about 5 weeks or more, or about 6 weeks or more. For example, treatment can be every 2-4 weeks or every 4-8 weeks. Suitable formulations and routes of administration are described above.

[0157] In a preferred embodiment, the antibodies as described herein may be for use in methods of treating cancer. [Brief explanation of the drawings]

[0158] Drawing List [Figure 1] Elevated LILRA3 mRNA expression in cancer tissues compared to normal tissues. Analysis was performed using The Cancer Genome Atlas (TCGA). Cancer acronyms as used by the TCGA database and are publicly available. [Figure 2] LILRB1 and LILRB2 expression in immune cells across cancer types compared to normal tissue-resident macrophages. UMAP analysis of the Smart-seq2 dataset from Mulder et al. 2021 https: / / pubmed.ncbi.nlm.nih.gov / 34331874 / shows immune cell type classification and LILRB1 and LILRB2 status. Top panel: normal healthy subjects; bottom panel: cancer; A: LILRB1 expression; B: LILRB2 expression; C: LILRB1 and LILRB2 expression. [Figure 3] Mixed LILRB1 and LILRB2 expression in TAMs from melanoma cancer patients. Analysis of LILRB1 and LILRB2 detection in macrophages in melanoma patients (n=33) treated with pre- and post-anti-PD-1 and / or anti-CTLA-4 therapy is shown using Smart-seq2 data from Sade-Feldman et al. https: / / pubmed.ncbi.nlm.nih.gov / 30633907 / . [Figure 4]Binding to domains of human LILRB1. Plates were coated with recombinant full-length LILRB1 ectodomain (SEQ ID NO: 41) or a protein containing domains 1 and 2 of human LILRB1 protein (SEQ ID NO: 42), then incubated with 10 nM clones 1-5, reference Ab1, and an isotype control. Bound antibody was detected using an HRP-labeled secondary antibody, and the results were plotted graphically as absorbance measured at 450 nm. [Figure 5] Binding to LILRB1-overexpressing cell lines. HEK293 cells overexpressing full-length human LILRB1 (SEQ ID NO: 45) were incubated with various concentrations of test antibodies (Antibodies 1-5 and Reference Ab1). Bound antibody was detected using a PE-labeled secondary antibody and analyzed by flow cytometry. A) Plot showing mean fluorescence intensity (MFI), B) EC50 value calculated using the data from panel A. [Figure 6] Blockade of HLA-G tetramer binding to cells overexpressing LILRB1. HEK293 cells overexpressing full-length human LILRB1 (SEQ ID NO: 45) were incubated with PE-labeled HLA-G tetramer and test antibodies (Antibodies 1-5, Reference Antibody 1, and isotype control) at a concentration of 500 nM. Bound HLA-G was analyzed by flow cytometry and plotted as mean fluorescence intensity (MFI). [Figure 7] Binding to cell lines overexpressing LILRB2. HEK293 cells overexpressing full-length human LILRB2 (SEQ ID NO: 46) were incubated with various concentrations of test antibodies (Antibodies 1-5 and Reference Antibody 1). Bound antibody was detected using a PE-labeled secondary antibody and analyzed by flow cytometry; the plot shows the mean fluorescence intensity (MFI). [Figure 8]Binding to LILRA1 (SEQ ID NO: 47), LILRA2 (SEQ ID NO: 48), and LILRA3 (SEQ ID NO: 49). Plates were coated with recombinant LILRB1, LILRA1, LILRA2, LILRA3, and control proteins, and then incubated with 10 nM of each antibody 1-5, reference Ab1, and isotype control. Bound antibody was detected using an HRP-labeled secondary antibody and plotted as absorbance measured at 450 nm. [Figure 9] Binding to wild-type and allelic variants of LILRB1. Plates were coated with recombinant wild-type and allelic variants of LILRB1 (L68P-A93T (SEQ ID NO: 50) and I142T-S155I (SEQ ID NO: 51)) and then incubated with 10 nM of each of antibodies 1-5, reference antibody 1, and an isotype control. Bound antibody was detected using an HRP-labeled secondary antibody and plotted as absorbance measured at 450 nm. [Figure 10] Binding to non-human primate LILRB. Plates were coated with cynomolgus or rhesus LILRB protein and then incubated with 10 nM of each of Antibodies 1-5, Reference Ab1, and an isotype control. Bound antibody was detected using an HRP-labeled secondary antibody and plotted as absorbance measured at 450 nm. [Figure 11-1] Cytokine release assay. Human PBMCs were incubated with 20 μg / ml of one of the test antibodies (antibodies 1-5, reference and isotype), 100 ng / ml LPS, 10 μg / ml PHA, or 15 μg / ml OCT3 (anti-CD3 antibody) for 24 hours. Cytokines were measured in the medium using Luminex technology: A) INF-γ, B) IL-1b, C) IL-2. [Figure 11-2] Cytokine release assay. Human PBMCs were incubated with 20 μg / ml of one of the test antibodies (antibodies 1-5, reference and isotype), 100 ng / ml LPS, 10 μg / ml PHA, or 15 μg / ml OCT3 (anti-CD3 antibody) for 24 hours. Cytokines were measured in the culture medium using Luminex technology: D) IL-6, E) IL-8, F) IL-10. [Figure 11-3] Cytokine release assay. Human PBMCs were incubated with 20 μg / ml of one of the test antibodies (antibodies 1-5, reference and isotype), 100 ng / ml LPS, 10 μg / ml PHA, or 15 μg / ml OCT3 (anti-CD3 antibody) for 24 hours. Cytokines were measured in the culture medium using Luminex technology: G) IL-17A, H) MIP-1α, and I) TNFα. [Figure 12] Antibodies 1-5 binding in iPS-derived macrophages. A) The graph shows the percentage of cells binding to the target compared to the IgG4 isotype control. B) The graph shows the MFI (mean fluorescence intensity) of cells as delta fluorescence intensity (delta FI) subtracted by the IgG4 background binding level. Antibodies were tested at 10 μg / ml. "Binding" indicates that the signal obtained for the LILRB1 antibody in this assay was at least 2-fold higher than that observed for the IgG4 isotype control. [Figure 13] Antibodies 1-5 binding in the NK92 cell line. A) The graph shows the percentage of cells that bind to the target compared to the IgG4 isotype control. B) The graph shows the MFI of the cells, which is the delta fluorescence intensity (delta FI) minus the IgG4 background binding level. Antibodies were tested at 10 μg / ml. "Binding" means that the signal obtained for the LILRB1 antibody in this assay was at least 2-fold higher than that observed with the IgG4 isotype control. [Figure 14]Antibodies 1-5 enhance cancer-induced phagocytosis in iPS-derived macrophages. Induction of macrophage phagocytosis in the presence of A) the MHC class I-negative cell line DLD1, B) the JURKAT HLA-ABC-positive, HLA-G-negative cell line, and C) the JURKAT HLA-ABC-positive, HLA-G-positive cell line. iPSC-DMs were incubated in the presence of DLD1 and JURKAT cells. 10 μg / ml of our anti-LILRB1 / 2 or anti-LILRB1 antibodies (1-5), isotype control (IgG4), or anti-LILRB1 reference antibody 1 ("Reference") was added to the cell culture medium. Phagocytosis was then measured 7 hours after treatment using the Incucyte s3 live cell analysis system. Graphs show the 6-hour analysis time point for DLD1 and the 7-hour analysis time point for JURKAT. The total detected phagocytic and activated macrophage population percentages were calculated as the area under the curve over time (1, 2, 3, 5, n=6 for reference and Iso) (4, n=3 / 4 for reference and Iso). Results are given as the mean ± SD of the area under the curve. Treatments were compared to IgG4 using the Kruskal-Wallis multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Figure 15] LILRB1 expression in iPS-derived macrophages. LILRB1 expression in iPS-derived macrophages (isotype control and LILRB1 line) determined by flow cytometry. N=3. [Figure 16]Antibodies 1–5 enhance GM-CSF and TNFα production in iPS-derived macrophages after LPS stimulation. iPSC-DMs were preincubated with 10 μg / ml of our LILRB1 antibodies (1–5), an isotype control (IgG4), or a reference anti-LILRB1 antibody (Reference Antibody 1) for 1 hour. Next, 25 (A) or 1 ng / ml (B, C) of LPS was added directly to the cells, and the supernatants were collected 5 hours later. A. Graph shows the GM-CSF concentration (pg / ml) detected in the supernatant after each treatment with 25 ng / ml LPS. B. Graph shows the GM-CSF concentration (pg / ml) detected in the supernatant after each treatment with 1 ng / ml LPS. C. Graph shows the TNFα concentration (μg / ml) found in the supernatant after each treatment with 1 ng / ml LPS. Supernatants were diluted 1:100 in the corresponding ELISA diluent for TNFα measurement. Results are given as mean ± SD. Two-way ANOVA with Kruskal-Wallis multiple comparison test was performed comparing treatments to IgG4. *p<0.05, **p<0.01, ***p<0.001. [Figure 17] Antibodies 1-5 do not enhance GM-CSF and TNFα production after LPS stimulation in iPS-derived macrophages in the absence of costimulation. iPSC-DMs were incubated with 10 μg / ml of anti-LILRB1 antibodies 1-5, isotype control (IgG4), or anti-LILRB1 reference antibody 1 for 6 hours. A. GM-CSF ELISA was performed on undiluted supernatants. The graph shows the GM-CSF concentration (pg / ml) detected in the supernatant after each treatment. B. The graph shows the TNFα concentration (pg / ml) found in the supernatant after each treatment. For TNFα measurement, supernatants were diluted 1:50 in the corresponding ELISA diluent. Concentrations interpolated from the standard curve were multiplied by the dilution factor and then plotted. Results are presented as mean ± SD. A two-way ANOVA with a Kruskal-Wallis multiple comparison test was performed comparing treatments versus IgG4. No statistically significant differences were observed. [Figure 18]Antibodies 1–5 enhance GM-CSF and TNFα production in primary monocyte-derived macrophages after LPS stimulation. Donor-derived monocytes (n=5) were differentiated into macrophages in vitro by exposure to M-CSF. At the end of the culture period (day 8), cells were replated in 96-well plates and incubated overnight. The following day, macrophages were exposed to anti-LILRB1 antibodies (1–5), an isotype control (IgG4), and an anti-LILRB1 reference antibody (reference antibody 1) for 1 hour, followed by exposure to LPS at 1 ng / ml (5 h). A. GM-CSF ELISA was performed on undiluted supernatants. The graph shows the GM-CSF concentration (pg / ml) detected in the supernatant after each treatment. B. The graph shows the TNFα concentration (pg / ml) found in the supernatant after each treatment. Supernatants were diluted 1:10 in the corresponding ELISA diluent for TNFα measurement. Concentrations interpolated from the standard curve were multiplied by the dilution factor and then plotted. Graphs show pg / mL or μg / mL values ​​for each group. Data are presented as mean ± SD. Two-way ANOVA with Dunn's multiple comparison test was performed comparing treatments to IgG4. *p<0.05. [Figure 19] Sequence alignment of human LILRB1 (SEQ ID NO: 45), human LILRB2 (SEQ ID NO: 46) and human LILRA3 (SEQ ID NO: 49). [Figure 20] VH, VL and CDR sequences of antibodies 1 to 5 (Kabat). [Figure 21-1] Neutralization of HLA-A binding to LILRB1 and LILRB2 receptors. Neutralizing reference antibodies 1 to 3 (LILRB1-specific, LILRB2-specific, and LILRB1 / 2-specific, respectively) were used as positive controls. [Figure 21-2] Neutralization of HLA-E binding to LILRB1 and LILRB2 receptors. Neutralizing reference antibodies 1 to 3 (LILRB1-specific, LILRB2-specific, and LILRB1 / 2-specific, respectively) were used as positive controls. [Figure 21-3] Neutralization of HLA-G binding to LILRB1 and LILRB2 receptors. Neutralizing reference antibodies 1 to 3 (LILRB1-specific, LILRB2-specific, and LILRB1 / 2-specific, respectively) were used as positive controls. [Figure 22] TNFα secretion by monocyte-derived macrophages under basal (CSF-1 only) (A) and CSF-1, IL-10, and TGFβ immunosuppressed (B) conditions. Graphs represent results normalized to isotype controls from three independent experiments using MDMs from three different donors. Reference 1 is a LILRB1-specific antibody, reference 2 is a LILRB2-specific antibody, and reference 3 antibody has dual LILRB1 / LILRB2 specificity. [Figure 23-1] (A) Immunogenicity scores for antibody clones 1-5 obtained using Abzena's iTope-AI and TCED™ in silico methods. [Figure 23-2] (B) Comparison of scores for antibodies 1-5 (*) with other fully human therapeutic antibodies, humanized, chimeric and mouse Abs (●); and with those against non-human proteins (p). [Figure 24-1] (A) Fluorescence intensity upon binding to a linear peptide microarray spanning the D3-D4 region of human LILRB1, LILRB2, and LILRA3. [Figure 24-2] (B) Fluorescence intensity upon binding to a "conformation" peptide microarray spanning the D3-D4 region of human LILRB1, LILRB2, and LILRA3. [Figure 25] F(ab)'2 to reprogram MDMs. [Figure 26] Fab monomers to reprogram MDMs. [Figure 27] IFN-gamma production. The y-axis shows fold change relative to CTR. 1. IgG4 isotype, 2. OPDIVO, 3. Rat IgG2a, 4. Reference antibody 1, 5. Reference antibody 2; 6. Antibody 2 IgG4, 7. Isotype IgG4, 8. Antibody 1 IgG4, 9. Reference antibody 3, 10. Antibody 3 IgG4, 11. Antibody 2 IgG1 LALA, 12. Antibody 5 IgG4, 13. Isotype IgG1, 14. Reference antibody 4, 15. Antibody 2 IgG1, 16. Isotype IgG1 LALA, 17. Antibody 4 IgG4. [Figure 28]CD4+ T cell proliferation (▲) and expansion (●). The y-axis shows fold change relative to CTR. 1. IgG4 isotype, 2. OPDIVO, 3. Rat IgG2a, 4. Reference antibody 1, 5. Reference antibody 2; 6. Antibody 2 IgG4, 7. Isotype IgG4, 8. Antibody 1 IgG4, 9. Reference antibody 3, 10. Antibody 3 IgG4, 11. Antibody 2 IgG1 LALA, 12. Antibody 5 IgG4, 13. Isotype IgG1, 14. Reference antibody 4, 15. Antibody 2 IgG1, 16. Isotype IgG1 LALA, 17. Antibody 4 IgG4. [Figure 29] A. Wild-type Jurkat cells. Anti-LILRB1 / 2 antibodies enhanced NKL cytolytic activity compared to isotype control in wild-type Jurkat cells. B. Jurkat HLA-G cells. The increase in cytolytic activity was greater in Jurkat co-cultures overexpressing HLA-G. Fold change is shown on the y-axis. 1. Untreated. 2. IgG4 isotype. 3. Antibody 1. 4. Antibody 2. 5. Antibody 3. 6. Antibody 4. 7. Antibody 5. [Figure 30] Anti-LILRB1 / 2 and reference antibodies enhanced NKL cytolytic activity compared to isotype control in wild-type Jurkat cells. Fold change is shown on the y-axis. 1. Untreated. 2. IgG4 isotype. 3. Reference antibody 3. 4. Reference antibody 4. 5. Reference antibody 5. 6. Reference antibody 6. 7. Reference antibody 1. 8. Reference antibody 2. 9. Antibody 1. 10. Antibody 2. 11. Antibody 3. 12. Antibody 4. 13. Antibody 5. [Figure 31A] Binding of antibodies 1-5 to all LILR family members tested by single-point ELISA. Recombinant LILRB1-5 and LILRA1 (A) were coated onto ELISA plates. These plates were then blocked with milk in PBST. Plates were then incubated with 1 nM of clones 1-5, references 1-3, or an IgG4 isotype control. Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. Antibodies 1-5 bound to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6. [Figure 31B]Binding of antibodies 1-5 to all LILR family members tested by single-point ELISA. Recombinant LILRB1 and LILRA2-6 (B) were coated onto ELISA plates. These plates were then blocked with milk in PBST. Plates were then incubated with 1 nM of clones 1-5, references 1-3, or an IgG4 isotype control. Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. Antibodies 1-5 bound to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6. [Figure 32A] Binding of clones 1 to 5 to selected LILR family members was tested by multipoint ELISA. Recombinant LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 or an unrelated control protein was coated onto ELISA plates. These plates were then blocked with milk in PBST. The plates were then incubated with serial dilutions (12.5 nM to 0.02 nM) of clones 1 to 5 (A to E). Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. [Figure 32B] Binding of clones 1 to 5 to selected LILR family members was tested by multipoint ELISA. Recombinant LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 or an unrelated control protein was coated onto ELISA plates. These plates were then blocked with milk in PBST. The plates were then incubated with serial dilutions (12.5 nM to 0.02 nM) of clones 1 to 5 (A to E). Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. [Figure 32C]Binding of clones 1 to 5 to selected LILR family members was tested by multipoint ELISA. Recombinant LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 or an unrelated control protein was coated onto ELISA plates. These plates were then blocked with milk in PBST. The plates were then incubated with serial dilutions (12.5 nM to 0.02 nM) of clones 1 to 5 (A to E). Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. [Figure 32D] Binding of clones 1 to 5 to selected LILR family members was tested by multipoint ELISA. Recombinant LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 or an unrelated control protein was coated onto ELISA plates. These plates were then blocked with milk in PBST. The plates were then incubated with serial dilutions (12.5 nM to 0.02 nM) of clones 1 to 5 (A to E). Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. [Figure 32E] Binding of clones 1 to 5 to selected LILR family members was tested by multipoint ELISA. Recombinant LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6 or an unrelated control protein was coated onto ELISA plates. These plates were then blocked with milk in PBST. The plates were then incubated with serial dilutions (12.5 nM to 0.02 nM) of clones 1 to 5 (A to E). Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. [Figure 33]Cross-competition between antibody (clone) 2 and other Abs in ELISA. Plates were coated with human LILRB1 and, after blocking, incubated with 5 nM biotinylated clone 2 and different concentrations of unlabeled clones 1–5 and reference Abs 1–3. Bound biotinylated clone 2 was detected using HRP-conjugated streptavidin and a chromogenic substrate. Unlabeled clone 2 competed with its biotinylated counterpart at a low nM IC50, while clones 1, 3, 4, and 5 each yielded IC50s of approximately 10 nM. Neither the reference Ab nor the isotype control was able to compete with antibody 2 at concentrations up to 250 nM. These data indicate that clone 2 binds to a unique epitope on LILRB1 that is distant from the epitopes of all reference Abs but close to the epitopes of antibodies (clones) 1, 3, 4, and 5. [Figure 34] Epitope mapping of antibody (clone) 2 on human LILRB1. The antibody was incubated with recombinant human LILRB1-avi-his in DO, 20 mM Na-phosphate, 150 mM NaCl, pH 7.4 for various times. The protein mixture was then digested with protease and analyzed on a Leap HDX autosampler and a Waters Cyclic IMS MS instrument. (A) The relative changes in deuterium uptake (brighter = smaller, darker = larger) in proteolytic peptides after 2, 10, and 60 minutes (top-to-bottom bars below the sequence) are shown in a heat map below the LILRB1 sequence. The putative epitope for clone 2 (sequence AEFPMGPVTSAHAGT ​​(SEQ ID NO: 78)) is underlined with a solid line, and another region potentially involved in antibody (clone) 2 binding (sequence LTHPSDPLEL (SEQ ID NO: 79)) is underlined with a dashed line. [Figure 34B] (B) Alignment of all 11 human LILR family members with the putative epitopes identified by HDX underlined, and other regions potentially involved in clone 2 binding are outlined with dotted lines. The LILR family members bound by clones 1–5 are in dashed boxes. [Figure 34C](C) Aligned 3D structures (surface views) of human LILRB1 (light gray) and LILRB2 (dark gray) with putative epitopes in domain 4 highlighted in black. [Figure 34D] (D) Ribbon diagram of the LILRB1 (left) and LILRB2 (right) 3D structures, with the putative clone 2 epitope in domain 4 highlighted in black. [Figure 35A] Antibody (clone) 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDMs) upon induction with various stimuli. M0-polarized MDMs were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with compound: (A) 10 ng / ml of IL-1β. Data shown are normalized to background. [Figure 35B] Antibody (clone) 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDMs) upon induction with various stimuli. M0-polarized MDMs were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with compounds: (B) 0.5 μg / ml of the TLR7 / TLR8 agonist R848. Data shown are normalized to background. [Figure 35C] Antibody (clone) 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDM) upon induction with various stimuli. M0-polarized MDM were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with compound (C) 10 μg / ml of the TLR3 ligand LMW poly(I:C). Data shown are normalized to background. [Figure 35D] Antibody (clone) 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDM) upon induction with various stimuli. M0-polarized MDM were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with compound (D) 10 μg / ml of the TLR3 ligand HMW poly(I:C). Data shown are normalized to background. [Figure 35E]Antibody (clone) 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDMs) upon induction with various stimuli. M0-polarized MDMs were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with compound (E), the STING agonist c-di-AMP, at 10 μg / ml. Data shown are normalized to background. [Figure 35F] Antibody (clone) 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDM) upon induction with various stimuli. M0-polarized MDM were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with compound (F), a TRL4 agonistic HMGB1-derived peptide, at 30 μg / ml. Data shown are normalized to background. [Figure 36] Antibody (clone) 2 enhances TNFα secretion by MDM polarized to either M0 or M2 phenotypes (by IL-10 and TGFβ) cocultured with A375 melanoma cells upon induction with LPS. MDM cocultured with A375 cells for 4.5 hours were incubated with 10 μg / ml of clone 2 for 1 hour and then stimulated with LPS (1 ng / ml and 10 ng / ml for M0 and M2 MDMs, respectively) for 3.5 hours. The data shown are normalized to background. DETAILED DESCRIPTION OF THE INVENTION

[0159] Example Generation of anti-LILRB1 antibodies Immunization. Fully human antibodies were generated by immunizing ATX transgenic mice (Alloy Therapeutics) with the human LILRB1 ectodomain (aa24-459) (SEQ ID NO: 41) fused to mouse IgG2a Fc. Animals with the highest antigen-specific serum native titers against human LILRB1 and rhesus monkey LILRB proteins were used for hybridoma generation, immune single-chain Fab (scFab) phage library generation, and B cell selection. Lymphocytes were obtained from the spleen and / or draining lymph nodes. Pooled lymphocytes (from each collection) were dissociated from lymphoid tissues by trituration in an appropriate medium (e.g., Dulbecco's Modified Eagle's Medium (DMEM)).

[0160] Hybridoma generation and screening. B cells were selected and / or expanded using standard methods and fused with an appropriate fusion partner using techniques known in the art. Hybridoma supernatants with binding to human LILRB1 were then selected for further characterization.

[0161] Immune scFAb phage library generation and screening. B cells were selected, and the variable regions of heavy and light Ab chains were used to construct a single-chain Fab library cloned into a phagemid vector using techniques known in the art. This library was then panned against human LILRB1-mIgG2a (SEQ ID NO: 41) and / or rhesus LILRB-mIgG2a (SEQ ID NO: 43) proteins. Periplasmic extracts of phage clones producing scFabs with binding to human LILRB1 were then selected for further characterization.

[0162] Antigen-specific B cell sorting. B cells were selected and stained with human LILRB1-mIgG2a protein (SEQ ID NO: 41), followed by a fluorescently labeled anti-mouse IgG secondary antibody. B cells expressing antibodies binding to human LILRB1 were then individually sorted using techniques known in the art.

[0163] Recovery of anti-LILRB1 antibody sequences. Hybridoma and B cell lysates were used to amplify antibody heavy and light chain variable region (V) genes using reverse transcription-mediated cDNA synthesis followed by polymerase chain reaction (RT-PCR). Phagemid DNA preps were used to amplify antibody heavy and light chain variable region (V) genes using polymerase chain reaction (PCR)-mediated DNA synthesis. Amino acid sequences were deduced from the corresponding nucleic acid sequences using bioinformatics. The resulting amino acid sequences were then analyzed to determine the germline sequence origin of the antibody and identify deviations from the germline sequence. Amino acid sequences corresponding to the complementarity-determining regions (CDRs) of the sequenced antibodies were aligned, and these alignments were used to group clones by similarity.

[0164] Biochemical characteristics of anti-LILRB1 antibodies Antibody Expression. Antibodies were expressed in CHO suspension cells and purified using Protein-A affinity chromatography followed by buffer exchange into phosphate buffer pH 7.4.

[0165] Binding to human LILRB1 and LILRB2. HEK293 cells stably expressing full-length human LILRB1 (SEQ ID NO: 45) or LILRB2 (SEQ ID NO: 46) or wild-type cells were detached from culture plates using methods known in the art. The cells were then incubated with different concentrations of anti-LILRB1 Ab clones, followed by incubation with a fluorescently labeled secondary antibody. The extent of antibody binding to cells stably expressing full-length human LILRB1 or full-length human LILRB2 was determined by flow cytometry and quantified using the geometric mean (GEOM) of the fluorescent signal.

[0166] Binding to allelic forms of LILRB1. There are four major allelic variants of the human LILRB1 protein ectodomain. LILRB1 variants were expressed as recombinant proteins (SEQ ID NOS: 50 and 51) containing a modified LILRB1 ectodomain and a mouse IgG2 Fc region in suspension CHO cells and purified using techniques known in the art. These recombinant proteins were then used to determine the binding potential of antibodies to different allelic forms of LILRB1 in a direct enzyme-linked immunosorbent assay (ELISA). Briefly, in a Maxisorp (Nunc) or similar immunoassay, plates were coated with a solution of the target protein in PBS overnight at 4°C. The plates were then washed with PBS, blocked with 5% milk in PBS, and washed again. Test antibodies diluted in PBS were then added to the plates and incubated at room temperature for at least 1 hour. The plates were then washed with PBS / 0.05% Tween-20 (PBST) and incubated with a solution of horseradish peroxidase (HRP)-conjugated secondary antibody for at least 1 hour at room temperature. After washing with PBST and PBS, bound antibody was detected using a 3,3',5,5-tetramethylbenzidine (TMB) color development solution. After 5–10 minutes, the reaction was stopped with 1% sulfuric acid, and absorbance was read at 450 nm.

[0167] Binding to non-human primate LILRB proteins. The ectodomains of the rhesus and cynomolgus homologs of human LILRB1 and / or LILRB2 proteins were expressed as fusions to mouse IgG2a Fc (SEQ ID NO: 43 and SEQ ID NO: 44) and used to test the cross-reactivity of anti-LILRB1 antibodies in a direct ELISA, as described above.

[0168] Binding to LILRA and LILRB family members. To determine the potential of antibodies to bind to LILRA receptors, recombinant LILRA1 (SEQ ID NO: 47), LILRA2 (SEQ ID NO: 48), and LILRA3 (SEQ ID NO: 49) proteins from commercial sources were used in direct ELISAs as described above.

[0169] Blockade of HLA binding to LILRB1 and LILRB2. HEK293 cells expressing human LILRB1 or LILRB2 receptors were incubated with fluorescently labeled HLA-A, HLA-E, or HLA-G oligomers in the presence or absence of antibodies, and then analyzed by flow cytometry as described above.

[0170] Epitope binning. Using techniques known in the art, such as surface plasmon resonance (SPR), cross-neutralization of binding of individual anti-LILRB1 antibodies to LILRB1 fused to mouse IgG2a Fc was examined. Recombinant human LILRB1 ectodomain (SEQ ID NO: 41) fused to mouse IgG2a Fc was first immobilized on an SPR chip, and then two different anti-LILRB1 antibodies were injected sequentially over the chip. If injection of the second antibody did not lead to an increase in the SPR signal, it was concluded that the two antibodies bind to similar regions on the LILRB1 molecule.

[0171] Antibody binding to iPSC-derived macrophages iPSC-derived macrophages were detached using enzyme-free PBS cell dissociation buffer (Life Technologies Cat# 13151014) according to the manufacturer's instructions. Cells were then blocked with MACS FcR Blocking Reagent (Cat# 130-059-901) for 20 minutes on ice according to the manufacturer's instructions. Cells were washed with PBS 2% BSA, 5 mM EDTA DPBS (FACS buffer). For each antibody test, 2x10 cells were cultured using the appropriate test antibody or IgG4 isotype control at a concentration of 10µg / ml. 5The cells were incubated on ice for 30 minutes. The macrophages were then washed once with FACS buffer and stained with a secondary antibody (PE mouse anti-human IgG4-pFC, Southern Biotech Cat#9190-09) at a concentration of 1 μg / ml for 30 minutes on ice. The macrophages were then washed once with FACS buffer and resuspended in 200 μl of FACS buffer and DAPI (0.1 μg / ml). The cells were kept on ice prior to data acquisition using an LSR Fortessa Analyser (BD). FCS Expression was used as the analysis software.

[0172] NK92 binding assay procedure: 1. Prepare FACS buffer: 2% BSA, 5 mM EDTA-DPBS. 2. Harvest cells: If suspension cells are used, harvest and resuspend in FACS buffer for counting and proceed to step 5. 3. Pipette in and out to detach the cells. 4. Count the number of cells: 2 x 10 5 Pieces per specimen required. 5. Spin down cells at 200G for 3 minutes. If blocking is desired, proceed to step 6; otherwise, proceed to step 10. 6. Resuspend each sample (up to 10 cells in 80 μl of 2% BSA-PBS-EDTA). 7 (Up to 1.5ml tubes are used). 7.<10 7 Add 20 µL of FcR blocking reagent per 100 total cells. 8. Incubate on ice for 20 minutes. Add 9.1 ml of FACS buffer and spin down at 200 G for 3 minutes. 10. Aspirate the supernatant and add a volume of FACS buffer to ensure the cells are finally at 200,000 cells (2 x 10 cells) in 100 μl. 6 The concentration should be (pieces / ml). 11. Take a 96 U bottom well plate and label: a) unstained b) isotype control c) test antibody. 12. Add 200,000 cells / well (100 μl). 13. Add the corresponding primary antibody (as indicated in the table below) and mix. 14. Incubate in the dark at 4°C for 30 minutes. 15. Add 100 μl of FACS buffer to each well. 16. Spin down cells at 200G for 3 minutes and remove supernatant. If a secondary antibody is used, proceed to step 17, otherwise proceed to step 23. 17. Resuspend cells in 100 μl FACS buffer. 18. Add the corresponding secondary antibody (as specified in the table below) and mix. * Note, some secondary antibodies need to be premixed. 19. Incubate in the dark at 4°C for 30 minutes. 20. Add 100 μl of FACS buffer to each well. 21.Spin down cells at 200g for 3 minutes. 22. Remove the supernatant. 23. Resuspend in 200 μL of FACS buffer. 24. Add DAPI (1:1000, 0.2 μl) to each well. 25. Transfer each sample to a FACS tube. 26.Submit to flow cytometry.

[0173] [Table 10]

[0174] iPSC method for the generation of genetically modified human iPSC-derived macrophages. WT iPSC 55 strain published protocol: https: / / www.jove.com / t / 61038 / production-characterization-human-macrophages-from-pluripotent-stem

[0175] Generation and characterization of human macrophages from pluripotent stem cells Lopez-Yrigoyen et al. (2020), M., May, A., Ventura, T., Taylor, H., Fidanza, A., Cassetta, L., Pollard, JW, Forrester, LMProduction and Characterization of Human macrophages from Pluripotent Stem Cells.J.Vis.Exp.(158),e61038,doi:10.3791 / 61038(2020).

[0176] The method for generating macrophages from human induced pluripotent stem cells and their subsequent characterization was performed as described in Lopez-Yrigoyen et al. (2020). The phenotype and function of these iPSC-derived macrophages were evaluated using cell surface marker expression, gene expression, and functional assays. The optimized protocol described in Lopez-Yrigoyen et al. (2020) enabled the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) expressed human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and live cell imaging functional assays revealed that they were capable of robust phagocytic activity. Cultured iPSC-DMs can be activated by the addition of LPS and IFNg, IL4, or IL10 into different macrophage states that change gene expression and exhibit phagocytic activity, providing a platform for generating human macrophages carrying genetic modifications that model specific human diseases and a source of cells for drug screening or cell therapy to treat these diseases.

[0177] A serum- and feeder-free protocol for the maintenance, freezing, and thawing of human iPSCs and for the differentiation of these iPSCs into functional macrophages is described below. The protocol is very similar to that described by Van Wilgenburg et al. (2014), with minor modifications including: 1) iPSC maintenance medium; 2) ROCK inhibitor was not used during the EB formation stage; 3) a mechanical approach rather than an enzymatic approach was used to generate uniform EBs from iPSC colonies; 4) the method for EB collection and seeding was different; 5) suspension cells were collected twice a week instead of once a week; and 6) collected suspension cells were cultured under CSF1 for macrophage maturation for 9 days instead of 7 days. The protocol used to characterize iPSC-derived macrophage phenotype and function included analysis of gene expression (qRT-PCR), cell surface marker expression (flow cytometry), and functional assays to assess phagocytosis and polarization.

[0178] Protocol Notes: All reagents and equipment used in this protocol are listed in the Table of Materials. Media should be at 37 °C for cell culture. Media and reagents used in the differentiation protocol must be sterile.

[0179] [Table 11]

[0180] [Table 12]

[0181] [Table 13]

[0182] 1. Freezing and Thawing and Maintenance of Human iPSC Lines 1. Cell maintenance medium, growth factors and other reagents were prepared. 1. Prepare hESC-serum-free medium (hESC-SFM; see Table of Materials) by supplementing Dulbecco's Modified Eagle Medium F12 (DMEM / F12) with hESC supplement, 1.8% w / v bovine serum albumin (BSA) and 0.1 mM 2-mercaptoethanol. 2. Human basic fibroblast growth factor (bFGF) stock solution (10 μg / mL) was prepared by dissolving bFGF in sterile 0.1% human serum albumin(I)-phosphate buffered saline (PBS) solution. The stock solution was dispensed into 200 μL aliquots in cryotubes. The stock solution was stored at -20°C for up to 1 year. Once frozen and thawed, stock bFGF was stored at 4°C for up to 7 days. 3. Rho kinase inhibitor (ROCK inhibitor) - Y27632 stock solution (1 mg / mL) was prepared by dissolving it in sterile water. The stock solution was dispensed as 50 μL aliquots in cryotubes. The stock solution was stored at -20°C for up to 1 year. Once frozen and thawed, the stock ROCK inhibitor was stored at 4°C for up to 7 days. 2. Stem cell substrate (see Table of Materials) was diluted 1:50 in Dulbecco's phosphate buffered saline with calcium and magnesium. 3. Place the diluted stem cell substrate solution into the culture plate so that the final volume per surface area is 78 µL / cm 2 750 μL of the solution was added to coat the wells of a 6-well plate. 4. The coated plates were incubated for 1 hour at 37°C and 5% CO2 in a humidified atmosphere. 5. The stem cell substrate coating was aspirated and 1 mL of hESCs supplemented with 20 ng / mL bFGF and 10 μM ROCK inhibitor was added. 6. Thaw a vial of frozen human iPSC cells by incubating the vial at 37°C until frozen, then transfer the cells to 5mL hESC-SFM medium. 7. Cells were centrifuged at 100 x g for 3 minutes. The cell pellet was resuspended in 0.5 mL hESC-SFM supplemented with 8.20 ng / mL bFGF and 10 μM ROCK inhibitor. The cells were transferred to the coated wells. 9. The cells were cultured for 24 hours. The medium was changed to hESC-SFM supplemented with 10.20 ng / mL bFGF but without ROCK inhibitor. 11. To maintain the cells, the medium was changed daily until the cells reached 80% confluency. Undifferentiated iPSCs usually require 3–4 days to reach 80% confluency. 12. When the cells reached 80% confluency, they were passaged. 1. Replace the spent culture medium with 1.5 mL of fresh hESC-SFM (without ROCK inhibitor) supplemented with 20 ng / mL bFGF. 2. Holding the culture vessel with one hand, roll a disposable cell passaging tool (see the Table of Materials) across the plate in one direction (i.e., from left to right). Ensure that all blades of the roller are in contact with the plate. Maintain uniform pressure while rolling. 3. Roll repeatedly in the same direction until all wells are covered. 4. The culture vessel was rotated 90° and rolled repeatedly as described in steps 1.12.2 and 1.12.3. 5. After use, the passaging tool was discarded. 6. Using a sterile pipette, the medium in the well was used to remove the excised colony. 7. Transfer cells onto pre-coated stem cell substrate wells (steps 1.2-1.5) at a 1:4 ratio to a final medium volume of 1.5 mL / well (hE-C-SFM supplemented with 20 ng / mL bFGF).

[0183] 2. Freezing Human iPSC Lines 1. To freeze iPSC cells, replace the medium in a 70%-80% confluent well of a 6-well plate with hESC-SFM supplemented with 20 ng / mL bFGF and 10 μM ROCK inhibitor. The wells were incubated for 2.1 hours at 37° C. and 5% CO 2 . 3. Use a cell passaging tool to cut the colonies and place the removed colonies into a centrifuge tube. 4. The cells were centrifuged at 100xg for 3 minutes. 5. Aspirate the medium and resuspend the cells in 1 mL of cell cryopreservation medium (see Table of Materials). 6. The cells were divided equally into two cryovials and placed in a pre-cooled cell cryopreservation container at 4°C. 7. Cells were stored at -80°C for 24 to 48 hours. 8. The vials were transferred to either a -135°C freezer or a liquid nitrogen tank.

[0184] 3. Differentiation of Human iPSCs into Macrophages 1. Preparation of Cell Differentiation Growth Factors and Other Reagents 1. Prepare hESC-SFM medium (see previous section). 2. A 0.1% w / v solution of porcine gelatin was prepared by dissolving the gelatin in sterile water. The gelatin solution was stored at 4°C for up to 2 years. A human BMP4 stock solution (25 μg / mL) was prepared by dissolving BMP4 in 3.4 mM hydrogen chloride (HCl)-0.2% BSA PBS solution. The stock solution was dispensed in 50 μL aliquots in cryotubes. The stock solution was stored at -20°C for up to 1 year. After thawing, stock BMP4 was stored at 4°C for up to 5 days. 4. A human VEGF stock solution (100 μg / mL) was prepared by dissolving VEGF in 0.2% BSA PBS solution. The stock solution was dispensed in 10 μL aliquots in cryotubes. The stock solution was stored at -20°C for up to 1 year. Once frozen and thawed, stock VEGF was stored at 4°C for up to 7 days. 5. A human SCF stock solution (100 μg / mL) was prepared by dissolving SCF in 0.2% BSA PBS solution. The stock solution was dispensed in 5 μL aliquots in cryotubes. The stock solution was stored at -20°C for up to 1 year. Once frozen and thawed, stock SCF was stored at 4°C for up to 10 days. 6. Human IL3 stock solution (10 μg / mL) was prepared by dissolving IL3 in 0.2% BSA PBS solution. The stock solution was dispensed in 500 μL aliquots in cryotubes. The stock solution was stored at -20°C for up to 2 years. Once frozen and thawed, stock SCF was stored at 4°C for up to 15 days. 7. Human CSF1 stock solution (10 μg / mL) was prepared by dissolving CSF1 in 0.2% BSA PBS solution. The stock solution was dispensed in 1 mL aliquots in cryotubes. The stock solution was stored at -20°C for up to 2 years. Once frozen and thawed, stock SCF was stored at 4°C for up to 15 days. 8. Separate 10 μg / mL stock solutions of interferon-gamma (IFNg), interleukin 4 (IL4), and interleukin 10 (IL10) were prepared by dissolving in 0.2% BSA PBS. Lipopolysaccharide (LPS) was prepared as a (100 U / mL) stock solution by dissolving in 0.2% BSA PBS. Each stock solution was dispensed in 35 μL aliquots. These were stored at -80°C for up to 2 years. After thawing, the stock solutions were stored at 4°C for up to 7 days.

[0185] 2. Step 1: Embryoid Body (EB) Generation (Day 0-3) 1. On day 0, 2.25 mL of stage 1 medium (hESC-SFM supplemented with 50 ng / mL BMP4, 50 ng / mL VEGF and 20 ng / mL SCF) was added to two wells of an ultra-low attachment 6-well plate. 2. Replace the maintenance medium of one 80% confluent well of iPSCs in a 6-well plate with 1.5 mL of Stage 1 medium. 3. Use a cell passaging tool to cut out colonies and transfer the cut colonies with a pipette into two wells of an ultra-low attachment 6-well plate (see Table of Materials). 4. On day 2, 0.5 mL of hESC-SFM medium was used to bring the cytokines to a final concentration of 50 ng / mL BMP4, 50 ng / mL VEGF and 20 ng / mL SCF. Note: IPSC colonies develop into EBs.

[0186] 3. Stage 2: Emergence of hematopoietic cells in suspension 1. On day 4, four wells of a 6-well tissue culture plate were coated with 0.1% w / v gelatin and incubated for at least 10 minutes. 2. Remove the gelatin and add 2.5 mL of stage 2 medium (X-VIVO15 supplemented with 100 ng / mL CSF1, 25 ng / mL IL-3, 2 mM glutamax, 1% penicillin-streptomycin, and 0.055 mM 2-mercaptoethanol). 3. The formed EBs were collected in a 50 mL centrifuge tube, allowing them to settle to the bottom of the tube by gravity. The medium was carefully aspirated. EBs were resuspended in 4.2 mL of stage 2 medium. 5. Transfer 10-15 EBs (not more than 15) to gelatin-coated wells containing 2.5 mL of stage 2 medium. 6. EBs were incubated at 37°C and 5% CO2 in air. 7. The medium on the seeded EBs was changed every 3-4 days for 2-3 weeks. 8. After 2-3 weeks, the EBs began to release non-adherent hematopoietic cells into suspension. The duration of floating cell release varies and is cell line dependent. Cells in this suspension were collected and allowed to mature into macrophages (see step 3).

[0187] 4. Stage 3: Final macrophage maturation 1. Floating hematopoietic cells were collected and the medium was replenished onto EB plates (stage 2 medium). 2. The suspended cells were centrifuged at 200xg for 3 minutes. 3. Resuspend the floating cells in stage 3 medium (X-VIVO15 supplemented with 100 ng / mL CSF1, 2 mM Glutamax, and 1% penicillin-streptomycin). 4. Recover and 0.2x10 6 The spun cells were seeded at a density of 10 cells / mL into untreated plastic 10 cm bacteriological grade plates (10 mL) or uncoated 6-well tissue culture plates (3 mL). Cells were maintained in stage 3 medium for 9-11 days, with medium changes every 5.5 days. Steps 3 through 3.4.1-3.4.5 could be repeated every 3-4 days, and floating cells were collected from the original EB plate for up to 3 months.

[0188] 5. Macrophage Polarization 1. To activate macrophages to the M(LPS+IFNg) phenotype, cells were stimulated with LPS (final concentration: 100 ng / mL) and IFNg (final concentration: 10 U / mL) for 48 hours. To activate cells to the M(IL4) phenotype, cells were stimulated with IL4 (final concentration: 20 ng / mL). To activate cells to the M(IL10) phenotype, macrophages were stimulated with IL10 (final concentration: 5 ng / mL).

[0189] 4. Quality Control Check of iPSC-derived Macrophages 2. The number of hematopoietic floating cells produced per 6-well plate of EBs was determined by counting the floating hematopoietic cells using a hemocytometer. 3. Macrophage morphology was assessed as previously described (e.g., according to Lopez-Yrigoyen, M., et al. (2019), Lopez-Yrigoyen, M., et al. (2018) using commercially available staining kits). 4. Detection of the expression of macrophage-specific and polarization markers using gene expression analysis and flow cytometry as previously described (Lopez-Yrigoyen, M., et al. (2019), Lopez-Yrigoyen, M., et al. (2018)). 1. For flow cytometry experiments in wells of a 6-well plate of macrophages, cells were collected by aspirating their maturation medium, washed with 2 mL of PBS, and incubated with 2 mL of enzyme-free cell dissociation buffer for 5 min at room temperature (RT). Macrophages were detached and collected by repeated pipetting. 2. Cells were counted using a hemocytometer and resuspended in 80 μL of 2% BSA, 0.5 mM ethylenediaminetetraacetic acid (EDTA)-PBS solution. 3. 20 μL of MACS human Fc blocking agent was added. 4. Cells were incubated on ice for 20 minutes, protected from light. 5. Add an appropriate volume of 2% BSA, 0.5 mM EDTA PBS solution to bring the cell concentration to 1 x 10 macrophages. 6 The concentration was calculated as 1 / mL. 6. 1x10 antibodies in 100 µL of 2% BSA, 0.5 mM EDTA PBS solution with the corresponding antibody (see notes below). 5 Cells were stained and incubated for 15 min at RT protected from light. 7. Wash the celled once with at least 100 μL of 2% BSA, 0.5 mM EDTA PBS. 8. Cells were resuspended in 200 μL of 2% BSA, 0.5 mM EDTA PBS. 9.4',6-diamidino-2-phenylindole (DAPI, 1:1,000 dilution) was added as a viability dye, and the suspension was incubated for 3 minutes. 5. For flow cytometry analysis, cells were gated on the primary population, then single cells, and then viable cells. Macrophage-associated marker expression was evident in the viable cell population. Antibodies were carefully titrated for each cell line used to derive macrophages. The dilution ratios for SFCi55-derived macrophage flow cytometry assays are also included.

[0190] 6. High-throughput Phagocytosis Assay 1. iPSC-derived macrophages (iPSC-DMs) were collected by aspirating the medium, adding ice-cold enzyme-free cell dissociation buffer, and incubating for 5 minutes. Macrophages were collected by repeated pipetting. 2. Place 8x10 cells in wells of an imaging tissue culture grade 96-well plate (e.g., Cellcarrier Ultra, Perkin Elmer) at least 2 days prior to high-throughput imaging in 200 µL of stage 3 medium. 4 iPSC-DMs were seeded. pHrodoGreen Zymosan-A bioparticles were prepared by resuspending one vial in 3.2 mL of PBS ("Solution 1") and vortexing the solution for 10 seconds. 4.2 mL of PBS bead suspension was diluted 1:5 with more PBS ("Solution 2"). 5. Solution 2 was sonicated for 8 seconds, the solution was vortexed for 10 seconds, and then kept at 4° C. This solution was used in step 5.11. 6. The medium on the seeded iPSC-DMs was removed and they were washed with PBS. iPSC-DMs were stained with a PBS solution containing 7.1:20 diluted Hoechst 33342. Incubation was at 37°C for 20 minutes. 8. The cells were washed with PBS. Cells were stained with a PBS solution containing a deep red plasma membrane stain diluted 9.1:1,000 (see Table of Materials). Incubation was at 37 °C for 30 min. 10. The cells were washed with PBS. 100 μL of the bead solution, maintained at 11.4° C., was added to each well of the iPSC-DMs. The plate was now ready for imaging. 12. Plates were imaged using a high content imaging system, with at least three fields across the wells to get a good view of the wells. 13. Phagocytosis was quantified using Columbus software (High Content Image Analysis System software). A specific algorithm was developed for unambiguous image batch analysis: 1. The intensity of the blue color was measured and the software determined that the blue signal represented the nucleus. 2. The intensity of the red color was measured and the software determined that the red signal represented the cytoplasm. 3. It was determined that the nucleus and cytoplasm together correspond to a cell. 4. The intensity of the green color was measured in the cells and a stringent cutoff / threshold was established to consider cells as phagocytic. 5. The fraction of phagocytic cells was quantified to obtain the average phagocytic index per cell. The color intensity of the beads is proportional to the number of beads, and therefore the phagocytic activity can be measured by the number of beads ingested. 6. The algorithm / pipeline is applied to all images within the entire field of view and at all time points acquired, allowing for a robust and unbiased batch processing approach to determine the phagocytic capacity of cells. Note: Columbus is a high-content analysis software that provides cell segmentation analysis for cell phenotyping and functional testing.

[0191] Induction of Macogreen16 GFP lineage The MacoGreen16GFP lineage was derived using a lentiviral plasmid encoding a 0.7 kb sequence encompassing the CBX3 gene, EF1 alpha promoter, eGFP gene, T2A, and puromycin resistance genes. 24 h prior to infection, four wells of a six-well plate of early-passage WT iPSC 55 iPSC cells were treated with Rock inhibitor (Merck Cat# SCM075) at a final concentration of 3.33 μg / ml. On the day of lentiviral infection, iPSC cells were detached by washing once with PBS, adding 0.5 ml of Stempro Accutase (Gibco Cat# A110501), and incubating for 3 minutes. One ml of Stempro hESC SFM medium (Thermo Fisher Cat# A1000701) was added to the well, and the cells were detached by gentle pipetting. iPSC cells were resuspended in Stempro hESC medium supplemented with 10 μg / ml (Sigma Cat# PHG0021) and ROCK inhibitor at a concentration of 3.33 μg / ml. 5x10 cells in single cell suspension were cultured in a 10 cm dish pre-coated with CTS CellStart (Invitrogen Cat# A1014201). 6iPSC cells were seeded. Lentiviral particles were added to the dish to reach an MOI of 0.5. 48 hours after infection, 30-40% of the cells were GFP-positive, and treatment with puromycin at 0.5µg / ml was initiated. After 8 days of puromycin selection, 48 colonies were picked and individually transferred to wells of a 24-well plate. The colonies were fed every other day, and when confluent, they were transferred to wells of a 6-well plate. From the 6-well plate system, 23 clones were expanded, frozen, and tested for GFP expression by flow cytometry. Clone 16 appeared to be a monoclonal population of GFP+ cells. Cells from this clone were successfully expanded and differentiated into a macrophage line. The number of MacoGreen16 macrophages produced was comparable to that of macrophages produced from SFCi55, and these were also a single, pure population of GFP-expressing cells. Furthermore, MacoGreen16 macrophages expressed human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169 (expression was comparable to that of SFCi55 macrophages); were capable of phagocytosing cancer cells, and had the ability to change their phenotype after the addition of LPS plus IFNg, IL4, or IL10.

[0192] Phagocytosis assay GFP-expressing iPSC-derived macrophages were imaged in 100ul of macrophage maturation medium: X-VIVO15 medium (Lonza, Cat#BE02-060F) supplemented with 100ng / ml recombinant human CSF1 (Biolegend Cat#574808), 2mM Glutamax (Invitrogen Cat#35050038), and 1% penicillin-streptomycin (Gibco Cat#15140-122). 2x10 cells were plated in TC-treated 96-well plates (CellCarrier-96 Ultra Microplates, Perkin Elmer Cat#6055300). 4 The cells were seeded at a density of 100 cells / well.

[0193] On the day of the assay, 24-48 hours after macrophage seeding, Jurkat WT cells or Jurkat-HLA-G overexpressing cells were stained for phagocytosis with the IncuCyte® pHrodo® Orange Cell Labeling Kit (Sartorius Cat#4766). Briefly, cells were harvested, centrifuged at 200g for 4 minutes, and washed with IncuCyte pHrodo Cell Wash Buffer (1x10 6 The cells were then spun down at 200g for 4 minutes and the pellet was then resuspended in IncuCyte pHrodo Cell Labelling Buffer to a density of 1x10 6 The cells were then centrifuged at 1300 rpm for 7 minutes to remove excess IncuCyte pHrodo Labeling Dye. Then, 1.2x10 cells were cultured in macrophage maintenance medium. Solubilized IncuCyte pHrodo Orange Cell Labeling Dye was added to the cell suspension at a final concentration of 600 ng / ml. The cell suspension was mixed and then incubated at 37°C for 1 hour (cells were mixed every 20 minutes of the incubation period). Cells were centrifuged at 1300 rpm for 7 minutes to remove excess IncuCyte pHrodo Labeling Dye. Next, 1.2x10 cells were cultured in macrophage maintenance medium. 6 The cell pellet was resuspended at a density of 1000 cells / ml. 50 μl of the cell suspension (60,000 cells) was added to each macrophage-containing well (macrophage:target cell ratio of 1:3). Test antibodies or IgG4 isotype controls were added at a final concentration of 10 μg / ml in triplicate. The plates were immediately placed in an Incucyte S3 live imaging system. Acquisitions of 4 fields / well were performed every 30 minutes for 7 hours. Image analysis was performed using the Cell by Cell pipeline from Incucyte. Briefly, macrophages were segmented based on GFP expression and size. Total orange fluorescence in the macrophage population was the first output obtained after Cell by Cell analysis. The phagocytic macrophage fraction was determined by setting an orange intensity threshold, and the mean orange intensity in the phagocytic macrophage population was determined.

[0194] Reprogramming assay Cell seeding and treatment Macrophages were harvested by aspirating the medium and adding 1.5 ml of enzyme-free cell dissociation buffer (Life Technologies, Cat. 13151014) to each well (6-well plate). Cells were incubated at room temperature for 4 minutes and vigorously pipetted to detach. The cell suspension was collected and centrifuged at 200 g for 3 minutes. The supernatant was aspirated, and cells were resuspended in macrophage maturation medium (X-VIVO15 (Lonza, Cat. BE02-060F) supplemented with 100 ng / mL CSF1 (BioLegend, Cat. 574808), 2 mM glutamax (Life Technologies, Cat. 35050038), and 1% penicillin / streptomycin (Life Technologies, Cat. 15140122). 1.25 x 10 cells were plated in a final volume of 375 μl / well onto an uncoated 24-well tissue culture plate. 5 Cells were seeded at a density of 1000 cells / well. Macrophages were incubated overnight at 37°C and 5% CO2. The following morning, the cell culture medium was replaced with fresh medium, and 10 μg / ml of each antibody / control was added in a final volume of 375 μl and incubated for 1 hour at 37°C and 5% CO2. LPS diluted in macrophage maturation medium was then added to a final concentration of 1 ng / ml or 25 ng / ml and the cells were returned to the incubator for 5 hours. After the incubation period, the macrophage supernatant was collected, transferred to a labeled 1.5 ml Eppendorf tube, and spun in a microcentrifuge at 200 x g for 3 minutes. 350 μl of the supernatant was carefully transferred to a new, labeled Eppendorf tube.

[0195] ELISA for cytokine quantification ELISAs were performed according to the manufacturer's instructions. GM-CSF: Human GM-CSF DuoSet ELISA (R&D Systems, Cat. DY215-05). Undiluted supernatants were used for the GM-CSF ELISA. TNFα: Human TNFα ELISA MAX Deluxe Set (BioLegend, Cat. #430204). The following supernatant dilutions were used for the TNFα ELISA: 1:200 for macrophages treated with 25 ng / ml LPS, 1:100 for macrophages treated with 1 ng / ml LPS, and 1:50 for macrophages not treated with LPS.

[0196] ELISA result calculation Results are analyzed using four parameter logistic (4-PL) regression using GraphPad Prism or custom R scripts.

[0197] result. Heterologous expression of MHC class I binders LILRB1, LILRB2, and LILRA3. Members of the LILRB and LILRA families have both distinct and overlapping tissue expression patterns, ligands, and biological functions. For example, both LILRB1 and LILRB2 can bind to HLA-G and HLA-A, while LILRB2 expression is restricted to the myeloid compartment, whereas LILRB1 is not. Furthermore, some family members possess intracellular ITIM domains (e.g., LILRB1, LILRB2, and LILRB3) and some ITAM domains (e.g., LILRA1). This raises the possibility of both redundant and non-redundant biology. Of particular interest are MHC class I binders, a family of molecules expressed on tumors where immunomodulation is relevant. To further explore this, we performed a bioinformatics analysis of a single-cell RNA sequencing dataset (Mulder et al. 2021), which showed that both LILRB1 and LILRB2 were upregulated in TAMs from multiple cancer types, along with the detection of LILRB1, LILRB2, or LILRB2 and LILRB1 double-positive TAMs in Figure 2. As expected, LILRB1 was also expressed on NK cells, B cells, and T cells. Further bioinformatics analysis of RNA sequencing data from TAMs in melanoma cancer patients (Mulder et al. 2021) showed that both LILRB1 and LILRB2 were detected in 58% of TAMs, LILRB1 alone in 7% of TAMs, and LILRB2 alone in 25% of TAMs (Figure 3). Because both LILRB1 and LILRB2 are immunosuppressive, this suggests that all three populations must be targeted to maximize therapeutic activity. Separately, to explore the role of LILRA3 in cancer biology, we performed a bioinformatics analysis using TCGA to compare LILRA3 RNA expression between multiple normal and cancer tissues, which shows that LILRA3 is overexpressed (p<0.05) in head and neck squamous sarcoma, esophageal carcinoma, renal cell carcinoma, gastric adenocarcinoma, thymic carcinoma, and endometrial carcinoma (Figure 1).

[0198] Therapeutic approaches targeting both LILRB1 and LILRB2 have the advantage of increasing the percentage of activated macrophages in tumor microenvironments where expression of these molecules is heterogeneous, and of reducing potential redundancy between highly homologous molecules, resulting in overlapping expression and ligand binding. Human genetic data suggesting that LILRA3 is anti-inflammatory and that its expression is elevated in tumors suggests that targeting LILRA3 may also be advantageous.

[0199] Example - MHCI deregulation in cancer and LILRB1 / 2 antibodies in the art Ligands for LILRB1, LILRB2, and LILRA3 include classical and non-classical MHC class I molecules. HLA-G is an example of a non-classical MHC class I molecule, while HLA-A, B, and C are examples of classical MHC class I molecules. Classical MHC class I downregulation is a known tumor immune evasion mechanism, reducing tumor antigen presentation and T cell activation (https: / / pubmed.ncbi.nlm.nih.gov / 32630675 / ). For example, classical MHC class I downregulation is observed in approximately one in three melanoma patients and is associated with natural and acquired resistance to TGF-beta signaling and T cell checkpoint therapy. In contrast, upregulation of non-classical MHC class I molecules, such as HLA-G, is also a known tumor immune evasion mechanism.

[0200] Antibodies that bind to LILRB1, LILRB2, or LILRB1 and LILRB2 have been described in the art (e.g., as described in the Background to the Invention and shown in Tables 1-4). However, these antibodies block the ligand interaction of LILRB1 and / or LILRB2, and blocking was used to identify antibodies with desired properties. Furthermore, it has also been reported that non-blocking antibodies have no activity in functional macrophage assays. Ligand blocking has been determined through the prevention of ligand-receptor interaction. "Non-blocking activity" or "non-blocking" or "no blocking" means that in the assays described herein, the assay signal is greater than 10% of the signal observed for the isotype control. The isotype control is 100% signal, blocking is less than 10% of the signal observed for the isotype control, and non-blocking is greater than 10% of the signal observed for the isotype control. To date, no human antibodies have been described that bind to human LILRB1, human LILRB2, and human LILRA3 and do not block the interaction of LILRB1 or LILRB2 with their respective ligands (e.g., LA-G / A / E).

[0201] Example - Identified LILRB1 Non-Blocking Antibodies result Surprisingly, despite the representation of ligand-blocking and non-ligand-blocking antibodies in the mode of action in our larger antibody panel, all five of the most active antibodies in our assay cascade binding to human LILRB1, human LILRB2, and human LILRA3 were non-ligand-blocking agents.

[0202] Identification of LILRB1 nonligand blocking antibodies. LILRB1 binds to its ligands, classical and non-classical HLA molecules, and can thereby transmit immunosuppressive signals to tumor-associated macrophages and other immune cells. This signaling occurs via ITIM domains present in various immunoregulatory receptors expressed on immune cells, such as Fc receptors, PD-1, TIGIT, and PECAM-1. Although ITIM domains are generally considered inhibitory domains, they can also transmit activating signals under some circumstances (Coxon et al. Blood (2017) 129(26):3407-3418). Antibodies whose activity can positively regulate immune cells in the immunosuppressive tumor microenvironment independent of target receptor-ligand interactions are preferred. Furthermore, because LILRB1 is widely expressed on TAMs in multiple cancer types, only a portion of these TAMs are involved in direct interaction with cells expressing their targets, and antibodies that can exert biological activity on target cells without inhibiting ligand-induced signaling are preferred.

[0203] HLA binding to LILRB1 occurs via domain 1 (D1) and domain 2 (D2), amino acids 24 to 224 (SEQ ID NO: 41) of the human LILRB1 ectodomain.

[0204] Antibodies obtained by immunization of humanized mice with human LILRB1 protein were tested in ELISA for their ability to bind to the full-length LILRB1 ectodomain (SEQ ID NO: 41) and to a truncated form of the human LILRB1 protein containing domains 1 and 2 (SEQ ID NO: 42). Five antibodies (antibodies 1, 2, 3, 4, and 5) were selected that were able to bind to the full-length LILRB1 ectodomain (SEQ ID NO: 41) of LILRB1 but not to the truncated form (D1-D2) of the LILRB1 ectodomain (SEQ ID NO: 42). Unlike the reference Ab (Reference Antibody 1), the five selected clones bound only to the full-length LILRB1 protein but not to its truncated form (Figure 4).

[0205] The binding ability of selected anti-LILRB-1 antibodies to the LILRB1 receptor in its native context on the cell membrane was examined by flow cytometry. HEK293 cells overexpressing full-length human LILRB1 (SEQ ID NO: 45) were incubated with increasing amounts of antibody, and cell-bound antibodies were detected using a flow cytometer with a PE-labeled secondary antibody. As shown in Figure 5, all five clones exhibited strong binding to LILRB1 embedded in the cell membrane, with calculated EC50 values ​​ranging from 0.37 nM to 2.24 nM; the reference antibody (Reference Antibody 1) exhibited an intermediate EC50 of 0.73 nM. "LILRB1 binding" indicates that the EC50 in this assay was lower than 100 nM.

[0206] HLA-G is the major ligand of LILRB1, which is known to be overexpressed in various tumors. We therefore tested the ability of selected anti-LILRB1 Abs to block the receptor's binding to its ligand. To do so, HEK293 cells overexpressing the human LILRB1 receptor were incubated with human HLA-G PE-labeled tetramer in the absence or presence of 500 nM test antibody. Cell-bound HLA-G was quantified by flow cytometry. As shown in Figure 6 , antibodies 1, 2, 3, 4, and 5 did not exhibit blocking activity at this high concentration. In contrast, the presence of a reference antibody (Reference Antibody 1) blocked HLA-G binding to cells. "Non-blocking activity" indicates that the signal in this assay was greater than 10% of that observed for the isotype control.

[0207] Binding to LILR family members. The binding ability of the selected antibodies to human LILRB2 was examined by flow cytometry. HEK293 cells overexpressing the full-length human LILRB2 receptor (SEQ ID NO: 46) were incubated with increasing amounts of anti-LILRB1 clones, and bound antibodies were detected by flow cytometry as described below in "Identification of LILRB1 non-neutralizing antibodies." Unlike Reference Antibody 1, all five selected antibodies of the present invention bind to LILRB2 with low nM affinity (Figure 7).

[0208] Recombinant full-length ectodomains of LILRA1 (SEQ ID NO: 47), LILRA2 (SEQ ID NO: 48), and LILRA3 (SEQ ID NO: 48) were used to test binding to the most similar LILRA family members by ELISA. As shown in Figure 8, antibodies 1-5 bind to LILRA3, unlike reference antibody 1. In this preliminary experiment, only antibody 3 was observed to bind to LILRA1, but subsequent ELISA revealed that antibodies 1-5 also bind to LILRA1. None of antibodies 1-5 bound to LILRA2. In this context, "binding" indicates that the signal obtained for the LILRA protein in this assay was at least three-fold higher than that observed for the control protein. To gain a more detailed understanding of the binding of clones 1-5 to individual family members, multipoint ELISAs were performed to assess the EC for binding. 50 It was decided that:

[0209] Binding to LILRB1 allelic forms. There are four major allelic variants within the ectodomain of LILRB1 in the human population [Human Molecular Genetics, 2005, Vol. 14, 2469-2480]: c.203T>C (L68P), c.277G>A (A93T), c.425T>C (I142T), and c.464G>T (S155I). Although they are located in domains 1 and 2, they may still affect the overall tertiary structure of the protein, thereby reducing the target population for therapy. Therefore, antibodies that bind equally well to all major allelic forms are preferred. To examine the ability of selected anti-LILRB1 antibodies to bind to allelic forms of human LILRB1, recombinant protein variants of the human LILRB1 ectodomain with amino acid substitutions corresponding to the combined first two allelic forms (SEQ ID NO: 50) or the combined third and fourth allelic forms (SEQ ID NO: 51) fused to mouse IgG2a Fc were expressed in mammalian cells and purified by protein A chromatography. These proteins were then used in ELISA. As shown in Figure 9, unlike reference antibody 1, the binding of antibodies 1-5 to the allelic variants of human LILRB1 was similar to that of the wild-type (wt) protein. In this context, "binding" indicates that the signal obtained for the LILRB1 variants in this assay was at least three-fold higher than that observed for the control protein.

[0210] Binding to non-human primate LILRB proteins. There are single LILRB1 homologs in rhesus monkeys (Macaca mulatta) (SEQ ID NO: 52) and cynomolgus monkeys (Macaca fascicularis) (SEQ ID NO: 53), respectively. Antibodies cross-reactive with nonhuman primate homologous proteins are preferred because they can be used in these species to examine the pharmacological properties of the antibodies. Recombinant rhesus and cynomolgus LILRB1 homologs were generated as fusions to mouse IgG2a (SEQ ID NO: 43 and SEQ ID NO: 44, respectively), and these proteins were used in ELISA. As shown in Figure 10, unlike reference antibody 1, antibodies 1-5 bind equally well to rhesus and cynomolgus LILRB proteins and human LILRB1. In this context, "binding" indicates that the signal obtained for the LILRB protein in this assay is at least three-fold higher than that observed for the control protein.

[0211] Human PBMC cytokine release assay In rare cases, infusion of therapeutic antibodies into humans can result in uncontrolled cytokine production from immune cells in the blood, a condition known as cytokine release syndrome (CRS). Therefore, antibodies that do not cause spurious activation of immune cells, resulting in cytokine release, are preferred. Clones 1-5 were tested in a standard cytokine release storm assay, in which PBMCs from two donors were incubated with 20 μg / ml of the test antibody for 24 hours. Various cytokines were then measured in the medium from the treated cells. As shown in Figure 11, none of clones 1-5 induced significant amounts of cytokine release, indicating that clones 1-5 are safe for use in humans.

[0212] Antibodies 1-5 bind to iPS-derived macrophages and NK92 cells Antibodies 1 to 5 were able to bind to iPS-derived macrophages (FIG. 12), and antibodies 2 to 5 were able to bind to NK92 cells (FIG. 13).

[0213] Non-blocking LILRB1 antibodies promote cancer cell phagocytosis in macrophages. Antibodies that were ligand blockers and also non-ligand blockers were advanced through a functional assay cascade to determine the biological performance of the antibodies (i.e., macrophage phagocytosis and reprogramming) in therapeutically relevant assays.

[0214] The inventors performed phagocytosis assays using an MHCI-deficient cell line (DLD1), an MHCI (HLA-ABC)-positive cell line (JURKAT), and an HLAG-overexpressing cell line (JURKAT HLAG).

[0215] Surprisingly, the inventors observed that antibodies 1-5 were superior to reference antibody 1, an anti-LILRB1 ligand-blocking antibody, in inducing higher levels of cancer cell phagocytosis. As can be seen in Figure 14, the ligand-blocking reference antibody 1 did not induce a significant increase in phagocytosis of DLD colorectal cancer cells, a cell line negative for MHC class I expression, by macrophages, whereas three out of five of the inventors' non-ligand-blocking antibodies (antibodies 1, 3, and 5) were able to induce phagocytosis in the absence of MHC class I ligands.

[0216] Altered MHC class I ligand expression is common in cancer, including immunotherapy-resistant diseases, for which there is an unmet need; the non-ligand blocking antibodies of the present invention are not limited by MHC-I expression in cancer and may offer new treatment options for patients.

[0217] Our antibodies were also able to induce phagocytosis in MHCI-positive cells better (Antibody 1) or equivalently (Antibodies 2-5) to Reference Antibody 1 (FIG. 14).

[0218] Remarkably, the antibodies produced by clones 1 to 5 were able to promote high levels of phagocytosis in macrophages without the need for any additional co-treatment, such as anti-CD47 or anti-EGFR antibodies, which have been reported as necessary by other authors (Barkal et al. ibid.; WO 2021222544 (NGM)).

[0219] Antibodies 1 to 5 promote macrophage reprogramming. Some studies have reported that blocking LILRB2 in macrophages reprograms them to an anti-tumor phenotype; LILRB2 antagonism resistants macrophages to humoral cytokine-dependent STAT6 activation by IL-4, alleviates the suppressive effect of macrophages on T cell proliferation, and reprograms human macrophages from the A549 lung tumor model and primary human non-small cell lung carcinoma. Furthermore, when used in combination with anti-PD-L1, LILRB2 blockade altered the tumor microenvironment and promoted anti-tumor immunity (Chen et al., JCI 2018).

[0220] LILRB1 blockade has been associated with improved phagocytosis in macrophages (Barkal et al.) and NK cytotoxicity (Chen et al., JITC, 2020), as well as changes in macrophage activation markers after differentiation from monocytes in the presence of LILRB1 antibodies.

[0221] As shown in Figure 16, antibodies 1-5, but not the anti-LILBR1 reference antibody 1, were able to strongly induce GM-CSF release (a marker of macrophage reprogramming) in macrophages upon LPS stimulation; among all antibodies tested, antibody 2 promoted GM-CSF production by nearly 5-fold compared to the IgG4 isotype control.

[0222] Antibodies 2 and 4 were also able to induce significantly higher levels of the pro-inflammatory cytokine TNFα in iPS-derived macrophages upon LPS stimulation (FIG. 16).

[0223] When macrophages were incubated with antibodies 1 to 5 in the absence of LPS stimulation, no reprogramming effect was observed (Figure 17).

[0224] As shown in Figure 18, the reprogramming effect was also confirmed when primary monocyte-derived macrophages were used.

[0225] Non-blocking antibodies 1 to 5 were able to induce phagocytosis of MHCI-negative and -positive cancer cell lines and induce macrophage reprogramming.

[0226] Consideration Antibodies 1–5 were identified that specifically bind to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6, respectively, on human macrophages. LILRB1 and LILRB2 are key signaling receptors used by tumor-associated macrophages to mediate immunosuppression in solid tumors. LILRB1 and LILRB2 share a common ligand, and both receptors transmit inhibitory signals to immune cells via their ITIM motifs. Furthermore, the inhibitory function of LILRB3 in monocytes and macrophages has recently been elucidated (Yeboah et al. JCI 2020). Therefore, an approach to simultaneously targeting the three inhibitory receptors may offer the advantage of avoiding redundant escape mechanisms in the tumor microenvironment. Furthermore, dual-binding antibodies provide broader coverage of the TAM population because TAMs differ in their LILRB1 and LILRB2 expression patterns: our analysis of several solid tumor types (Figure 2) shows that although the majority of TAMs co-express LILRB1 and LILRB2, there are detectable TAMs that express only LILRB1 or LILRB2. Thus, the dual LILRB1 / 2-binding properties of antibodies 1-5 allow for complete coverage of single LILRB1-, single LILRB2-, or dual LILRB1- and LILRB2-expressing immune cells.

[0227] LILRA3 is a soluble factor with anti-inflammatory activity. Unlike LILRB1 and LILRB2, LILRA3 is biologically active in its soluble form. The antibodies of the present invention bind to LILRA3 and may benefit from targeting additional receptors in the LILR family member community.

[0228] The prior art contributes to the specific mechanisms required to achieve antitumor phagocytic activity and macrophage reprogramming. In particular, LILRB1 inhibition of its interaction with MHCI, primarily HLA-G and SS2M, is associated with enhanced phagocytic activity by macrophages. It is postulated that blocking the formation of the HLAG-SS2M-LILRB1 complex is required to achieve such activity.

[0229] Here, we report a novel mechanism for enhancing phagocytic activity by nonligand-blocking anti-LILRB1 antibodies: these antibodies enhance phagocytic activity in the absence of MHCI, as shown using the MHCI-deficient DLD1 cell line.

[0230] conclusion Finally, we report a novel class of human LILRB1 / LILRB2 / LILRB3 / LILRA1 / LILRA3 / LILRA4 / LILRA6-targeting antibodies that exhibit potent reprogramming activity without blocking ligand-receptor interactions.

[0231] Neutralization of HLA binding to LILRB1 and LILRB2 As demonstrated above, unlike the reference antibody, antibodies 1-5 did not efficiently neutralize HLA-G binding to LILRB1, even at high concentrations. To examine whether antibodies 1-5 could neutralize other HLA-A binding to LILRB1 or LILRB2, HEK293 cells overexpressing this receptor were incubated with a fixed concentration of PE-labeled HLA-A2, HLA-E, and HLA-G multimers and increasing concentrations of antibodies 1-5 or reference antibody. The ability to neutralize HLA binding to cells was determined by flow cytometry (Figure 21). None of antibodies 1-5 neutralized HLA-A(A), HLA-E(B), and HLA-E(C) binding to LILRB1 protein at concentrations up to 100 nM, whereas reference 1 antibody (specific for LILRB1) and reference 3 antibody (specific for LILRB1 / 2) showed IC values ​​in the sub-nM or low nM range. 50 Similarly, none of antibodies 1 to 5 efficiently neutralized the binding of HLA-A(E) and HLA-G(G). Antibody 1 neutralized the binding of HLA-E(F) to LILRB2, but antibody clones 2 to 5 did not neutralize the binding of HLA-E(F) to LILRB2. Reference antibody 2 (specific for LILRB2) and reference antibody 3 (specific for LILRB1 / 2) had IC values ​​in the sub-nM or low nM range. 50 inhibited binding to all HLA.

[0232] Reprogramming immunosuppressed MDMs Human normal (M0) or immunosuppressed (M2) monocyte-derived macrophages (grown in the presence of 50 ng / ml IL-10 and 50 ng / ml TGFβ) were incubated with 10 μg / ml of antibodies 1–5 or isotype control or reference antibodies 1–3 for 1 hour, and then stimulated with 1 ng / ml or 10 ng / ml of LPS for 5 hours for M0 and M2 conditions, respectively. TNFα levels were measured by ELISA (Figure 22). As previously shown, normal MDMs in the presence of CSF-1 (A) could be reprogrammed with reference antibody 2 (a LILRB2-specific Ab), reference antibody 3 (a dual LILRB1 / LILRB2-specific Ab), and antibodies 1–5, but not with the isotype control or reference antibody 1 (a LILRB1-specific Ab). In contrast, in the presence of two major immunosuppressive cytokines, CSF-1 and IL-10, and TGFβ (B), none of the reference Abs were able to induce further secretion of TNFα, whereas clones 2 and 4-5 increased TNFα levels, indicating that these clones can reprogram highly immunosuppressed macrophages, which are a model for TAMs present in the tumor microenvironment.

[0233] immunogenicity The immunogenicity of the VH and VL sequences of antibodies 1-5 was evaluated by the CRO Abzena using their proprietary in silico technologies: iTope-AI and TCED™ (Figure 23). The two methods estimated that antibodies 1-5 have an average risk of inducing immunogenicity in humans, comparable to other fully human therapeutic Abs and to a lesser extent than murine, chimeric, or humanized therapeutic antibodies.

[0234] Epitope mapping PEPperPRINT CRO used their proprietary PEPperCHIP® linear and "conformational" peptide microarrays to map putative epitopes on the D3-D4 region of the human LILRB1, LILRB2, and LILRA3 molecules for antibodies 1-5 ( FIG. 24 ). Strong binding to the peptide microarray was observed only for clone 2, suggesting that it binds to a relatively unstructured epitope. The two putative epitopes present in all three target proteins corresponded to the peptides: epitope 1 in LILRB1 (sequence FVLYKDGERDF (SEQ ID NO: 80)), sequence GYDRFVLYKEGERD (SEQ ID NO: 81) in LILRB2, and sequence YDRFVLYKEWGRD (SEQ ID NO: 82) in LILRA3) and epitope 2 (sequence SSEWSAPSDPLD (SEQ ID NO: 83) in LILRB1, sequence ECSAPSDPLDI (SEQ ID NO: 84) in LILRB2, and sequence SEWSAPSDPLD (SEQ ID NO: 85) in LILRA3). Binding was also observed to additional putative epitopes: epitope 3 (sequence LQCVSDVGYD (SEQ ID NO: 86) in LILRB2 and sequence FQCGSDAGYDRF (SEQ ID NO: 87) in LILRB3) and epitope 4 (sequence FLLTKEGAADDPW (SEQ ID NO: 88) in LILRB1 and sequence AADAPLRLRSIHEY (SEQ ID NO: 89) in LILRB2). Binding to similar peptides was observed for Antibody 1, albeit with weaker signals. Antibody 4 was seen to bind to two putative epitopes; epitope 5 (sequence RSYGGQYR (SEQ ID NO: 90) in LILRB1 and sequence PVSRSYGGQYRC (SEQ ID NO: 91) in LILRB2). Weak binding to the peptide array was observed for antibody 5, suggesting one putative epitope: epitope 6 (sequence LDILIAGQFYD (SEQ ID NO: 92) in LILRB1, sequence APSDPLDILI (SEQ ID NO: 93) in LILRB2, and sequence PSDPLDILI (SEQ ID NO: 94) in LILRA3. No significant binding to the peptide array was observed for antibody 3.

[0235] MDM reprogramming using F(ab')2 dimers and Fab monomers F(ab')2 dimers and Fab monomers were prepared from the IgG4P and IgG1 variants of Antibody 2 using the FabRICATOR and FabALACTICA kits, respectively, according to the manufacturer's protocols. Purified Ab fragments were used in macrophage reprogramming assays as previously described.

[0236] Treatment of M0 macrophages with the F(ab')2 fragment of Antibody 2 for 6 hours resulted in partial activation of the cells, as measured by TNFα release, when compared with the effects of intact Antibody 3Ab and an isotype control. Similar treatment of M2 macrophages did not result in activation of the cells beyond that observed with the isotype control (Figure 25).

[0237] Treatment of M0 macrophages with the Fab fragment of Antibody 3 for 6 hours did not result in enhanced activation (assessed as production of TNFα) when compared to isotype control production of TNFα (FIG. 26).

[0238] These results indicate that Antibody 2 requires bivalent binding and Fc receptor engagement on the macrophage membrane for optimal reprogramming activity, especially under immunosuppressive M2 conditions.

[0239] In the M2-like suppression assay, monocytes were isolated from three cryopreserved PBMC donors and cultured with M-CSF and a specific cytokine cocktail (IL-4, IL-10, and TGF-β) to obtain M2-like macrophages. For the final 4 hours of polarization, M2-like macrophages were activated with LPS. At the end of polarization / activation, expression of CD163, CD209, CD206, CD86, LILRB1, and LILRB2 was assessed by flow cytometry. Macrophages were then washed and plated in quintuplicate in 96-well plates. After overnight incubation, CD4+ T cells were added to the plates at a 1:5 macrophage:CD4+ T cell ratio. T cells in this coculture were then activated by the addition of CD3 / CD28 ImmunoCult™ (STEMCELL Technologies) in the presence of a single concentration (10 μg / mL) of the test antibody, one concentration (10 μg / mL) of OPDIVO, and a human IgG4 isotype control and reference antibody and corresponding isotype control. On day 5 of coculture, supernatants were collected and selected IFN-gamma was assessed by ELISA. T cell proliferation was assessed by flow cytometry (proliferation dye dilution).

[0240] Supernatants were tested by ELISA to measure IFN-gamma release. As reported in Figure 27, the isotype control showed a small but significant increase, while the inventors observed significant IFN-gamma release using Antibody 2, Antibody 3, and Antibody 4; interestingly, the significant IFN-gamma production observed with these three antibodies was superior to the reference antibodies tested. Antibody 2 was tested in different IgG versions (IgG1, IgG4, LALA), and no significant difference was observed between IgG1 and IgG4, while the LALA version showed a decrease in IFN-gamma production.

[0241] T cell proliferation and expansion were examined by flow cytometry. For T cell proliferation, T cells undergoing proliferation at the start of the assay were assessed. For expansion cultures, the total proliferation index was assessed, which included T cells that had not yet proliferated at the start of the assay. The T cell expansion results (Figure 28) showed that Antibody 2 significantly outperformed the remaining antibodies tested. Antibody 2 was tested in different IgG versions (IgG1, IgG4, LALA) and no significant differences were observed between the IgG1 and IgG4 versions, while the LALA version showed a decrease in T cell expansion.

[0242] NKL killing assay NKL is a human natural killer (NK) cell line established from the peripheral blood of a patient with CD3-, CD16+, CD56+, large granular lymphocytes (LGLs) and kindly provided by Professor Werner Held (University of Lausanne, Switzerland).

[0243] Jurkat and Jurkat HLA-G overexpressing cells were used as targets in the cytolytic cell assay. Target cells were labeled with CellTracker Deep Red (ThermoFisher) to distinguish them from NKL cells and grown at 5 × 10 in assay medium. 5 cells / ml.

[0244] 1x10 cells in assay medium (RPMI 1640 with GlutaMAX, 10% human AB serum, 1% penicillin / streptomycin, 1 mM sodium pyruvate, 1000 U / ml recombinant human IL-2 (rhIL-2)). 6NKL cells were suspended at 100 cells / ml, and 100 μl of the NKL cell suspension was added to each well of a 96-well plate. Anti-LILRB1 and / or anti-LILRB2 antibodies and isotype controls were simultaneously added to corresponding wells at a final concentration of 10 μg / ml in a final volume of 200 μl. NKL cells were incubated with different treatments at 37°C for 1 hour, followed by the addition of 100 μl of target cells to the corresponding wells, resulting in a target NKL:1 ratio. The plates were incubated at 37°C for 3 hours, then centrifuged at 200×g for 3 minutes at room temperature, and the medium was removed.

[0245] Each well was resuspended in 100 μL of FACS buffer (2% BSA, 5 mM EDTA-DPBS), and CD56-FITC antibody was added for identification of NKLs by flow cytometry and incubated for 30 minutes at 4°C. After antibody incubation, 100 μL of FACS buffer was added per well, the antibody was washed off, and the cells were spun down at 200×G for 3 minutes, and the supernatant was removed. Cells were then resuspended in FACS buffer containing a 1:1,000 dilution of Sytox Blue (ThermoFisher) to stain cells with defective membranes so that viable cells could be distinguished from dead or damaged cells.

[0246] As shown in Figures 29 and 30, the anti-LILRB1 and / or anti-LILRB2 antibodies of the present invention enhanced NKL cytolytic activity compared to the isotype control on wild-type Jurkat cells. However, this enhanced cytolytic activity was observed to be greater in the co-culture with Jurkat cells overexpressing HLA-G.

[0247] These results suggest that anti-LILRB1 and anti-LILRB1 / LILRB2 antibodies can block the interaction between LILRB1 on NKL cells and MHC I molecules on the surface of target cells, thereby enhancing the killing ability of NKL.

[0248] Binding of clones 1 to 5 to all LILR family members tested by single-point ELISA (Figure 31). Recombinant full-length ectodomains of LILRA1 (SEQ ID NO: 47), LILRA2 (SEQ ID NO: 48), LILRA3 (SEQ ID NO: 48), LILRA4 (SEQ ID NO: 95), LILRA5 (SEQ ID NO: 96), LILRA6 (SEQ ID NO: 97), LILRB1 (SEQ ID NO: 98), LILRB2 (SEQ ID NO: 99), LILRB3 (SEQ ID NO: 100), LILRB4 (SEQ ID NO: 101) and LILRB5 (SEQ ID NO: 102) were used to test binding to other LILR family members by ELISA.

[0249] Recombinant LILRB1-5 and LILRA1 (A) or LILRB1 and LILRA2-6 (B) were coated onto ELISA plates. Plates were then blocked with milk in PBST. Plates were then incubated with 1 nM of clones 1-5, references 1-3, or an IgG4 isotype control. Bound antibodies were detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate.

[0250] Clones 1 to 5 bind to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6.

[0251] [Table 14]

[0252] As shown in Figures 31A and B, antibody clones 1-5 each bound to LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6. At the same time, reference antibodies 1-3 showed more restricted binding specificity. In this context, "binding" indicates that the signal obtained for the LILR protein in this assay was at least three times higher than that observed for the control protein. To understand the binding of antibodies 1-5 to individual family members in more detail, multipoint ELISA was performed to measure the EC for binding. 50 It was decided that:

[0253] Binding of clones 1-5 to selected LILR family members tested by multipoint ELISA (Figure 32). Recombinant LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, LILRA6 or an unrelated control protein was coated onto an ELISA plate. The plate was then blocked with milk in PBST. The plate was then incubated with serial dilutions (12.5 nM to 0.02 nM) of clones 1-5 (A-E). Bound antibody was detected using an HRP-conjugated anti-human Fc antibody and a chromogenic substrate. The calculated apparent EC in nM for each of antibodies 1-5. 50 is shown in Table 11.

[0254] [Table 15]

[0255] Competition between Antibody 2 and other antibodies in ELISA (Figure 33) Antibodies 1-5 were all found to bind within the D3-D4 fragment of human LILRB1. To determine whether antibody 2 binds to an epitope similar to or different from that of the other antibodies (and reference antibodies), a competitive ELISA was performed. Plates were coated with human LILRB1, blocked, and then incubated with 5 nM biotinylated antibody 2 and different concentrations of each of unlabeled antibodies 1-5 and reference antibodies 1-3. Bound biotinylated antibody 2 was detected using HRP-conjugated streptavidin and a chromogenic substrate. Unlabeled antibody 2 showed a low nM IC 50 competed with their biotinylated counterparts at 1000 kJ / mL, whereas the IC of antibodies 1, 3, 4 and 5 50 The binding activity of antibody 2 was approximately 10 nM. Neither the reference Ab nor the isotype control was able to compete with antibody 2 at concentrations below 250 nM. These data indicate that antibody 2 binds to a unique epitope on LILRB1 that is distant from all epitopes of the reference Abs but close to the epitopes of antibodies 1, 3, 4, and 5.

[0256] Epitope mapping of Antibody 2 on human LILRB1 by HDX method (Figure 34) Experiments were performed using a Trajan automated platform and a Waters Cyclic IMS MS. For peptide mapping, recombinant LILRB1-avi-his was diluted to 10 μM in an HO-based buffer prior to the experiment. For peptide mapping experiments, 8.5 μL of protein solution was mixed with 41.5 μL of HO-based buffer. At the end of the reaction, 45 μL of sample was mixed with 45 μL of pre-dispensed quench buffer. The sample was further diluted with 90 μL of quench dilution solution. After dilution, 85 μL of sample was injected into the sample loop. Before capture and desalting, the sample was run through a Nap2 / pepsin or Pep / Protease XIII column for 210 seconds at a flow rate of 0.1 mL / min, followed by separation on an analytical C18 column at 0.035 mL / min.

[0257] For labeling experiments, three different time points were used (2, 10, and 60 min) in both the free and bound states. Both free and bound LILRB-avi-his samples were prepared at 10 μM.

[0258] Peptide libraries were prepared using PLGS, and DynamX 3.0 was used for deuterium uptake analysis.

[0259] H2O buffer: 20mM phosphate buffer, 150mM NaCl, pH7.4 DO / exchange buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.0 Quench buffer: 7.6M Ground HCl, 100mM phosphate buffer, 500mM TCEP, pH 2.3

[0260] Antibodies were incubated with recombinant human LILRB1-avi-his in DO, 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 for different times. The protein mixtures were then digested with protease and analyzed on a Leap HDX autosampler and a Waters Cyclic IMS MS instrument. (A) The relative changes in deuterium incorporation (lighter indicates less incorporation, darker indicates more incorporation) in proteolytic peptides after 2, 10, and 60 minutes (top-to-bottom bars below the sequence) are shown by a heat map below the sequence of LILRB1 (Figure 34). The putative epitope for Antibody 2 (sequence AEFPMGPVTSAHAGT ​​(SEQ ID NO: 78)) is underlined with a solid line, and another region potentially involved in Antibody 2 binding (sequence LTHPSDPLEL (SEQ ID NO: 79)) is underlined with a dashed line. (B) Alignment of all 11 human LILR family members with the putative epitope identified by HDX outlined in solid lines and other regions potentially involved in Antibody 2 binding outlined in dotted lines. LILR family members bound by Antibodies 1-5 are represented by dashed boxes. (C) Aligned 3D structures (surface view) of human LILRB1 (light gray) and LILRB2 (dark gray) with the putative epitope in domain 4 highlighted in black. (D) Ribbon representation of the LILRB1 (left) and LILRB2 (right) 3D structures with the putative Antibody 2 epitope in domain 4 highlighted in black.

[0261] MDM method Monocytes for monocyte-derived macrophage (MDM) differentiation were either isolated from fresh PBMCs by CD14-positive selection using the MACS isolation system (Miltenyi Biotec 130-050-201) or obtained commercially as cryopreserved peripheral blood monocytes (StemCell Technologies 70034 / 200-0166).

[0262] 1–2 in a 6-well UpCell plate (Thermo Scientific 174901) * 10 6MDMs were differentiated for 7–10 days in MDM medium (RPMI 1640 with Glutamax (Gibco 61870010) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies A3840402), 100 U / ml penicillin / streptomycin (Gibco 15140-122), and 100 ng / ml recombinant human M-CSF (Biolegend 574806)) by seeding at 10 monocytes / well. To polarize MDMs toward an immunosuppressive phenotype, 50 ng / ml each of recombinant human IL-10 (Peprotech 200-10) and TGF-β1 (Peprotech 100-21) were added at the time of seeding and for the entire duration of differentiation and subsequent assays.

[0263] For MDM reprogramming assays, after 6–12 days of differentiation, 31,000–94,000 cells / cm 2MDMs were replated in MDM medium at a density of 10 μg / ml. Immunosuppressed MDMs were supplemented with 50 ng / mL IL-10 and TGF-β1 as before. MDMs were pretreated with 10 μg / ml of monoclonal antibody for 1 hour before the addition of activating stimuli. The antibodies tested were Antibody 2, Reference Antibody 1, Reference Antibody 2, Reference Antibody 3, and Reference Antibody 7. Antibody 7 is an antibody specific for LILRB2. Human anti-HEL (hen egg lysozyme) IgG4P was used as an isotype control. Next, LPS was added at 1 ng / mL (resting MDM, M0) or 10 ng / mL (immunosuppressed MDM, M2), recombinant human IL-1β (Peprotech 200-01B) at 10 ng / mL, HMGB1 peptide (FKDPNAPKRLPSAFFLFCSE (SEQ ID NO: 105) generated by GenScript) at 30 μg / mL, c-di-AMP (Invivogen tlrl-nacda2r) at 10 μg / mL, poly(I:C) (Invivogen, LMW-tlrl-picw, HMW-tlrl-pic) at 10 μg / mL, and R848 / resiquimod (Invivogen tlrl-r848) at 0.5 μg / mL. Culture medium was collected 5 h after LPS addition or 24 h after the addition of all other stimuli. TNFα and GM-CSF release was measured by Duoset ELISA (R&D Systems).

[0264] Antibody 2 enhances TNFα secretion by human blood monocyte-derived macrophages (MDMs) upon induction with various stimuli (Figure 35). M0-polarized MDMs were incubated with 10 μg / ml of antibody 2 for 1 hour and then stimulated with various compounds: (A) 10 ng / ml of IL-1β, (B) 0.5 μg / ml of the TLR7 / TLR8 agonist R848, (C) 10 μg / ml of the TLR3 ligand LMW poly(I:C), (D) 10 μg / ml of the TLR3 ligand HMW poly(I:C), (E) the STING agonist c-di-AMP, and (F) 30 μg / ml of the TRL4 agonist HMGB1-derived peptide. Data shown are normalized to background.

[0265] Antibody 2 enhances TNFα secretion by MDM polarized to either M0 or M2 phenotypes (with IL-10 and TGFβ) co-cultured with A375 melanoma cells upon induction with LPS (Figure 36). A375 cells and MDMs were co-cultured for 4.5 hours, incubated with 10 μg / ml of antibody 2 for 1 hour, and then stimulated with LPS (1 ng / ml and 10 ng / ml for M0 and M2 MDMs, respectively) for 3.5 hours. Data presented are normalized to background.

[0266] References Barkal et al., “Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy”, Nat.Immunol.(2018)Jan;19(l):76-84. Chen et al JCI (2018)J Clin Invest.2018;128(12):5647-5662.https: / / doi.org / 10.1172 / JCI97570. Chen et al, JITC, (2020)Chen H, Chen Y, Deng M, et al.Antagonistic anti-LILRB1 monoclonal antibody regulates antitumor functions of natural killer cells.Journal for ImmunoTherapy of Cancer 2020;8:e000515. doi: 10.1136 / jitc-2019-000515. Coxon et al.Blood (2017)129 (26):3407-3418. Kontermann (2012)Mabs 4(2):182-97. Kuroki et al.Human Molecular Genetics, 2005, Vol.14, 2469-2480. Lopez-Yrigoyen, M., May, A., Ventura, T., Taylor, H., Fidanza, A., Cassetta, L., Pollard, J.W., Forrester, L.M.Production and Characterization of Human Macrophages from Pluripotent Stem Cells. J.Vis.Exp. (158), e61038, doi:10.3791 / 61038 (2020). Lopez-Yrigoyen, M., et al.A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen:A tool for the study and in vivo tracking of therapeutic cells.Philosophical Transactions of the Royal Society B:Biological Sciences.373, (1750), (2018). Lopez-Yrigoyen, M., et al.Genetic programming of macrophages generates an in vitro model for the human erythroid island niche.Nature Communications.10, (1), 881 (2019). Mulder et al.Immunity. 2021 Aug 10;54(8):1883-1900.e5. doi:10.1016 / j.immuni.2021.07.007. Epub 2021 Jul 30. Spiess et al.(2015)Mol Immunol 67:95-106. van Wilgenburg, B., Browne, C., Vowles, J., Cowley, SAEfficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partially-defined and fully-defined conditions.PLoS One.8, (8), 71098 (2013). Wang et al. (2020) Cell Mol Immunol. 2020 Sep;17(9):966-975. doi:10.1038 / s41423-019-0258-5. Epub 2019 Jul 4. Yeboah et al.JCI 2020 JCI Insight.2020 Sep 1;5(18):e141593.doi:10.1172 / jci.insight.141593.

[0267] Sequence Listing Information SEQ ID NO: 1 Antibody 1 HCDR1 DYYMS SEQ ID NO:2 Antibody 1 HCDR2 YISSSGSIKKYADSVKG SEQ ID NO:3 Antibody 1 HCDR3 TNWHFDY SEQ ID NO: 4 Antibody 1 LCDR1 RASQSVSSSYLA SEQ ID NO: 5 Antibody 1 LCDR2 GASTRAT SEQ ID NO: 6 Antibody 1 LCDR3 QQYYSTPFT SEQ ID NO:7 Antibody 1 VH QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSIKKYADSVKGRFTISRDNAKNSLYLQMNSLRGEDTAVYYCARTNWHFDYWGQGTLVTVSS SEQ ID NO:8 Antibody 1 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDVAVYYCQQYYSTPFTFGGPGTKVDIK SEQ ID NO: 9 Antibody 2 HCDR1 DYYMS SEQ ID NO: 10 Antibody 2 HCDR2 YISPSGSTIFYADSVKG SEQ ID NO: 11 Antibody 2 HCDR3 DRVRLFDY SEQ ID NO: 12 Antibody 2 LCDR1 RASQSVSSSYLA SEQ ID NO: 13 Antibody 2 LCDR2 GASTRAT SEQ ID NO: 14 Antibody 2 LCDR3 QQRSNWPIT SEQ ID NO: 15 Antibody 2 VH QVQLVESGGDLVKPGGSLRLSCAASGFTFRDYYMSWIRQAPGKGLEWVSYISPSGSTIFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKDRVRLFDYWGQGTLVTVSS SEQ ID NO: 16 Antibody 2 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTRLEIK SEQ ID NO: 17 Antibody 3 HCDR1 SYGIS SEQ ID NO: 18 Antibody 3 HCDR2 WISAYNGNTNYAQKLQG SEQ ID NO: 19 Antibody 3 HCDR3 SYSGSHWWFDP SEQ ID NO: 20 Antibody 3 LCDR1 KSSQSVLYSSNNNNYLA SEQ ID NO: 21 Antibody 3 LCDR2 WAFSRES SEQ ID NO: 22 Antibody 3 LCDR3 QQYYSTPPT SEQ ID NO: 23 Antibody 3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSYSGSHWWFDPWGQGTLVTVSS SEQ ID NO: 24 Antibody 3 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNNNYLAWYQRKPGQPPKLLINWAFSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPPTFGQGTKVEIK SEQ ID NO: 25 Antibody 4 HCDR1 SYGIS SEQ ID NO: 26 Antibody 4 HCDR2 WISAYNGNTNYAQKLQG SEQ ID NO: 27 Antibody 4 HCDR3 SYSGSYWWFDP SEQ ID NO: 28 Antibody 4 LCDR1 KSSQSVLYSSNNNNYLA SEQ ID NO: 29 Antibody 4 LCDR2 WASTRES SEQ ID NO: 30 Antibody 4 LCDR3 QQYYSTPLT SEQ ID NO: 31 Antibody 4 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSYSGSYWWFDPWGQGTLVTVSS SEQ ID NO: 32 Antibody 4 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNNNYLAWYQQKPGQPPKLLINWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK SEQ ID NO: 33 Antibody 5 HCDR1 DYYMT SEQ ID NO: 34 Antibody 5 HCDR2 YISISGITRYYADSVKG SEQ ID NO: 35 Antibody 5 HCDR3 DQTGYFDY SEQ ID NO: 36 Antibody 5 LCDR1 RASQSFSSNLA SEQ ID NO: 37 Antibody 5 LCDR2 GASTRAT SEQ ID NO: 38 Antibody 5 LCDR3 QQYINWPHT SEQ ID NO: 39 Antibody 5 VH QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWVSYISISGITRYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQTGYFDYWGQGTLVTVSS SEQ ID NO: 40 Antibody 5 VL DIVMTQSPATLSVSPGEKTTLSCRASQSFSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYINWPHTFGQGTKVEIK SEQ ID NO: 41 (human LILRB1 ectodomain fused to mIgG2a Fc) [ka] SEQ ID NO: 42 Human LILRB1 (D1-D2) fused to mouse IgG2a Fc [ka] SEQ ID NO: 43 Rhesus monkey (Macaca mulatta) LILRB ectodomain fused to mIgG2a Fc [ka] SEQ ID NO: 44 Cynomolgus monkey (Macaca fascicularis) LILRB ectodomain fused to mIgG2a [ka] SEQ ID NO: 45 Human full-length LILRB1 [ka] SEQ ID NO: 46 Human full-length LILRB2 [ka] SEQ ID NO: 47 Human LILRA1 ectodomain [ka] SEQ ID NO: 48 Human LILRA2 ectodomain [ka] SEQ ID NO: 49 Human full-length LILRA3 [ka] SEQ ID NO: 50 Human LILRB1 (L68P / A93T) ectodomain fused to mIgG2aFc [ka] SEQ ID NO: 51 Human LILRB1 (I142T / S155I) ectodomain fused to mIgG2aFc [ka] SEQ ID NO: 52 Rhesus monkey (Macaca mulatta) full-length LILRB [ka] SEQ ID NO: 53 Cynomolgus monkey (Macaca fascicularis) full-length LILRB [ka] Reference antibody 1 (anti-human LILRB1 Ab) SEQ ID NO: 54 Reference Antibody 1 VH DVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTSVTVSS SEQ ID NO: 55 Reference Antibody 1 VL DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK Reference antibody 2 (anti-human LILRB2 Ab) Reference antibody heavy chain comprising SEQ ID NO: 56 VH [ka] SEQ ID NO: 57 Reference antibody 2VL [ka] Uniprot reference sequences: human LILRB1 for B5 and human LILRA1 for A6 SEQ ID NO: 59 Human LILRB1 Uniprot D9IDM8 [ka] SEQ ID NO: 60 Human LILRB2 Uniprot Q8N423-2 [ka] SEQ ID NO: 61 Human LILRA3 Uniprot Q8N6C8-1 [ka] SEQ ID NO: 62 Human LILRA1 Uniprot O75019-1 [ka] SEQ ID NO: 63 Human LILRA2 Uniprot Q8N149-1 [ka] SEQ ID NO: 64 Human LILRB3 Uniprot O75022-1 [ka] SEQ ID NO: 65 Human LILRB4 Uniprot Q8NHJ6 (Isoform 1 is the canonical sequence) [ka] SEQ ID NO: 66 Human LILRB5 Uniprot O75023-1 [ka] SEQ ID NO: 67 Human LILRA4 Uniprot P59901-1 [ka] SEQ ID NO: 68 Human LILRA5 Uniprot A6NI73-1 [ka] SEQ ID NO: 69 Human LILRA6 Uniprot Q6PI73-1 [ka] Reference antibody 3 (anti-human LILRB1 and LILRB2 Ab) SEQ ID NO: 70 Reference antibody 3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGNVNPNDGGTTYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARREIYFYGTIYYYAMDYWGQGTLVTVSS SEQ ID NO: 71 Reference antibody 3 VL DIQLTQSPSFLSASVGDRVTITCRASESVDYYGNSFMYWYQQKPGKAPKLLIYFASNLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQNNEDPWTFGGGTKVEIK

[0268] Reference antibody 4 (anti-human LILRB1 Ab) SEQ ID NO: 72 Reference antibody 4 VH QVQLKESGPG LVAPSQSLSI TCTVSGFSLT SYGVSWVRQP PGKGLEWLGV IWGDGSTNYH SALISRLSIS KDNSKSQVFL KLNSLQTDDT ATYYCAKPRW DDYAMDYWGQ GTSVTVSS SEQ ID NO: 73 Reference antibody 4 VL DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQQ GNTLWTFGGG TKLEIK Reference antibody 5 (anti-human LILRB1 Ab) SEQ ID NO: 74 Reference antibody 5 VH QVQLQQPGAE LVKPGASVRM SCKASGYTFT SYWVHWVKQR PGQGLEWIGV IDPSDSYTSY NQNFKGKATL TVDTSSKTAY IHLSSLTSED SAVYFCARGE RYDGDYFAMD YWGQGTSVTV SS SEQ ID NO: 75 Reference antibody 5 VL DIVMTQSPAS LSVSVGETVT ITCRASENIY SNLAWYQQKQ GKSPQLLVYA ATNLADGVPS RFSGSRSGTQ YSLKINSLQS EDFGTYYCQH FWNTPRTFGG GTKLEIK Reference antibody 6 (anti-human LILRB1 Ab) SEQ ID NO: 76 Reference antibody 6 VH QVQLQQSGAE LVKPGASVRL SCKASGYTFT AHTIHWVKQR SGQGLEWIGW LYPGSGSIKY NEKFKDKATL TADKSSSTVY MELSRLTSED SAVYFCARHT NWDFDYWGQG TTLTVSS SEQ ID NO: 77 Reference antibody 6 VL NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YGGASYMNWY QQKPGQPPKL LIYAASNLES GIPARFSGSG SGTDLTLNIH PVEEEDAAMY YCQQSNEEPW TFGGGTKLEI K SEQ ID NO: 78 Epitope sequence in human LILRB1 AEFPMGPVTSAHAGT SEQ ID NO: 79 Epitope sequence in human LILRB1 LTHPSDPLEL SEQ ID NO: 80 Epitope sequence in human LILRB1 FVLYKDGERDF SEQ ID NO: 81 Epitope sequence in human LILRB2 GYDRFVLYKEGERD (SEQ ID NO: 81) in human LILRB2, and the sequence SEQ ID NO: 82 Epitope sequence in human LILRA3 YDRFVLYKEWGRD SEQ ID NO: 83 Epitope sequence in human LILRB1 SSEWSAPSDPLD SEQ ID NO: 84 Epitope sequence in human LILRB2 ECSAPSDPLDI SEQ ID NO: 85 Epitope sequence in human LILRA3 SEWSAPSDPLD SEQ ID NO: 86 Epitope sequence in human LILRB2 LQCVSDVGYD SEQ ID NO: 87 Epitope sequence in human LILRA3 FQCGSDAGYDRF SEQ ID NO: 88 Epitope sequence in human LILRB1 FLLTKEGAADDPW SEQ ID NO: 89 epitope sequence in human LILRB2; AADAPLRLRSIHEY SEQ ID NO: 90 Epitope sequence in human LILRB1 RSYGGQYR SEQ ID NO: 91 Epitope sequence in human LILRB2 PVSRSYGGQYRC in LILRB2, SEQ ID NO: 92 Epitope sequence in human LILRB1 LDILIAGQFYD SEQ ID NO: 93 Epitope sequence in human LILRB2 APSDPLDILI SEQ ID NO: 94 Epitope sequence in human LILRA3 PSDPLDILI SEQ ID NO: 95 LILRA4 recombinant, full length ectodomain (LILRA4 24-446) (Uniprot P59901) [ka] SEQ ID NO: 96 LILRA5 recombinant, full length ectodomain (LILRA5 42-268) (Uniprot A6NI73) [ka] SEQ ID NO: 97 LILRA6 recombinant, full length ectodomain (LILRA6 24-447) (Uniprot Q6PI73) [ka] SEQ ID NO: 98 LILRB1 recombinant, full length ectodomain [ka] SEQ ID NO: 99 LILRB2 recombinant, full length ectodomain (LILRB2 22-458) (Uniprot Q8N423.4) [ka] SEQ ID NO: 100 LILRB3 recombinant, full length ectodomain (LILRB3 24-443) (Uniprot aab68668) [ka] SEQ ID NO: 101 LILRB4 recombinant, full length ectodomain (LILRB4 17-257) (Uniprot AAH26309) [ka] SEQ ID NO: 102 LILRB5 recombinant, full length ectodomain (LILRB5 18-456) (Uniprot O75023) [ka] Reference antibody 7 (anti-human LILRB2 Ab) SEQ ID NO: 103 Reference Antibody 7 VH [ka] SEQ ID NO: 104 Reference antibody 7 VL [ka] SEQ ID NO: 105 HMGB1 peptide FKDPNAPKRLPSAFFLFCSE

Claims

1. An antigen-binding protein that can specifically bind to human LILRB1 and human LILRB2, which does not block the interaction between human LILRB1 and the HLA-G tetramer, and / or the interaction between human LILRB2 and the HLA-G tetramer, and is capable of reprogramming macrophages.

2. The antigen-binding protein according to claim 1, which can reprogram fully differentiated macrophages into an antitumor (pro-inflammatory) phenotype.

3. The antigen-binding protein according to claim 1, wherein reprogramming is indicated / detected by induction of a marker for macrophage reprogramming.

4. The antigen-binding protein according to claim 1, wherein reprogramming is indicated / detected by the release of pro-inflammatory cytokines from the macrophages after exposure of the macrophages to the antigen-binding protein and LPS stimulation.

5. The antigen-binding protein according to claim 1, wherein reprogramming is indicated / detected by the release of the pro-inflammatory cytokine TNF-alpha and / or GM-CSF from macrophages after exposure to the antigen-binding protein and LPS stimulation.

6. (a) Stimulating the production of GM-CSF and / or TNF-alpha in iPS-derived macrophages and / or primary monocyte-derived macrophages upon LPS stimulation. (b) Stimulating the production of GM-CSF and / or TNF-alpha in primary monocyte-derived macrophages expressing LILRB1 and LILRB2 upon LPS stimulation. (c) Stimulating the production of GM-CSF and / or TNF-alpha in human macrophages expressing LILRB1 and LILRB2 upon LPS stimulation. The antigen-binding protein according to claim 1, having one or more properties selected from the above.

7. (a) Induces phagocytosis, (b) Induce phagocytosis in the absence of the second signal, (c) Induce phagocytosis in the absence of a second antibody (e.g., anti-CD47 antibody or anti-EGFR antibody), (d) Induce phagocytosis in the absence of a second opsonized antibody (e.g., anti-CD47 antibody or anti-EGFR antibody), (e) Induce phagocytosis of cancer cells in the absence of a second opsonized antibody (e.g., a tumor-binding antibody), and (f) Induces phagocytosis of MHC class I-positive and / or MHC class I-negative cancer cells. The antigen-binding protein according to claim 1, having one or more properties selected from the ability to do so.

8. (a) Human LILRB1, human LILRB2, and human LILRA3; (b) Human LILRB1, human LILRB2, human LILRA3 and human LILRA1; (c) Human LILRB1, Human LILRB2, Human LILRB3, Human LILRA3, Human LILRA4 and Human LILRA6; and / or (d) Human LILRB1, Human LILRB2, Human LILRB3, Human LILRA1, Human LILRA3, Human LILRA4 and Human LILRA6 The antigen-binding protein according to claim 1, which is specifically capable of binding to the antigen.

9. The antigen-binding protein according to claim 1, which does not bind to human LILRB4, human LILRB5, human LILRA2, or human LILRA5.

10. The antigen-binding protein according to claim 1, wherein binding is evaluated by flow cytometry or ELISA.

11. The antigen-binding protein according to claim 1, which is capable of specifically binding to rhesus monkey and / or cynomolgus monkey homologs of the human LILRB1 or LIRB2 external domain.

12. (a) Human LILRB1, LILRB2, and LILRA3; (b) Human LILRB1, LILRB2, LILRA3 and LILRA1; (c) Human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4 and LILRA6; and / or (d) Human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4 and LILRA6 The antigen-binding protein according to claim 1, which binds to an epitope common to the above.

13. (a) Human LILRB1, LILRB2, and LILRA3; (b) Human LILRB1, LILRB2, LILRA3 and LILRA1; (c) Human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4 and LILRA6; and / or (d) Human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4 and LILRA6 The antigen-binding protein according to claim 1, which binds to an epitope common to the above.

14. Epitope, (a) Human LILRB1 sequence AEFPMGPVTSAHAGT ​​(SEQ ID NO: 78); (b) Human LILRB1 sequences AEFPMGPVTSAHAGT ​​(SEQ ID NO: 78) and LTHPSDPLEL (SEQ ID NO: 79) Formed by, The aforementioned epitopes are mapped using hydrogen-deuterium exchange (HDX) mass spectrometry. The antigen-binding protein according to claim 1.

15. Epitope, (a) Sequence FVLYKDGERDF in human LILRB1 (SEQ ID NO: 80), sequence GYDRFVLYKEGERD in human LILRB2 (SEQ ID NO: 81), and sequence YDRFVLYKEWGRD in human LILRA3 (SEQ ID NO: 82); (b) Sequence SSEWSAPSDPLD in LILRB1 (SEQ ID NO: 83), sequence ECSAPSDPLDI in LILRB2 (SEQ ID NO: 84), and sequence SEWSAPSDPLD in LILRA3 (SEQ ID NO: 85); (c) Sequence LQCVSDVGYD in LILRB2 (SEQ ID NO: 86) and sequence FQCGSDAGYDRF in LILRA3 (SEQ ID NO: 87); (d) Sequence FLLTKEGAADDPW (SEQ ID NO: 88) in LILRB1 and sequence AADAPLRRRSIHEY (SEQ ID NO: 89) in LILRB2; (e) Sequence RSYGGQYR in LILRB1 (SEQ ID NO: 90) and sequence PVSRSYGGQYRC in LILRB2 (SEQ ID NO: 91); or (f) Sequence LDILIAGQFYD in LILRB1 (SEQ ID NO: 92), sequence APSDPLDILI in LILRB2 (SEQ ID NO: 93), and sequence PSDPLDILI in LILRA3 (SEQ ID NO: 94) Formed by, The epitope is mapped using binding to a peptide microarray. The antigen-binding protein according to claim 1.

16. The antigen-binding protein according to claim 1, which is an antibody or an antigen-binding fragment thereof.

17. The antigen-binding protein according to claim 1, which is a human antibody or an antigen-binding fragment thereof.

18. The antigen-binding protein according to claim 1, which is a monoclonal antibody, for example, a human monoclonal antibody.

19. The antigen-binding protein according to claim 1, comprising Fc.

20. (a) Antibody 1 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; (b) Antibodies 2 of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; (c) Antibodies 3 of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22; (d) Antibodies 4 of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30; and (e) Antibodies 5 of SEQ ID NOs: 33, 34, 35, 36, 37 and 38 Six CDRs of antibodies selected from (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively) Including; The aforementioned array is defined using Kabat naming conventions. The antigen-binding protein according to claim 1.

21. (a) Antibody 1 of SEQ ID NO: 7 and SEQ ID NO: 8; (b) Antibodies 2 of SEQ ID NO: 15 and SEQ ID NO: 16; (c) Antibody 3 of Sequence ID No. 23 and Sequence ID No. 24; (d) Antibodies 4 of SEQ ID NO: 31 and SEQ ID NO: 32; and (e) Antibodies 5 of Sequence ID No. 39 and Sequence ID No. 40 It comprises VH and VL of the antibody selected from; The aforementioned array is defined using Kabat naming conventions. The antigen-binding protein according to claim 1.

22. An antigen-binding protein, such as a human antibody or its antigen-binding fragment, that is competitive with the antigen-binding protein described in claim 1, such as an antibody or its antigen-binding fragment, for binding to human LILRB1, human LILRB2 and / or human LILRA3.

23. The antigen-binding protein, e.g., a human antibody or its antigen-binding fragment, according to claim 22, wherein competition for binding is evaluated using a competitive assay selected from cell-based binding assays, cell-free binding assays, immunoassays, ELISA, HTRF, flow cytometry, fluorescence microvolume assay (FMAT) assays, Mirrorball, high-content imaging-based fluorescence immunoassays, radioligand binding assays, biolayer interferometry (BLI), surface plasmon resonance (SPR), and thermal shift assays.

24. A composition comprising the antigen-binding protein described in claim 1 and a diluent.

25. (a) For use as a medicine; (b) For use as a drug for the treatment of cancer; (c) For use in cancer treatment; (d) For use in the manufacture of drugs for the treatment of cancer; (Optionally, cancer (b), (c), or (d) is selected from the following: (i) Acute myeloid leukemia (AML), urothelial carcinoma of the bladder (BLCA), low-grade glioma of the brain (LGG), invasive breast cancer (BRCA), esophageal cancer (ESCA), glioblastoma multiforme (GBM), squamous cell carcinoma of the head and neck (HNSC), clear cell carcinoma of the kidney (KIRC), papillary renal cell carcinoma of the kidney (KIRP), hepatocellular carcinoma of the liver (LIHC), adenocarcinoma of the lung (LUAD), squamous cell carcinoma of the lung (LUSC), Pancreatic adenocarcinoma (PAAD), sarcoma (SARC), cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), endometrial carcinoma (UCEC), uveal melanoma (UVM), colorectal cancer, prostate cancer, childhood cancers, lymphomas and leukemias, such as DLBCL, NHL, multiple myeloma, and Hodgkin lymphoma; (ii) Cancer that is positive for LILRB1 or LILRB2, or positive for both LILRB1 and LILRB2; (iii) Cancers that are positive for immunosuppressive macrophages (as measured by CD163 or CD68 positivity) and / or tumor-infiltrating T cells; (iv) Cancers in which classical or non-classical MHC class I expression is increased or decreased; (v) Cancer that is positive for one or more of LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4, and LILRA6; (e) For use as a drug for the treatment of immunosuppressive diseases; (f) For use in the treatment of immunosuppressive diseases; or (g) For use in the manufacture of drugs for the treatment of immunosuppressive diseases, An antigen-binding protein according to any one of claims 1 to 23, or the composition according to claim 24.

26. An antigen-binding protein according to any one of claims 1 to 23 or a composition according to claim 24 for use in a method of treating cancer or an immunosuppressive disease, comprising administering the antigen-binding protein according to any one of claims 1 to 23 or the composition according to claim 24 to a target.

27. An isolated recombinant DNA or RNA sequence comprising a sequence encoding the antigen-binding protein described in claim 1.

28. The isolated recombinant DNA sequence according to claim 27, wherein the vector is optionally an expression vector.

29. The isolated recombinant DNA sequence according to claim 27, which encodes the antigen-binding protein under the control of a promoter.

30. A host cell comprising a DNA or RNA sequence according to any one of claims 27 to 29, wherein the host cell is optionally capable of expressing an antigen-binding protein according to any one of claims 1 to 23.

31. A method for producing an isolated antigen-binding protein, comprising culturing the host cells described in claim 30 under conditions suitable for the expression of an isolated antibody or its antigen-binding fragment.

32. A method for identifying an antigen-binding protein according to any one of claims 1 to 23, (a)(i) Human LILRB1, LILRB2 and / or LILRA3; (ii) Human LILRB1, LILRB2, and LILRA3 proteins; (iii) Human LILRB1, LILRB2, LILRA1 and LILRA3 proteins; (iv) Human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4 and LILRA6 proteins; and / or (v) Human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4 and LILRA6 proteins To provide one or more antigen-binding proteins capable of binding to the target; (b), (c), and (d): (b) Evaluate the ability of one or more antigen-binding proteins to modulate one or more biological activities / phenotypes of human macrophages, such as by promoting phagocytosis and / or the release of pro-inflammatory cytokines (e.g., TNF-alpha or GM-SCF) or the expression of macrophage activation markers (e.g., HLA-DR and / or CD80), (c) Evaluate the ability of one or more antigen-binding proteins to block the binding of LILRB1 and / or LILRB2 to cells expressing a ligand for LILRB1 and / or LILRB2, e.g., HLA-G, and select one or more antibodies that bind to LILRB1, LILRB2 and LILRA3 and do not block the binding of LILRB1 and / or LILRB2 to target cells expressing a ligand for LILRB1 and / or LILRB2, e.g., HLA-G; (d) Evaluate the ability of another antigen-binding protein to block the binding of a ligand (e.g., HLA-G) to cells expressing LILRB1 and / or LILRB2, and select one or more antibodies that do not block the binding of the ligand of LILRB1 and / or LILRB2, e.g., HLA-G, to cells expressing LILRB1 and / or LILRB2. To conduct one or more evaluations selected from the following: (e) Select one or more antigen-binding proteins that can specifically bind to human LILRB1 and human LILRB2, wherein the antigen-binding proteins do not block the interaction between human LILRB1 and the HLA-G tetramer and can reprogram macrophages, and optionally, (f) Formulating the one or more antigen-binding proteins into a composition together with one or more excipients, A method that includes this.

33. A method for identifying an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 23, (a)(i) Human LILRB1, LILRB2 and / or LILRA3 proteins; (ii) Human LILRB1, LILRB2, and LILRA3 proteins; (iii) Human LILRB1, LILRB2, LILRA1 and LILRA3 proteins; (iv) Human LILRB1, LILRB2, LILRB3, LILRA3, LILRA4 and LILRA6 proteins; and / or (v) Human LILRB1, LILRB2, LILRB3, LILRA1, LILRA3, LILRA4 and LILRA6 proteins To provide one or more antibodies or antigen-binding fragments thereof that can bind to the target; (b), (c), and (d): (b) Evaluate the ability of another antibody or its antigen-binding fragment to block the binding of LILRB1 and / or LILRB2 to cells expressing the ligand for LILRB1 and / or LILRB2, e.g., HLA-G, and select one or more antibodies that bind to LILRB1, LILRB2 and LILRA3 and do not block the binding of LILRB1 and / or LILRB2 to target cells expressing the ligand for LILRB1 and / or LILRB2, e.g., HLA-G; (c) Evaluate the ability of another antibody or its antigen-binding fragment to block the binding of a ligand (e.g., HLA-G) to cells expressing LILRB1 and / or LILRB2, and select one or more antibodies that do not block the binding of the ligand for LILRB1 and / or LILRB2, e.g., HLA-G, to cells expressing LILRB1 and / or LILRB2; (d) Evaluate the ability of one or more antibodies or their antigen-binding fragments to modulate one or more biological activity / phenotypes of human macrophages, for example, to promote phagocytosis and / or the release of pro-inflammatory cytokines (such as TNF-alpha or GM-SCF) or the expression of macrophage activation markers (such as HLA-DR and / or CD80). To conduct one or more evaluations selected from the following: Methods that include...

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