Cross-specific antigen-binding proteins (ABPs) targeting leukocyte immunoglobulin-like receptor subfamily Bl (LILB1) and LILB2, combinations and uses thereof

Abstract

The present invention relates to antigen-binding proteins, such as antibodies, that bind to both leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and LILRB2, but do not bind, or bind with significantly lower affinity, to leukocyte immunoglobulin-like receptor subfamily A (LILRA), which can also inhibit the interaction between LILRB1 and / or LILRB2 and the natural ligands of the LILRB receptors on immune cells (interacting proteins such as HLA-G); inhibition of such interactions can reduce the suppression of immune cells, thereby supporting anti-infection and anti-tumor immune responses in subjects suffering from such diseases. In particular, the present invention provides products, compositions, and methods for treating diseases using LILRB1 and / or LILRB2 antigen-binding proteins, particularly antigen-binding proteins that preferably bind specifically to LILRB1 and LILRB2, but do not further bind, or bind with lower affinity, to one or more LILRA receptor proteins (such as LILRA1 and / or LILRA3). Certain related aspects, including methods of reducing immunosuppression of cells involved in cell-mediated immune responses and / or methods of treating infectious and / or proliferative diseases, as well as detection, diagnostic and screening methods, using LILRB1 and / or LILRB2 antigen binding proteins, such as antibodies that bind both LILRB1 and / or LILRB2, are also provided.

Description

[Technical Field]

[0001] The present invention relates to antigen-binding proteins, such as antibodies, that bind to both leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and LILRB2, but do not bind, or bind with significantly lower affinity, to leukocyte immunoglobulin-like receptor subfamily A (LILRA), which can also inhibit the interaction between LILRB1 and / or LILRB2 and the natural ligands of the LILRB receptors on immune cells (interacting proteins such as HLA-G); inhibition of such interactions can reduce the suppression of immune cells, thereby supporting anti-infection and anti-tumor immune responses in subjects suffering from such diseases. In particular, the present invention provides products, compositions, and methods for treating diseases using LILRB1 and / or LILRB2 antigen-binding proteins, particularly antigen-binding proteins that preferably bind specifically to LILRB1 and LILRB2, but do not further bind, or bind with lower affinity, to one or more LILRA receptor proteins (such as LILRA1 and / or LILRA3). Certain related aspects, including methods of reducing immunosuppression of cells involved in cell-mediated immune responses and / or methods of treating infectious and / or proliferative diseases, as well as detection, diagnostic and screening methods, using LILRB1 and / or LILRB2 antigen binding proteins, such as antibodies that bind both LILRB1 and / or LILRB2, are also provided. [Background technology]

[0002] In the treatment of cancer, there are several approaches that can result in the elimination of tumor cells, including those that involve or utilize one or more components of the immune system, either directly or indirectly. One limitation associated with such treatments is that cancerous cells often utilize immune checkpoints to evade the patient's immune system, such as by preventing immune recognition or downregulating tumor-specific cytotoxic T cell (CTL) responses, thereby developing resistance to the immune response (Rabinovich et al. 2007, Annu Rev Immunol 25:267; Zitvogel et al. 2006, Nat Rev Immunol 6:715). Under normal conditions, such immune regulatory checkpoints are important for maintaining self-tolerance under physiological conditions, but there is increasing recognition of the important role they may also play in cancer (Hanahan and Weinberg 2011, Cell; 144:646), and cancerous cells can take over these mechanisms to evade and suppress the immune system in order to develop into tumors (Drake et al 2006, Adv Immunol 90:51).

[0003] Current state-of-the-art cancer treatments involve blockade of a small number of known immune regulatory checkpoints, the mechanisms of which are understood. For example, blocking antibodies against surface-expressed immune regulatory proteins such as CTLA4 and PD-L1 (Chambers et al., 2001, Annu Rev Immunol 19:565; Blank et al., 2004, Cancer Res 64:1140) can boost antitumor immunity and have shown clinical success against many cancer types (Page et al., 2014, Annu Rev Med 65:185). However, the majority of cancer patients do not respond to such checkpoint blockade therapy (Bu et al., 2016, Trends Mol Med 22:448; Hugo et al., 2016, Cell 165:35; Topalian et al., 2012, New Engl J Med 366:2443), indicating that other immune checkpoint pathways may be active. Indeed, synergistic cooperation among several immunoregulatory pathways maintains immune tolerance to tumors, which may explain why blocking only one immunoregulatory checkpoint node can still result in tumor escape (Woo et al., 2012, Cancer Res 72:917; Berrien-Elliott et al., 2013, Cancer Res 73:605). However, little is known about the molecular factors central to the mechanisms of action of such immunoregulatory pathways. Indeed, successful cancer immunotherapy requires a systematic delineation of the entire immunoregulatory circuitry ("immunomodulators") expressed by tumors. Therefore, today, there remains an unmet need for identifying additional molecular targets that may function as immunoregulatory checkpoints, and for means and methods for modulating, detecting, and otherwise exploiting such potential checkpoint targets, particularly in medicine, diagnostics, and research.

[0004] Human leukocyte immunoglobulin-like receptors (LILRs), also known as the immunoglobulin-like transcript (ILT) family, belong to a superfamily of paired receptors that have the potential to transmit stimulatory or inhibitory signals according to the presence or absence of tyrosine-based signaling motifs in their cytoplasmic tails. Human LILRs consist of six stimulatory receptors (LILRA1-6) and five inhibitory receptors (LILRB1-5). LILRs are primarily expressed on myeloid and lymphoid cells, as well as some non-immune cells, and expression patterns vary among receptors. Polymorphisms and copy number variation contribute to intrahuman diversity. Receptor binding leads to intracellular phosphorylation of tyrosine-based motifs within the receptor (LILRB) or on associated adaptor molecules (LILRA). Downstream signaling events can be mediated by phosphatases such as SHP1, SHP2, and SHIP. In general, LILR activity can result in the up- or down-regulation of both innate and adaptive immune functions, with diverse effects on different cell types. Certain LILRs also play a regulatory role in neuronal activity and osteoclast development.

[0005] LILRB1 is broadly expressed on myeloid cells, as well as on subsets of B and T cells and natural killer (NK) cells. LILRB2-5 are restricted to myeloid cells and dendritic cells (DCs). Several of the ligands and signaling pathways for LILRB have been identified. LILRB1 and LILRB2 are the best-characterized receptors and bind to both classical (HLA-A, HLA-B, and HLA-C) and non-classical (HLA-E, HLA-F, HLA-G, and HLA-H) MHC class I or HLA class I molecules, as well as members of the angiopoietin-like protein family. Because the immunosuppressive function of LILRB is similar to that of the classical immune checkpoint proteins CTLA-4 and PD-1, the interaction between LILRB and its ligands has been proposed to function as an immune checkpoint. For example, engagement of LILRB1 and LILRB2 by HLA-G on cancer cells inhibits immune cell activation and generates regulatory T cells (Tregs) and suppressive antigen-presenting cells (APCs), which can indirectly support tumorigenesis. Furthermore, interaction of P2-microglobulin (P2M)-associated MHC class I on cancer cells with LILRB2 on macrophages results in loss of immune surveillance. It is unclear whether the interaction between LILRB2 and its ligands also functions as a phagocytic checkpoint. LILRBs may also represent targets for inducing transplant tolerance to prevent allograft rejection. LILRBs (especially LILRB2 and LILRB4) are important for inducing a tolerogenic phenotype of APCs and initiating the T cell suppressive cascade that leads to immune tolerance. LILRB1 and LILRB2 can also mediate transplant tolerance by binding to HLA-G. In addition to immune cells, LILRBs are expressed by cancer cells and may support malignant transformation and recurrence, as well as cancer stem cell activity. Collectively, these findings reveal the dual role of LILRB as an immune checkpoint molecule and a tumor-maintaining factor. The development of drugs useful for modulating signaling from LILRB may be highly beneficial in diseases involving immune system dysregulation, including cancer, inflammatory and autoimmune diseases, and transplant rejection.

[0006] Therefore, in view of one or more of the above points, there is a need for novel approaches to reduce the immunosuppressive function of LILRB receptors while avoiding any inhibition of immunostimulatory LILRA receptors on immune cells involved in defense against certain disorders (e.g., infections or tumors). The present invention seeks to provide, in particular, novel therapeutic approaches and methods comprising novel compounds, e.g., compounds and ABPs, that reduce the immunosuppressive function of LILRB receptors but do not inhibit any immunostimulatory function of LILRA receptors. Furthermore, the present invention seeks to provide novel strategies for diagnosing, prognosing, and / or monitoring immune cells based on LILRB expression. It is therefore an object of the present invention to provide alternative, improved, simpler, less expensive, and / or integrated means or methods that address one or more of these or other problems. Such objectives underlying the present invention are solved by the subject matter disclosed or defined elsewhere herein, for example, by the itemized embodiments and / or the subject matter of the appended claims. Summary of the Invention

[0007] The present invention is based on the surprising discovery that certain antigen-binding proteins, such as the antibodies of the present invention, do not bind to any of the LILRA receptors, or do so with relatively less selectivity and specificity, but selectively and specifically bind to both LILRB1 and LILRB2. In certain preferred aspects of the present invention, the antibodies of the present invention maintain their cross-specificity for the targets LILRB1 and LILRB2, while binding significantly less to the off-target proteins LILRA1 and LILRA3. Maintaining cross-specific binding only to LILRB on-target proteins without off-target binding to LILRA is one of several surprising features of the antibodies of the present invention. [Means for solving the problem]

[0008] Thus, broadly and by way of brief description, the main aspects of the present invention can be described as follows:

[0009] In a first aspect, the present invention relates to an antigen-binding protein (ABP) that specifically binds to a LILRB1 and / or LILRB2 protein (e.g., the extracellular domain (ECD) of a LILRB1 and / or LILRB2 protein) and, optionally, can inhibit the binding of a ligand of a LILRB1 and / or LILRB2 protein or a variant thereof to the LILRB1 and / or LILRB2 protein or a variant thereof. Preferably, the ABP does not specifically bind to, or binds significantly less to, leukocyte immunoglobulin-like receptor subfamily A (LILRA) type proteins.

[0010] In a second aspect, the invention relates to an ABP that competes with the ABP of the first aspect for binding to a LILRB1 and / or LILRB2 protein (e.g., the ECD of a LILRB1 and / or LILRB2 protein). In a related aspect, the invention relates to an ABP that binds to the same epitope as the ABP of the first aspect.

[0011] In another aspect, the present invention relates to the antigen binding domain (ABD) of the ABP of the present invention.

[0012] In a third aspect, the present invention relates to nucleic acids encoding the ABP or ABD of the invention or components thereof, and in a related aspect, the present invention relates to nucleic acid constructs (NACs) comprising such nucleic acids, and to host cells comprising the nucleic acids or NACs of the invention.

[0013] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising an ABP, ABD, nucleic acid, NAC or host cell of the invention, or a compound that is a modulator of the expression, function, activity and / or stability of leukocyte immunoglobulin-like receptor subfamily Bl (LILRB1) and / or LILRB2 type proteins, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0014] In a fifth aspect, the present invention relates to a method for treating a particular disease, disorder, or condition in a subject by administering to the subject a product, wherein the product is selected from the list consisting of an ABP, ABD, nucleic acid, NAC, and host cell of the present invention, or a compound that is a modulator of the expression, function, activity, and / or stability of LILRB1 and / or LILRB2. In a related aspect, the present invention relates to a product for use in medicine and to the use of a product for the manufacture of a medicament, wherein the product is selected from the list consisting of an ABP, ABD, nucleic acid, NAC, or host cell of the present invention, or a compound that is a modulator of the expression, function, activity, and / or stability of LILRB1 and / or LILRB2.

[0015] The present invention also relates in other aspects to various methods for producing the recombinant cell lines or ABPs of the invention, hybridomas or host cells capable of producing the ABPs of the invention, as well as various assay and / or diagnostic methods or methods of use, and kits useful for such assay and / or diagnostic methods, and various methods for identifying and / or characterizing compounds, such as compounds suitable for use in medicine. [Brief explanation of the drawings]

[0016] A brief description of the drawings follows:

[0017] [Figure 1] Figure 1 shows the domain structure of the leukocyte immunoglobulin-like receptor family members LILRA (A) and LILRB (B). Ig-like = Ig-like domain, EC = extracellular, PM = plasma membrane, IC = intracellular, white box = ITIM. Modified from Hirayasu K. & Arase H. (2016, Nat Microbiol.)

[0018] [Figure 2]FIG. 2 shows the binding affinity of antibodies of the invention to LILRB1 and LILRB2, and counter-targets LILRA1 and LILRA3. Y-axis = KD(M), open triangle = LILRA1, open triangle = LILRA3, closed triangle = LILRB1, closed circle = LILRB2, X-axis = antibody, 1 = A-001, 2 = A-004, 3 = A-005, 4 = A-006, 5 = A-007, 6 = A-008, 7 = A-009, 8 = A-010, 9 = A-011, 10 = A-012, 11 = A-013, 12 = A-014, 13 = A-015, 14 = A-016, 15 = A-017, 16 = A-018, 17 = A-019, 18 = A-020, 19 = A-021, 20 = A-045, 21 = A-046, 22 = A-003, 23 = A-022, 24 = A-023, 25 = A-024, 26 = A-025, 27 = A-026, 28 = A-027, 29 = A-028, 30 = A-029, 31 = A-030, 32 = A-031, 33 = A-032, 34 = A-033, 35 = A-047, 36 = A-048, 37 = A-034, 38 = A-035, 39 = A-036, 40 = A-037, 41 = A-038, 42 = A-039, 43 = A-040, 44 = A-041, 45 =[Ref47, MK-4830, LILRB2 specific], 46=[Ref051, BND-22, LILRB1 specific], 47=[Ref062, NGM707, cross-specific].

[0019] [Figure 3]Figure 3 shows inhibition of the interaction of (A) LILRB1 or (B) LILRB2 and their ligand HLA-G using the antibody of the present invention, clone A-001, and its mature variants. Y-axis = residual binding (%), X-axis = antibody concentration (nM); 1 = A-001, 2 = A-004, 3 = A-005, 4 = A-006, 5 = A-007, 6 = A-008, 7 = A-009, 8 = A-010, 9 = A-011, 10 = A-012, 11 = A-013, 12 = A-014, 13 = A-015, 14 = A-016, 15 = A-017, 16 = A-018, 17 = A-019, 18 = A-020, 19 = A-021, 20 = A-045, 21 = A-046.

[0020] [Figure 4] Figure 4 shows the inhibition of the interaction of (A) LILRB1 or (B) LILRB2 and their ligand HLA-G using antibodies of clone A-003 of the present invention and its mature variants. Y-axis = residual binding (%), X-axis = antibody concentration (nM); 22 = A-003, 23 = A-022, 24 = A-023, 25 = A-024, 26 = A-025, 27 = A-026, 28 = A-027, 29 = A-028, 30 = A-029, 31 = A-030, 32 = A-031, 33 = A-032, 34 = A-033, 35 = A-047, 36 = A-048.

[0021] [Figure 5] Figure 5 shows the inhibition of the interaction of LILRB1 or LILRB2 with their ligand, HLA-G, using reference antibodies. (A) Inhibition of LILRB1 and HLA-G; (B) Inhibition of LILRB2 and HLA-G. Y-axis = residual binding (%); X-axis = antibody concentration (nM); open circles = Ref065 [10-108, LILRB2-specific], diamonds = Ref062 [NGM707, cross-specific], filled squares = Ref051 [BND-22, LILRB1-specific], filled circles = Ref047 [MK-4830, LILRB2-specific], filled stars = Ref001 (isotype control).

[0022] [Figure 6-1] FIG. 6 shows the effect of anti-LILRB2 / 1 blockade on the polarization of human M2 macrophages in a functional myeloid assay (AC) and on the polarization of human M2 macrophages and cytotoxic T cells in a myelosuppression assay (DH). Such effects were assessed by measuring (A and D) surface M2-marker CD163 and (F) early activation marker CD69 (Y-axis = median Fl of viable cells; B = background), as well as cytokine secretion of (B) CCL13, (C) CCL23, (E) CCL18, (G) GM-CSF, and (H) IFNγ (Y-axis = cytokine concentration (pg / ml); X-axis = antibodies at the indicated concentrations; 0 = Ref001 (isotype control); 1 = A-010; 2 = A-026; 3 = Ref047 [LILRB2-specific MK-4830]; 4 = Ref062 [NGM707, cross-specific]; 5 = A-045; X = isotype control). [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 6-4] Same as above

[0023] [Figure 7-1] Figure 7 shows target binding of LILRB2 / 1 antibodies (A = A-045, B = Ref047 [LILRB2-specific MK-4830], and C = Ref062 [NGM707, cross-specific]) to transfected Expi293 cells, (D) to primary T cells, and (E) to monocytes; Y-axis = % positive (of CD8+ cells) [for D only]; otherwise, Y-axis = median FI of GFP+ cells (RL1-H) (pg / ml); X-axis = antibody concentration (nM); X = isotype control. For A-C: circle = non-transfected, square = LILRB1, triangle = LILRA1, inverted triangle = LILRB2, diamond = LILRA2, hexagon = LILRA3. For D and E: circle = A-045, square = Ref047 [LILRB2-specific MK-4830], triangle C = Ref062 [NGM707, cross-specific]. [Figure 7-2] Same as above

[0024] [Figure 8-1] Figure 8 shows the following: (A) Triple culture assay principle; AB = antibody addition (antibody present throughout the assay), T = T cell addition, TC = tumor cell addition, X = monocyte differentiation with M-CSF, Y = macrophage polarization with addition of IL-4, IL-10, and TGF-β; Z = autologous T cell activation with addition of anti-CD3 and anti-CD28; (B) reversal of T cell suppression by optimized A-010 (Y-axis = IL-2 [pg / ml]) and (C) tumor cell killing (Y-axis = tumor cell signaling [RLU] with optimized A-010); X-axis: 1 = unstimulated T cells only, 2 = stimulated T cells only, 3 = stimulated T cells + M2-polarized macrophages + A-010; 4 = stimulated T cells + M2-polarized macrophages + Ref001 (isotype control); 5 = tumor cells only, 6 = tumor cells. + unstimulated T cells only, 7 = tumor cells + stimulated T cells only, 8 = tumor cells + stimulated T cells + M2-polarized macrophages + A-010, 9 = tumor cells + stimulated T cells + M2-polarized macrophages + Ref001 (isotype control); ac = assay control. [Figure 8-2] Same as above

[0025] [Figure 9] 9 shows BLI measurements demonstrating the displacement of LILRB2 from the HLA-G interaction site and the inhibition of de novo LILRB2 and HLA-G complex formation using antigen-binding fragments obtained from antibodies of the invention (A) A-010, (B) A-045, (C) A-047, (D) A-048, (E) antigen-binding fragments obtained from the reference molecule Ref062 [NGM707, cross-specific], or (F) assay buffer. Y-axis = wavelength shift (nm), X-axis = time (sec).

[0026] [Figure 10-1]10 shows BLI measurements demonstrating the displacement of LILRB1 from the HLA-G interaction site and the inhibition of de novo LILRB1 and HLA-G complex formation using antigen-binding fragments obtained from antibodies of the invention (A) A-010, (B) A-045, (C) A-047, (D) A-048, (E) antigen-binding fragments obtained from the reference molecule Ref062 [NGM707, cross-specific], or (F) assay buffer. Y-axis = wavelength shift (nm), X-axis = time (sec). [Figure 10-2] Same as above

[0027] [Figure 11] Figure 11 shows BLI measurements demonstrating antibody binding to LILRB2 in the presence of different concentrations of LILRA1 and LILRA3. The panels show binding to LILRB2 using the antibody A-045 of the invention in the presence of (A) LILRA1 and (B) LILRA3, and using the reference molecule Ref062 [NGM707, cross-specific] in the presence of (C) LILRA1 and (D) LILRA3. Y-axis = normalized binding response, X-axis = time (seconds). The start of the dissociation phase is indicated by the vertical dashed line at 600 seconds; X = no addition of LILRA1 or LILRA3, respectively; Y = addition of 20 nM of LILRA1 or LILRA3, respectively; Z = addition of 100 nM of LILRA1 or LILRA3, respectively.

[0028] [Figure 12]Figure 12 shows BLI measurements demonstrating antibody binding to LILRB1 in the presence of different concentrations of LILRA1 and LILRA3. The panels show binding to LILRB1 using the antibody of the invention A-045 in the presence of (A) LILRA1 and (B) LILRA3, using the reference molecule Ref062 [NGM707, cross-specific] in the presence of (C) LILRA1 and (D) LILRA3, and using the reference molecule Ref051 [Biond / Sanofi BND-22] in the presence of (E) LILRA1 and (F) LILRA3. Y-axis = normalized binding response, X-axis = time (seconds). The start of the dissociation phase is indicated by the vertical dashed line at 600 seconds; X = no addition of LILRA1 or LILRA3, respectively; Y = addition of 20 nM of LILRA1 or LILRA3, respectively; Z = addition of 100 nM of LILRA1 or LILRA3, respectively.

[0029] [Figure 13] Figure 13 shows target binding of antibody A-045, a cross-specific reference antibody (Ref062 [NGM707]), and the appropriate isotype control (Ref001) to primary T cells (A), neutrophils (B), and monocytes (C), as well as in vitro differentiated M1 (D)- and M2-like (E) macrophages. Binding curves show results for one representative donor. X-axis = antibody concentration (nM); y-axis = median FI (RL-1) of CD8+ cells for T cells (A), CD66B+ cells for neutrophils (B), or CD66B+ cells for live cells (CE); circles = A-045, squares = Ref062 [NGM707, cross-specific], triangles = Ref001 [isotype control].

[0030] [Figure 14]Figure 14 shows the activity of antibody A-045 on the repolarization of M1-like and M2-like macrophages compared to a cross-specific reference antibody. Surface expression of different M1- and M2-markers was assessed by flow cytometry. (A) Shows a dose-dependent decrease in surface expression of the M2-like marker CD206 on in vitro differentiated M1-like macrophages. (B) Shows a dose-dependent increase in surface expression of the M1-marker CD86 on in vitro differentiated M2-like macrophages, while (C) and (D) show a dose-dependent decrease in surface expression of the M2-markers CD163 and CD209 on in vitro differentiated M2-like macrophages. X-axis = antibody concentration; y-axis = absolute mean fluorescence intensity value of each macrophage population gated on live cells; X = isotype control.

[0031] [Figure 15] Figure 15 shows LILRB2 T cell reporter activation upon treatment with antibody A-045, a cross-specific reference antibody (Ref062 [NGM707]), or an isotype control (Ref001). The figure shows (A) the assay principle and (B) the results for one representative donor. X-axis = antibody concentration (nM); y-axis = absolute luminescence subtracted from background; circles = A-045, squares = Ref062 [NGM707, cross-specific], triangles = Ref001 [isotype control].

[0032] [Figure 16] Figure 16 shows the dose-dependent increase in phagocytic activity of in vitro differentiated M0 macrophages upon treatment with antibody A-045, a cross-specific reference antibody (Ref062 [NGM707]), and the appropriate isotype control (Ref001). Results from one representative donor are shown. X-axis = antibody concentration (nM); y-axis = increase in phagocytic activity measured by CD45 / CFTR double-positive cells, normalized to the isotype control (=100%) by flow cytometry; circles = A-045, squares = Ref062 [NGM707, cross-specific], triangles = Ref001 [isotype control].

[0033] [Figure 17-1] Figure 17 shows the sustained and robust activity of antibody A-045 in the presence of recombinant LILRA3 on macrophage phenotype and cytokine profile, compared to treatment with a cross-specific reference antibody (Ref062 [NGM707]) or the appropriate isotype control (Ref001). Panels A and B show the effect of LILRB1 / 2 blockade in the presence of recombinant LILRA3 on M1-like macrophage phenotype, compared to the appropriate isotype control (Ref001). CD163 (A) and CD209 (B) expression was assessed by flow cytometry. X-axis = concentration of recombinant LILRA3 (nM); 1 = 5 nM Ref001 + 150 nM rLILRA3; 2 = 1 nM Ref062 + 150 nM rLILRA3; 3 = 1 nM Ref062 + 37.5 nM rLILRA3; 4 = 1 nM A-045 + 150 nM rLILRA3; 5 = 1 nM A-045 + 37.5 nM rLILRA3. Y-axis = % of CD163 or CD209 positive cells as a percentage of viable cells. C-E show cytokine secretion of antibody-treated M1-like macrophages in the presence of recombinant LILRA3 for GM-CSF (C), IFNγ (D), and IL-9 (E). X-axis = concentration of recombinant LILRA3 and antibody (nM), 1 = 5nM Ref001 + 150nM rLILRA3, 2 = 1nM Ref062 + 150nM rLILRA3, 3 = 1nM Ref062 + 37.5nM rLILRA3, 4 = 1nM A-045 + 150nM rLILRA3, 5 = 1nM A-045 + 37.5nM rLILRA3. Y-axis = % cytokine release normalized to isotype control (=100%). [Figure 17-2] Same as above

[0034] The present invention, and non-limiting aspects and / or embodiments thereof, can be described in further detail as follows:

[0035] In a first aspect, and as may be further described, defined, or disclosed herein, the present invention relates to an antigen binding protein (ABP) that specifically binds to a LILRB1 and / or LILRB2 protein (e.g., the extracellular domain (ECD) of a LILRB1 and / or LILRB2 protein), and optionally the ABP is capable of inhibiting (e.g., inhibiting) the interaction between a natural ligand (e.g., HLA-G) of a LILRB1 and / or LILRB2 protein or a variant thereof and a LILRB1 and / or LILRB2 protein or a variant thereof, e.g., the ABP is capable of selectively inhibiting binding of a LILRB1 and / or LILRB2 protein or a ligand of LILRB1 and / or LILRB2. Whenever, in the context of that aspect and embodiment of the present invention, an antigen binding protein of the present invention is referred to as binding to LILRB1 and / or LILRB2, in its most preferred variation, the binding of such antigen binding proteins of the present invention, such as antibodies, is suitably such that they bind to both LILRB1 and LILRB2. Preferably, such antigen binding proteins do not bind, or bind with lower affinity, to LILRA1 and LILRA3.

[0036] In a further preferred embodiment, the present invention relates to an antigen binding protein (ABP) that specifically binds to LILRB1 and LILRB2 proteins (e.g., the extracellular domain (ECD) of LILRB1 and LILRB2 proteins), and optionally, the ABP can inhibit (e.g., inhibits) the interaction between a natural ligand of LILRB1 and a natural ligand of LILRB2 protein (or HLA-G, etc.). Preferably, the ABP of the present invention binds to LILRB1 and LILRB2 with significantly higher affinity than the binding of ABPs to LILRA1 and LILRA2, and optionally, the ABP cannot inhibit (e.g., does not inhibit) the interaction between a natural ligand of LILRA1 and a natural ligand of LILRA3 protein (or HLA-G, etc.).

[0037] In certain aspects and embodiments of the present invention, the ABP of the present invention is not the antibody disclosed as ADA-011 in WO 2023 / 225626, more specifically, is not an antibody comprising the heavy chain CDR1-CDR3 domain sequence of ADA-011 disclosed in WO 2023 / 225626 and / or does not comprise the light chain CDR1-CDR3 domain sequence of ADA-011 disclosed in WO 2023 / 225626. Thus, an ABP of the present invention is in particular not an antibody comprising: (a) a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 213 of WO 2023 / 225626; (b) a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 223 of WO 2023 / 225626; (c) a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 233 of WO 2023 / 225626; (d) a light chain complementarity determining region 1 (L-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 243 of WO 2023 / 225626; (e) a light chain complementarity determining region 2 (L-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 253 of WO 2023 / 225626; and / or (f) a light chain complementarity determining region 1 (L-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 253 of WO 2023 / 225626. Light chain complementarity-determining region 3 (L-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 263 of 2023 / 225626.

[0038] "Antigen-binding proteins that target LILRB1 and / or LILRB2, preferably target both LILRB1 and LILRB2"

[0039] As used herein, "antigen binding protein" ("ABP") refers to a protein that specifically binds to a target antigen, such as one or more epitopes presented by or present on the target antigen. The antigen of the ABP of the present invention is LILRB1 and / or LILRB2; and the ABP can selectively bind to one or more extracellular domains of said LILRB1 and / or LILRB2 (e.g., an epitope may be presented by or present on one or more extracellular domains of said LILRB1 and / or LILRB2). Typically, the antigen binding protein is an antibody (or fragment thereof), although other forms of antigen binding proteins are also contemplated by the present invention. For example, the ABP can be another (non-antibody) receptor protein derived from a small, robust non-immunoglobulin "scaffold," such as one equipped with binding functionality, e.g., by using methods of combinatorial protein design (Gebauer & Skerra, 2009; Curr Opin Chem Biol, 13:245).Specific examples of such non-antibody ABPs include afibrins based on the Z domain of protein A (Nygren, 2008; FEBS J 275:2668); affilins based on gamma-B crystals and / or ubiquitin (Ebersbach et al, 2007; J MoI Biol, 372:172); afibrins based on cystatins (Johnson et al, 2012; Anal Chem 84:6553); affitins based on Sulfolobus acidocaldarius (Krehenbrink et al, 2008; J MoI Biol 383:1058); alphabodies based on triple-helical coiled-coils (Desmet et al, 2014; Nature Comms 5:5237); anticalins based on lipocalins (Skerra, 2008; FEBS J 275:2677); and domains of various membrane receptors (Silverman et al, 2005; Nat Biotechnol 23: 1556); DARPins based on ankyrin repeat motifs (Strumpp et al, 2008; Drug Discov Today, 13:695); Fynomers based on the SH3 domain of Fyn (Grabulovski et al, 2007; J Biol Chem 282:3196); Kunitz domain peptides of various protease inhibitors (Nixon et al, Curr opin Drug Discov Devel, 9:261), and centrins and monobodies based on the tenth type III domain of fibronectin (Diem et al., 2014; Protein Eng Des Sel 27:419 doi: 10.1093 / Protein / gzu016; Koide & Koide, 2007; Methods Mol Biol 352:95).In the context of the ABPs of the present invention that specifically bind to LILRB1 and / or LILRB2 (and do not bind to LILRA1 and / or LILRA3 or bind with significantly lower affinity), such ABPs are not proteins that are natural ligands of LILRB1 and / or LILRB2 (in particular, they are not ligand proteins that have more than 70%, 80% or 90% sequence identity to the amino acid sequence of HLA-G).

[0040] The term "epitope" includes any determinant capable of being bound by an antigen-binding protein such as an antibody. An epitope is a region of an antigen that is bound by an antigen-binding protein that targets the antigen, and, if the antigen is a protein, includes specific amino acids that bind to the antigen-binding protein (e.g., via the antigen-binding domain of the protein). Epitopes can include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antigen-binding protein specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0041] As used herein, the term "extracellular domain" ("ECD" or "EC" domain) refers to the region or regions of a protein that are exposed to the extracellular space and are typically involved in ligand binding. Immunoglobulin (Ig) superfamily genes typically have an immunoglobulin-like ECD, such as an Ig-like C2-type domain.

[0042] An antigen-binding protein is "specific" if it binds preferentially (e.g., more strongly or more extensively) to one antigen (e.g., LILRB1 and / or LILRB2; e.g., human LILRB1 and / or LILRB2, orthologs, and other variants thereof) over its binding to another antigen, preferably a LILRA protein (e.g., LILRA1 and LILRA3). As used herein, in the context of an ABP, the term "specifically binds" (or "binds specifically," etc.) means that the ABP binds preferentially to a desired antigen (e.g., LILRB1 and / or LILRB2, particularly the ECD of LILRB1 and / or LILRB2) over its binding to other proteins (or other molecules), such as preferentially binding to LILRB1 and / or LILRB2 compared to one or more of the leukocyte immunoglobulin-like subfamily A (LILRBA) proteins. Thus, preferably, the binding affinity of the ABP for a LILRB antigen (e.g., LILRB1 and / or LILRB2) is at least 2-fold, 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 5000-fold, at least 1 ... 5 times, or even at least 10 6 times, most preferably at least twice as much.

[0043] Leukocyte Ig-like receptors (LIRs) are a family of immunoreceptors expressed primarily on monocytes and B cells, and at lower levels on dendritic cells and natural killer (NK) cells. All members of LIR subfamily B, such as LILRB2, contain a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and have inhibitory functions. Upon binding of an LIR subfamily B member by MHC class I or other ligands and tyrosine phosphorylation of the ITIM, intracellular protein tyrosine phosphatase, such as SHP1 (PTPN6), is recruited, and an inhibitory signal cascade ensues. Most members of LIR subfamily A (e.g., LILRA1) have a short cytoplasmic domain lacking an ITIM, a transmembrane domain containing a charged arginine residue, and can initiate a stimulatory cascade. One subfamily A member, LILRA3, lacks a transmembrane domain and is predicted to be a soluble receptor (summarized by Borges et al., 1997).

[0044] The interaction between LILRB1 and / or LILRB2 and their natural ligands (such as HLA-G) has subsequently been described independently (De Louche et al 2022 JCI Insight. 2022;7(2):el51553).

[0045] The human LILRB1 gene is located at chromosomal position 19ql3.42 and has orthologs (e.g., conserved) in many species. In some embodiments of the present invention, the term LILRB1 may also refer to variants of the human LILRB1 protein that are substantially identical to, or have at least 70%, 75%, or 80%, preferably 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., at least 90% or 95% sequence identity) to, the amino acid sequence set forth in any of SEQ ID NOs: 385-389, as determined, for example, using the "Blast 2 sequence" algorithm described by Tatusova & Madden 1999 (FEMS Microbiol Lett 174: 247-250). (Preferably) retain the same or substantially the same biological activity as the respective reference LILRB1 protein (e.g., binds to HLA-G) and / or modulates macrophage (or other immune cell) function / activity. The term LILRB1 may refer to a LILRB1 protein (such as those described above) or an mRNA molecule encoding such a LILRB1 protein, where applicable in the context (unless more specifically indicated).

[0046] The human LILRB2 gene is located at chromosomal location 19q13.42 and has orthologs (e.g., conserved) in many species. In some embodiments of the present invention, the term LILRB2 may also refer to variants of the human LILRB2 protein that are substantially identical to, or have at least 70%, 75%, or 80%, preferably 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., at least 90% or 95% sequence identity) to, the amino acid sequence set forth in any of SEQ ID NOs: 390-393, as determined, for example, using the "Blast 2 sequence" algorithm described by Tatusova & Madden 1999 (FEMS Microbiol Lett 174: 247-250). (Preferably) they retain the same or substantially the same biological activity as the respective reference LILRB1 protein (e.g., binds to HLA-G) and / or modulates macrophage (or other immune cell) function / activity. The term LILRB2 may refer to a LILRB2 protein (such as those described above) or an mRNA molecule encoding such a LILRB2 protein, where applicable in the context (unless more specifically indicated).

[0047] In one embodiment, an ABP of the present invention that binds to the ECD of human LILRB1 and / or LILRB2 protein is cross-reactive with the ECD of an orthologous protein, such as the ECD of cynomolgus monkey LILRB1 and / or LILRB2 protein and / or the ECD of mouse LILRB1 and / or LILRB2 protein and / or the ECD of rat LILRB1 and / or LILRB2 protein.

[0048] As used herein, the term "ortholog" refers to variants that originate from the same ancestral gene but exist in different organisms due to speciation events. Orthologs of LILRB1 and / or LILRB2 are typically expected to retain the same function (or have similar function) as human LILRB1 and / or LILRB2.

[0049] As used herein, the term "variant" in the context of a protein refers to any naturally occurring or non-naturally occurring form of such a protein that contains one or more amino acid mutations compared to the reference protein, but shares significant amino acid sequence identity with the reference protein, e.g., at least 70% or 75% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, and most preferably at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Preferably, a protein variant possesses and / or maintains at least one function / activity that is identical, essentially identical, or similar to that of the reference protein. These variants of LILRB1 and / or LILRB2 may include orthologs of human LILRB1 and / or LILRB2 as well as naturally occurring variants of human LILRB1 and / or LILRB2. A "functional variant" of LILRB1 and / or LILRB2 (e.g., a functional fragment of the LILRB1 and / or LILRB2 protein) is a variant of the LILRB1 and / or LILRB2 protein that provides, possesses, and / or maintains one or more of the functions / activities described herein of the non-mutant LILRB1 and / or LILRB2 protein. For example, such a functional variant may be capable of binding to HLA-G protein and / or inhibiting T cell (or other immune cell) function / activity as the LILRB1 and / or LILRB2 protein, such as having the same, essentially the same, or similar specificity and / or receptor function as the LILRB1 and / or LILRB2 protein. In other embodiments, such functional variants may possess an activity other than that possessed by the non-mutant LILRB1 and / or LILRB2 protein, preferably so long as they provide, possess, and / or maintain at least one function / activity that is the same, essentially the same, or similar to that of the LILRB1 and / or LILRB2 protein.In a more preferred embodiment, functional variants of LILRB1 and / or LILRB2 may act as immune checkpoint inhibitors, such as by inhibiting one or more cell-based immune responses against tumor or cancer cells expressing such functional variants.

[0050] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides are exemplified by the following: Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48: 1073.

[0051] When calculating percent identity, the sequences being compared are typically aligned to maximize the match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acids or nucleotides (the "matching span" determined by the algorithm). A gap opening penalty (3x, calculated as the average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used, and "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm.

[0052] Standard comparison matrices (for the PAM 250 comparison matrix see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919; for the BLOSUM 62 comparison matrix see) can also be used by the algorithm.

[0053] Examples of parameters that can be used when determining percent identity of polypeptide or nucleotide sequences using the GAP program are: (i) Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; (ii) Comparison Matrix: BLOSUM 62 from Henikoff et al., 1992, see above; (iii) Gap Penalty: 12 (but no penalty for end gaps); (iv) Gap Length Penalty: 4; (v) Threshold of Similarity: 0.

[0054] A preferred method for determining similarity between a protein or nucleic acid and human LILRB1 and / or LILRB2 (or between them), paralogs, orthologs, or other variants thereof is provided by a Blast search supported by Uniprot supra (e.g., http: / / www.Uniprot.org / Uniprot / Q8NHL6 for LILRB1 and http: / / www.Uniprot.org / Uniprot / Q8N423 for LILRB2); particularly for amino acid identity, using the following parameters: blastp; Matrix: blosum62; Threshold: 10; Filtered: false; Gapped: true; Maximum hits reported: 250.

[0055] Certain alignment schemes for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this small aligned region may have very high sequence identity despite the lack of significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) can be adjusted, if desired, to result in an alignment spanning at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, or other number of consecutive amino acids of the target polypeptide or region thereof.

[0056] In certain embodiments of the present invention, LILRB1 is a protein comprising an amino acid sequence selected from the group consisting of human LILRB1, preferably SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, and SEQ ID NO: 389 (particularly SEQ ID NO: 385), or a protein having no more than 2, 4, 6, 8, or 10, for example no more than 1, 2, or 3, for example no more than 1 amino acid substitution, insertion, or deletion compared to these sequences.

[0057] In certain embodiments of the invention, LILRB2 is human LILRB2, preferably a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, and SEQ ID NO: 393 (particularly SEQ ID NO: 390), or a protein having no more than 2, 4, 6, 8, or 10, for example no more than 1, 2, or 3, for example no more than 1 amino acid substitution, insertion, or deletion compared to these sequences.

[0058] In the context of variants of LILRB1 and / or LILRB2 (these variants), the present invention includes embodiments in which these variants of LILRB1 and / or LILRB2 are proteins comprising an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, or 97% sequence identity (particularly at least 92% or 95% sequence identity) to any one of the sequences of SEQ ID NOs: 385 to 393 (preferably SEQ ID NO: 353 (LILRB1) or SEQ ID NO: 358 (LILRB2)).

[0059] In the context of other variants of LILRB1 and / or LILRB2, the present invention also includes embodiments in which those variants of LILRB1 and / or LILRB2 are selected from the group consisting of orthologs (or paralogs) of LILRB1 and / or LILRB2 and functional fragments of LILRB1 and / or LILRB2 proteins. In some such embodiments, such functional fragments of LILRB1 and / or LILRB2 proteins bind to natural ligands of LILRB1 and / or LILRB2 proteins, such as human leukocyte antigen (HLA) type proteins (such as those described elsewhere herein).

[0060] In certain embodiments, the ABPs of the present invention can inhibit (e.g., inhibit) the interaction between the natural ligand of the LILRB1 and / or LILRB2 protein and the LILRB1 and / or LILRB2 protein (e.g., HLA-G, etc.). For example, the ABPs can selectively inhibit (e.g., inhibit) the binding of the LILRB1 and / or LILRB2 protein to a ligand of the LILRB1 and / or LILRB2 protein (e.g., HLA-G).

[0061] Protein sequences of the canonical isoforms and additional isoforms of off-target LILRA1 and LILRA3 can also be derived from the UniProt database. For LILRA1, the protein sequence and additional information are available under accession number: 075019. The canonical protein sequence is provided herein as SEQ ID NO: 394. For LILRA1, the protein sequence and additional information are available under accession number: 075019. The canonical protein sequence is provided herein as SEQ ID NO: 395.

[0062] As used herein, the term "HLA-G" refers to a protein also known as human leukocyte antigen G or HLA-G histocompatibility antigen class G. This protein was first discovered in the extravillous trophoblast (EVT) present at the maternal-fetal interface during pregnancy and is a heterologous material expressed only in cell membranes by selective conjugation of HLA-G mRNA (membrane-bound HLA-G, e.g., HLA-G1, G2, G3, and G4, exist), and also has a soluble single-molecule form secreted to the outside of cells (HLA-G5, G6, and G7 exist as soluble HLA-G). The amino acid sequence and further information of human HLA-G can be obtained from the December 2022 edition of the UniProt database, which has accession number P17693.

[0063] In certain embodiments, the ABPs of the present invention can inhibit (e.g., inhibit) the interaction between the LILRB1 and LILRB2 proteins and their natural ligands. For example, the ABPs can selectively inhibit (e.g., inhibit) the binding of the LILRB1 and LILRB2 proteins to their ligands (e.g., HLA-G). The ABPs of the present invention further do not inhibit the interaction between LILRA1 and LILRA3 and their natural ligands.

[0064] "Modulators of LILRB1 and / or LILRB2 Expression, Function, Activity and / or Stability"

[0065] In certain embodiments of the titled aspect, the ABP is a modulator of the expression, function, activity and / or stability of LILRB1 and / or LILRB2, or a variant of LILRB1 and / or LILRB2, e.g., the ABP inhibits the expression, function, activity and / or stability of LILRB1 and / or LILRB2, or a variant of LILRB1 and / or LILRB2, or in particular, the ABP is an inhibitor of the function and / or activity of LILRB1 and / or LILRB2, or a variant of LILRB1 and / or LILRB2, In one such embodiment, the ABP of the present invention is an inhibitor of the interaction between LILRB1 and / or LILRB2, or a variant thereof of LILRB1 and / or LILRB2, and its endogenous receptor or ligand, such as an HLA-type protein; in particular, the ABP of the present invention can inhibit (e.g., inhibits or is an inhibitor of) the binding of a natural ligand of the LILRB1 and / or LILRB2 protein to the LILRB1 and / or LILRB2 protein. Thus, the ABP of the present invention can be a "modulator."

[0066] The term "modulator," as used herein, refers to a molecule that alters, modifies, or changes one or more characteristics, properties, and / or capabilities of another molecule, or that alters, modifies, or changes an immune response, such as, for example, a cell-mediated immune response (an "immune modulator"). For example, a modulator (e.g., an inhibitory or antagonistic modulator) can impair, prevent, or reduce the expression, function, activity, and / or stability, e.g., the magnitude of a particular activity or function, of a molecule compared to the magnitude of such characteristic, property, or capability observed in the absence of the modulator. In an alternative example, a modulator (e.g., an activating or agonistic modulator) can enhance, promote, or increase the expression, function, activity, and / or stability, e.g., the magnitude of a particular activity or function, of a molecule compared to the magnitude of such characteristic, property, or capability observed in the absence of the modulator. Specific exemplary characteristics, properties, or capabilities of a molecule include, but are not limited to, expression, function, activity, and / or stability, e.g., binding ability or affinity, enzymatic activity, and signal transduction; e.g., any of the functions or activities of LILRB1 and / or LILRB2 described herein.

[0067] Modulatory molecules (particularly, regulatory ABPs) can act as "inhibitors" ("antagonists") for receptors such as LILRB1 and / or LILRB2, e.g., by impairing (e.g., blocking) ligand binding to such receptors by inhibiting the interaction between LILRB1 and / or LILRB2 and their ligands. Alternatively, modulatory molecules (particularly, regulatory ABPs) can act as "activators" ("agonists") for receptors such as LILRB1 and / or LILRB2, e.g., by enhancing or promoting the function and / or activity of such receptors, e.g., by mimicking the binding of endogenous ligands to such receptors, e.g., by triggering the signaling pathway of such receptors.

[0068] A particular embodiment of a modulator of LILRB1 and / or LILRB2 is a "inhibitor of LILRB1 and / or LILRB2" (or "LILRB1 and / or LILRB2 inhibitor"), which is meant to include any moiety that inhibits LILRB1 and / or LILRB2, which may refer to the inhibition of LILRB1 and / or LILRB2, particularly the expression (e.g., amount), function, activity, and / or stability of LILRB1 and / or LILRB2 protein. In one particular such embodiment, the inhibitor of LILRB1 and / or LILRB2 can reduce the function (and / or activity) of LILRB1 and / or LILRB2 protein, and in another such embodiment, the inhibitor of LILRB1 and / or LILRB2 can reduce the expression of LILRB1 and / or LILRB2 mRNA and / or protein.

[0069] General and specific examples of LILRB1 and / or LILRB2 inhibitors (including those that are ABPs of the present invention) are described elsewhere herein, including those that may be characterized by applicable functional and / or structural features described herein.

[0070] Thus, in certain embodiments of the present invention, the ABP of the present invention is capable of specifically binding (e.g., specifically binding to) LILRB1 and / or LILRB2 and inhibiting (e.g., reducing or blocking) the interaction between the LILRB1 and / or LILRB2 protein (or a variant thereof, such as those described above) and its natural ligand (e.g., an HLA protein or other ligand). In particular, such an ABP is capable of inhibiting (e.g., inhibiting) the binding of an HLA-G protein (or a variant thereof, such as those described above) to the LILRB1 and / or LILRB2 protein (or a variant thereof, such as those described above).

[0071] Methodologies for determining the interaction (e.g., binding) between LILRB1 and / or LILRB2 and an HLA protein such as HLA-G protein (or between variants thereof) are known to those skilled in the art and include techniques such as ELISA assays (as described in the Examples below), and flow cytometry, surface plasmon resonance, surface acoustic waves, and microscale thermophoresis, among others. Such determination methods can be used (or adapted) not only to detect the presence of such interaction / binding, but also to measure (e.g., quantitatively) the degree of binding between the interaction partners LILRB1 and / or LILRB2 and their ligands, such as HLA-G protein (or variants thereof). Such (quantitative) measurement of interaction (binding) can be determined or measured in the presence of a competing (e.g., inhibiting) ABP of the present invention, and thus the ability of the ABP of the present invention to inhibit (e.g., block) such interaction can be measured and reported, for example, as an IC50.

[0072] Such IC50 values ​​can be determined, for example, using an ELISA method (e.g., using an assay corresponding to or substantially similar to the ELISA described in Example 3) using surface-bound LILRB1 and / or LILRB2 in the presence of an appropriate concentration of HLA-G protein (or a variant thereof) in solution.

[0073] In certain such embodiments of the present invention, the ABPs of the present invention (e.g., those that bind to one or more epitopes presented by the extracellular domain(s) of LILRB1 and / or LILRB2, or a paralog, ortholog, or other variant) can inhibit (e.g., inhibit) the binding of a natural ligand of the LILRB1 and / or LILRB2 protein, or variant thereof, to the LILRB1 and / or LILRB2 protein, or variant thereof, with an IC50 of 100 nM, 50 nM, or preferably 20 nM or less, e.g., 15 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 500 pM or less, 250 pM or less, or 100 pM or less. In such an embodiment, in particular, the ABP of the present invention can inhibit (e.g., inhibit) the binding of an HLA-G protein, or a variant thereof, to a LILRB1 and / or LILRB2 protein, or a variant thereof, with an IC50 of 5 nM or less, preferably 2 nM or less.

[0074] In other embodiments, a modulator of the invention that is an inhibitor or antagonist (e.g., an ABP that binds to LILRB1 and / or LILRB2) may instead or also: * inhibit, impair, reduce or reverse LILRB1 and / or LILRB2-mediated inhibition of cell-mediated immune responses (e.g., in an in vitro assay or in a subject, such as a subject in need thereof); and / or * inhibiting, impairing, reducing or reversing LILRB1 and / or LILRB2-mediated inhibition of humoral immunity (e.g., in an in vitro assay or in a subject, such as a subject in need thereof); * inhibiting, impairing, reducing or reversing LILRB1 and / or LILRB2-dependent macrophage (e.g., tumor-associated macrophage (TAM)) polarization, e.g., immune cell polarization towards immunosuppressive phenotypes such as M2-like macrophages or myeloid-derived suppressor cells (MDSCs); * Increase, support and / or enhance macrophage-dependent phagocytosis; * Increasing, supporting and / or enhancing the polarization or repolarization of immune cells towards immune enhancing phenotypes such as M1-like macro(co)phages.

[0075] The term "cell-mediated immune response" as used herein may include, but is not limited to, a response in a host organism that utilizes and / or promotes any one or combination of T-cell maturation, proliferation, activation, migration, infiltration, and / or differentiation, and / or activation / regulation / migration / infiltration of macrophages, natural killer cells, T lymphocytes (or T cells), helper T lymphocytes, memory T lymphocytes, suppressor T lymphocytes, regulator T lymphocytes, and / or cytotoxic T lymphocytes (CTLs), and / or the production, release, and / or effect of one or more cell-secreted or cell-secreted factors, such as cytokines or autologous factors (particularly pro-inflammatory cytokines), and / or any one or more components of such processes (cytokines or autologous factors, particular pro-inflammatory cytokines, etc.). The term "cell-mediated immune response," as used herein, may include a cellular response involving genetically engineered, in vitro cultured, autologous, xenogeneic, modified, and / or transferred T lymphocytes, or it may include cell-secreted or cell-secreted factors (e.g., cytokines or autologous cytokines, particularly pro-inflammatory cytokines) produced by genetic engineering. The cell-mediated immune response is preferably not a humoral immune response, e.g., not an immune response involving the release of antibodies. In certain embodiments, particularly when the proliferative disorder is cancer or tumor, the cell-mediated immune response is an anti-tumor cell-mediated immune response. For example, one that results in a reduction in tumor (cell) proliferation, such as a cytotoxic cell-mediated immune response (e.g., exposure of cytotoxic T cells) that kills cancer or tumor cells.

[0076] In certain embodiments, cells mediating a cell-mediated immune response may be mediated by cells such as immune cells that are capable of secreting (e.g., secrete) inflammatory cytokines, such as those selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-12, IL-17 and IL-18, tumor necrosis factor (TNF) [alpha], interferon gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor.

[0077] In certain embodiments, the cell-mediated immune response may be mediated by lymphocytes (e.g., T cells), particularly cytotoxic T lymphocytes (CTLs), or proinflammatory cytokine-secreting cells such as natural killer cells (NK cells).

[0078] In certain embodiments, the cell-mediated immune response may induce the death of cells associated with or involved in a disease, disorder, or condition, such as a proliferative disorder (eg, cancer).

[0079] The term "humoral immunity" (or "humoral immune response") is also readily understood by those skilled in the art and includes aspects of the immune response mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Humoral immunity is so named because it involves body fluids or substances found in body fluids. Aspects that include antibodies may be referred to as antibody-mediated immunity.

[0080] As used herein, a "subject" includes all mammals, including, but not limited to, humans, but also non-human primates such as cynomolgus monkeys. It also includes dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents (such as mice and rats). It will be understood that particularly preferred subjects according to the present invention are human subjects, e.g., human patients, such as humans suffering from (or at risk of suffering from) a disorder, disease, or condition.

[0081] In yet other embodiments, the modulator of the present invention (e.g., an ABP that binds to LILRB1 and / or LILRB2) is an inhibitor or antagonist, preferably an inhibitor or antagonist of the interaction of LILRB1 and / or LILRB2 with its natural ligand, such as HLA-G, and may alternatively or also: * activating and / or reducing the suppression of immune signaling pathways, such as AKT and ERK signaling, in cells expressing LILRB1 and / or LILRB2; * Depolarize or repolarize immunosuppressive macrophages, such as M2 macrophages; * Depolarizing or repolarizing the tolerogenic dendritic cell (DC) phenotype; * promoting adaptive immune responses, such as cellular or humoral immune responses elicited against antigens or cells or therapeutic antibodies (e.g., in in vitro assays or in subjects in need thereof); * enhancing humoral immune responses elicited by therapeutic or prophylactic vaccines (e.g., in in vitro assays or in subjects in need thereof); * Reduce tumor cell immune evasion mediated by HLA-G expression; *Mediating any one or a combination of at least one of the following effects: (i) an increase in immune response; (ii) increased T cell activation; (iii) increased activity of cytotoxic T cells; (iv) increased NK cell activity; (v) alleviation of T-cell suppression; (vi) increased proinflammatory cytokine secretion; (vii) increased IL-2 secretion; (viii) increased interferon-γ production; (ix) increased Th1 response; (x) decreased Th2 response; (xi) reducing or eliminating the number and / or activity of at least one of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes; (xii) a decrease in regulatory cell activity and / or activity of at least one of myeloid-derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes; (xiii) reducing or eliminating M2 macrophages; (xiv) reduced M2 macrophage oncogenic activity; (xv) reducing or eliminating N2 neutrophils; (xvi) reduction of N2 neutrophil oncogenic activity; (xvii) reducing the inhibition of T cell activation; (xviii) reducing the inhibition of CTL activation; (xix) Reduces the inhibition of NK cell activation; (xx) Reverse T cell exhaustion, (xxi) Increased T cell responses, (xxii) increasing the activity of cytotoxic cells; (xxiii) stimulating antigen-specific memory responses; (xxiv) inducing apoptosis or lysis of cancer cells; (xxv) stimulating cytotoxic or cytostatic effects on cancer cells; (xxvi) induce direct killing of cancer cells; (xxvii) increasing Th17 activity, and / or (xxviii) Induce complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity (e.g., in an in vitro assay or in a subject in need thereof), under any condition, the modulator may induce an effect opposite to one or more of (i) to (xxviii).

[0082] "ABPs of the Invention Comprising One or More Complementarity-Determining Regions"

[0083] In certain embodiments, the ABP of the present invention may comprise at least one complementarity determining region (CDR), e.g., preferentially derived from an antibody (particularly derived from a human antibody), and in certain embodiments, the ABP may comprise a CDR having an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity (preferably at least 90% sequence identity) or has no more than three or no more than two, preferably no more than one amino acid substitution, deletion or insertion, compared to the CDR sequence set out in Table 1 herein.

[0084] The term "complementarity determining region" (or "CDR" or "hypervariable region"), as used herein, broadly refers to one or more hypervariable or complementarity determining regions (CDRs) found in the variable region of an antibody light or heavy chain. See, e.g., "IMGT", Lefranc et al., 2003, Dev Comp Immunol 27:55; Honegger & Pluckthun, 2001, J Mol Biol 309:657; Abhinandan & Martin, 2008, Mol Immunol 45:3832; Kabat, et al. (1987): Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD. These expressions include the hypervariable regions, or hypervariable loops, in the three-dimensional structure of antibodies as defined by Kabat et al. (1983) Sequences of Proteins of Immunological Interest, US Dept. of Health and Human Services (Chothia and Lesk, 1987; J Mol Biol 196:901). The CDRs in each chain are held in close proximity by framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site. Within the CDRs are select amino acids described as selectivity-determining regions (SDRs), which represent important contact residues used by the CDRs in antibody-antigen interactions (Kashmiri, 2005; Methods 36:25).

[0085] In all of its aspects and embodiments, the framework sequences of the ABPs of the invention may contain one or more mutations, for example, to improve the isoelectric point of the molecule. One preferred mutation in the ABPs of the invention is at position E81, e.g., the E81M mutation according to the Kabat nomenclature.

[0086] As noted above, in certain embodiments of the invention, the ABP may comprise at least one complementarity determining region (CDR). In certain such embodiments, the ABP of the invention has at least one complementarity determining region 3 (CDR3), e.g., a sequence selected from the heavy and light chain CDR3 sequences set forth in Table 1 (e.g., SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 146, 147, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, and 383).

[0087] The ABPs of the invention may alternatively, or as well as, comprise at least one CDR1 and / or at least one CDR2 (such as from an antibody, particularly a human antibody) as a CDR3 sequence. Preferably, the ABPs of the invention comprise at least one such CDR3, and at least one such CDR1 and at least one CDR2, more preferably each of such CDRs having at least 80%, 85%, 90% or 95% (preferably at least 90%) sequence identity, or having no more than three, preferably no more than two amino acid substitutions, deletions or insertions, compared to a sequence selected from the corresponding (heavy and light chain) CDR1, CDR2 and CDR3 sequences shown in Table 1.

[0088] In certain preferred embodiments, the antibodies of the invention are derived from three parent antibody sequences, designated A-001, A-002, and A-003. The invention therefore presents binding profiles of the parent sequences as well as mature derivative antibody heavy and / or light chain sequences. The invention therefore also relates, in certain embodiments, to antibody chain combinations of any light chain sequence with any heavy chain sequence of the antibodies of the invention.

[0089] In certain embodiments, the ABP of the present invention may be an antibody or an antigen-binding fragment thereof.

[0090] As used herein, the term "antibody" can be understood in the broadest sense as any immunoglobulin (Ig) capable of binding to its epitope. Such antibodies are a species of Ig. Full-length "antibodies" or "immunoglobulins" are generally heterotetrameric glycoproteins of approximately 150 kDa composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, with the number of disulfide bonds varying among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an amino-terminal variable domain (VH) followed by three carboxy-terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Other forms of antibodies include heavy-chain antibodies, which consist of only two heavy chains and lack the two light chains typically found in antibodies. Heavy-chain antibodies include hcIgG (IgG-like) antibodies from camelids, such as starfish, camels, llamas, and alpacas, and IgNAR antibodies from cartilaginous fish (e.g., sharks). Yet other forms of antibodies include single-domain antibodies (sdAbs, called Nanobodies by Ablynx, Developer), which are antibody fragments consisting of a single monomeric variable antibody domain. Single domain antibodies are typically produced from heavy chain antibodies, but may also be derived from conventional antibodies.

[0091] Antibodies (or antibodies from which fragments can be isolated) can include, for example, chimeric antibodies, humanized antibodies, (fully) human antibodies, or hybrid antibodies with dual or multiple antigen or epitope specificities, antibody fragments and antibody subfragments such as Fab, Fab' or F(ab')2 fragments, single chain antibodies (scFv), etc. (described below), and include any hybrid fragment of an immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0092] Thus, in certain embodiments, the ABP of the present invention may comprise an antibody heavy chain, or an antigen-binding fragment thereof, and / or an antibody light chain, or an antigen-binding fragment thereof.

[0093] In a further embodiment, the ABP of the present invention may comprise an antibody heavy chain variable region, or an antigen-binding fragment thereof, and / or an antibody light chain variable region, or an antigen-binding fragment thereof, and in yet a further embodiment, the ABP of the present invention may comprise antibody heavy chain variable region CDR1, CDR2, and CDR3, and / or antibody light chain variable region CDR1, CDR2, and CDR3.

[0094] In certain embodiments of the present invention, when the ABP comprises an antibody heavy chain sequence and / or an antibody light chain sequence, or an antigen-binding fragment thereof, the antibody heavy chain sequence or fragment thereof is selected from the group consisting of heavy chain CDR3 sequences shown in Table 1 (e.g., SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283, 291, 299, 307, 315, 323, 330, 340, 351, 360, 370, 380, 390, 410, 420, 430, 440, 451, 460, 470, 480, 490, 512, 520, 530, 540, 551, 560, 570, 580, 590, 600, 610, 620, 630, 640, 651, 660, 670, 680, 690, 700, 710, 720, 730, 740, 751, 760, 770, 780, 790, 800, 810, 820, 830, 8 and / or the antibody light chain sequence or fragment thereof can comprise a CDR3 having at least 80%, 85%, 90%, or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion, of, a sequence selected from the list consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 159, 167, 175, 183, 191, 199, 207, 215, 220, 225, 230, 231, 239, 347, 345, 363, 371, and 379; or 95% (preferably at least 90%) sequence identity to a CDR3 sequence selected from the group consisting of: 223, 231, 239, 247, 255, 263, 271, 279, 287, 295, 303, 311, 319, 327, 335, 343, 351, 359, 367, 375, and 383), or a CDR3 having no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion.

[0095] In certain embodiments of the invention, when the ABP comprises an antibody heavy chain sequence and / or an antibody light chain sequence, or an antigen-binding fragment thereof, the antibody heavy chain sequence or fragment thereof may have at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity, or a CDR3 with three or two or fewer, preferably one or fewer, amino acid substitutions, deletions or insertions, to a CDR3 sequence selected from the heavy chain CDR3 sequences selected from the list consisting of SEQ ID NOs: 75, 115, and 195, and / or the antibody light chain sequence or fragment thereof may have at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity, or a CDR3 with three or two or fewer, preferably one or fewer, amino acid substitutions, deletions or insertions, to a light chain CDR3 sequence selected from the list consisting of SEQ ID NOs: 287, 311, and 319. Any combination of these heavy chain CDR3s and light chain CDR3s, in particular combinations selected from SEQ ID NOs: 75 and 287, SEQ ID NOs: 115 and 287, SEQ ID NOs: 195 and 311 and SEQ ID NOs: 195 and 319, are included in the ABPs of the present invention.

[0096] In a further embodiment of the invention, when the ABP comprises an antibody heavy chain or an antigen-binding fragment thereof, the antibody heavy chain sequence or fragment thereof is selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369 and 377 (e.g., heavy chain CDR1 sequences disclosed in Table 1), or may further comprise a CDR1 having three or two or fewer, preferably one or fewer, amino acid substitutions, deletions, or insertions; and / or SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226, 234, 242, 250, 258, The CDR2 may further comprise a CDR2 having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to a sequence selected from 266, 274, 282, 290, 298, 306, 314, 322, 330, 338, 346, 354, 362, 370, and 378 (e.g., a CDR2 sequence disclosed in Table 1), or having no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion.

[0097] In yet a further embodiment of the invention, the ABP of the invention comprises an antibody light chain or an antigen-binding fragment thereof, wherein the antibody light chain sequence or fragment thereof is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, and 381 and 381 (e.g., a light chain CDR1 sequence disclosed in Table 1), or further comprising a CDR1 having at least 80%, 85%, 90%, or 95% (preferably at least 90%) sequence identity to a sequence selected from SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142, 150, 158, 166, 174, 182, 190, 198, 206, 214, 222, 230, 238, 246, and further comprising a CDR2 that is at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identical to, or has no more than three or two amino acid substitutions, deletions, or insertions, compared to a sequence selected from: 254, 262, 270, 278, 286, 294, 302, 310, 318, 326, 334, 342, 350, 358, 366, 374, and 382 (e.g., the light chain CDR2 sequences disclosed in Table 1).

[0098] In other embodiments of the invention, the ABP of the invention is selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380 and 384 (e.g., the VH or VL sequences disclosed in Table 1), or have at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity, or have no more than 10, 9, 7, 6, 4, 3, 2, or 1 amino acid substitutions, deletions, or insertions.

[0099] In certain embodiments of the invention, the ABP of the invention comprises an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises CDR1, CDR2 and CDR3. In certain such embodiments, CDR1 is selected from those disclosed in Table 1, CDR2 is selected from those disclosed in Table 1, and CDR3 is selected from those disclosed in Table 1 (e.g., CDR1, CDR2, and CDR3 are selected from CDR1, CDR2, and CDR3 sequences having the following respective amino acid sequences: SEQ ID NOs: 1, 2, 3; or 9, 10, 11; or 17, 18, 19; or 25, 26, 27; or 33, 34, 35; or 41, 42, 43; or 49, 50, 51; or 57, 58, 59; or 65, 66, 67; or 73, 74, 75; or 81, 82, 83; or 89, 90, 91; or 97, 98, 99; or 105, 106, 107; or 113, 114, 115; or 121, 122, 123; or 129, 130, 131; or 137, 138, 139; or 145, 146, 147; or 153, 154, 155; or 161, 162, 163; or 169, 170, 171; or 177, 178, 179; or 185, 186, 187; or 193, 194, 195; or 201, 202, 203; or 209, 210, 211; or 217, 218, 219; or 225, 226, 227; or 233, 234, 235; or 241, 242, 243; or 249, 250, 251; or 257, 258, 259; or 265, 266, 267; or 273, 274, 275; or 281, 282, 283; or 289, 290, 291; or 297, 298, 299; or 305, 306, 307; or 313, 314, 315; or 321, 322, 323; or 329, 330, 331; or 337, 338, 339; or 345, 346, 347; or 5, 6, 7; or 13, 14, 15;or 21, 22, 23; or 29, 30, 31; or 37, 38, 39; or 45, 46, 47; or 53, 54, 55; or 61, 62, 63; or 69, 70, 71; or 77, 78, 79; or 85, 86, 87; or 93, 94, 95; or 101, 102, 103; or 109, 110, 111; or 117, 118, 119; or 125, 126, 127; or 133, 134, 135; or 141, 142, 143; or 149, 150, 151; or 157, 158, 159; or 165, 166, 167; or 173, 174, 175; or 181, 182, 183; or 189, 190, 191; or 197, 198, 199; or 205, 206, 207; or 213, 214, 215; or 221, 222, 223; or 229, 230, 231; or 237, 238, 239; or 245, 246, 247; or 253, 254, 255; or 261, 262, 263; or 269, 270, 271; or 277, 278, 279; or 285, 286, 287; or 293, 294, 295; or 301, 302, 303; or 309, 310, 311; or 317, 318, 319; or 325, 326, 327; or 333, 334, 335; or 341, 342, 343; or 349, 350, 351; or 353, 354, 355; or 357, 358, 359; or 361, 362, 363; or 365, 366, 367; or 369, 370, 371; or 373; 374, 375; or 377, 378, 379; or 381, 382, ​​383); each independently and selectively having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to the sequences described above;

[0100] In further specific embodiments of the invention, the ABP of the invention can comprise antibody heavy chain variable regions CDR1, CDR2, and CDR3, and / or antibody light chain variable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence of a heavy or light chain CDR1 shown in Table 1 (e.g., SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, and 353, 357, 361, 365, 369, 373, 377, and 381), wherein the CDR2 has the amino acid sequence of a heavy chain or light chain CDR2 set forth in Table 1 (e.g., SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210,214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294,298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, and 382), and wherein the CDR3 has the amino acid sequence of a heavy chain or light chain CDR3 set forth in Table 1 (e.g., SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, and 383); and, independently in each case, optionally, having no more than three or two, and preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences.

[0101] In preferred such embodiments, the ABP may be an antibody or antigen-binding fragment thereof composed of at least one, and preferably two, antibody heavy chain sequences and at least one, and preferably two, antibody light chain sequences, wherein at least one, and preferably both, of the antibody heavy chain sequences and at least one, and preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the heavy chain CDR combinations shown in Table B and / or selected from any of the light chain CDR combinations shown in Table B (in each case, combinations CDR-A-001 to CDR-A-044); in each case independently and selectively having no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences. Preferably, both the heavy chain and light chain CDR combinations are selected from the list marked by any one of the combinations CDR-A-001 to CDR-A-044, and each CDR optionally and independently has no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences. Table B: Preferred combinations of heavy chain CDRs and preferred combinations of light chain CDRs (Table A does not exist, shown below as Table X) [Table X-1] [Table X-2] [Table X-3]

[0102] In another preferred embodiment of the present invention, the ABP may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise a variable region sequence in a heavy chain and light chain variable domain combination shown in Table C (e.g., selected from any of variable chain combinations Chain-A-001 to Chain-A-044); in each case, independently and selectively, they may have no more than 10, 9, 8, 7, 6, 5, or 4 amino acid substitutions, insertions, or deletions compared to these sequences, preferably no more than 3, 2, or 1 amino acid substitutions, insertions, or deletions. Table C: Preferred combinations of heavy and light chain variable domains (Table C is shown below as Table Y) [Table Y-1] [Table Y-2]

[0103] All ABPs of the present invention are characterized by advantageous and surprising on-target and off-target binding affinity profiles. Accordingly, the present invention relates to several embodiments of ABPs that bind to LILRB1 and LILRB2 but not to LILRA1 and not to LILRA3. More specifically, the ABPs of the present invention are the following ABPs, where the binding of the ABP to either the target LILRB1 or LILRB2 is at least two-fold or three-fold higher than the binding to either the off-target LILRA1 or LILRA3, preferably with the binding affinity measured by BLI for KD (monovalent) under the conditions described in the Examples section of the present disclosure. More preferably, the present invention provides novel, advantageous binding profiles, where the binding of the ABP to either the target LILRB1 or LILRB2 is at least five-fold, or preferably ten-fold, higher than the binding to either the off-target LILRA1 or LILRA3, preferably with the binding affinity measured by BLI for KD (monovalent) under the conditions described in the Examples section of the present disclosure. Additionally, in some embodiments, the ABP has a variable heavy chain sequence that is at least 90%, preferably 95%, 96%, 97%, 98% or 99% identical to the variable heavy chain sequence set forth in any one of ABPs A-001 to A-048; and / or the ABP has a variable light chain sequence that is at least 90%, preferably 95%, 96%, 97%, 98% or 99% identical to the variable light chain sequence set forth in any one of ABPs A-001 to A-048.

[0104] In a particularly preferred embodiment, the ABP of the present invention may comprise a combination of light chain CDR1, CDR2 and CDR3 sequences and a combination of heavy chain CDR1, CDR2 and CDR3 sequences in a combination as shown by antibody A-010, for example, as shown in Table B by column CDR-A-010 (e.g., heavy chain CDR1, CDR2 and CDR2 having the sequences set forth in SEQ ID NOs: 73, 74 and 75, respectively, and light chain CDR1, CDR2 and CDR2 having the sequences set forth in SEQ ID NOs: 77, 78 and 79, respectively), with each CDR independently and optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences. In another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof, consisting of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1-CDR3 sequence, respectively, in the combination CDR-A-010, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1-CDR3 sequence, respectively, shown in the combination shown in the row of Table B marked by CDR-A-010, with, independently and optionally, no more than one amino acid substitution, insertion or deletion in each CDR compared to these sequences. In yet another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise the variable region sequences in the heavy and light chain variable domain combinations shown in the row of Table B marked by chain A-010. In each of such particularly preferred embodiments of the ABP, optionally, the ABP is capable of inhibiting the binding of an HLA-G protein, or a variant thereof, to a LILRB1 and / or LILRB2 protein, or a variant thereof, with an IC50 of 20 nM or less, or 10 nM or less, for example, 5 nM or less, or preferably 2 nM or less. Such an IC50 can be determined using methods described elsewhere herein.

[0105] In a particularly preferred embodiment, the ABP of the present invention comprises a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences in the combination shown by antibody A-010, e.g., shown in column CDR-A-026 of Table B (e.g., heavy chain CDR1, CDR2 and CDR2 having the sequences shown by SEQ ID NOs: 201, 202 and 203, respectively, and light chain CDR1, CDR2 and CDR2 having the sequences shown by SEQ ID NOs: 205, 206 and 207, respectively). Independently in each CDR, combinations may optionally include those having no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences. In another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1-CDR3 sequence, respectively, in the combination CDR-A-026, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1-CDR3 sequence, respectively, in CDR-A-026, as shown in the combination shown in the row of Table B marked by CDR-A-026, and wherein each CDR independently and optionally has no more than one amino acid substitution, insertion or deletion compared to these sequences. In yet another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise the variable region sequences in the heavy and light chain variable domain combinations shown in the row of Table B marked by chain A-026. In each of such particularly preferred embodiments of the ABP, optionally, the ABP is capable of inhibiting binding of an HLA-G protein, or a variant thereof, to a LILRB1 and / or LILRB2 protein, or a variant thereof, with an IC50 of 20 nM or less, or 10 nM or less, for example, 5 nM or less, or preferably 2 nM or less. Such an IC50 can be determined using methods described elsewhere herein.

[0106] In a particularly preferred embodiment, the ABP of the present invention may comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences, a combination represented by antibody A-045, shown in column CDR-A-045 of Table B (e.g., heavy chain CDR1, CDR2 and CDR2 having the sequences represented by SEQ ID NOs: 353, 354, and 355, respectively, and light chain CDR1, CDR2 and CDR2 having the sequences represented by SEQ ID NOs: 357, 358, and 359, respectively), and may independently and selectively contain no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion in each CDR compared to these sequences. In another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1-CDR3 sequence, respectively, in the combination CDR-A-045, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1-CDR3 sequence, respectively, in the combination shown in the row of Table B marked by CDR-A-045, with each CDR independently and optionally having no more than one amino acid substitution, insertion or deletion compared to these sequences. In yet another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise the variable region sequences in the heavy and light chain variable domain combinations shown in the row of Table B marked by chain A-045. In each of such particularly preferred embodiments of the ABP, optionally, the ABP is capable of inhibiting binding of an HLA-G protein, or a variant thereof, to a LILRB1 and / or LILRB2 protein, or a variant thereof, with an IC50 of 20 nM or less, or 10 nM or less, e.g., 5 nM or less. Such an IC50 can be determined using methods described elsewhere herein (see Example 3). Preferably, the ABP of this embodiment has a binding affinity for LILRB1 and / or LILRB2 measured by KD (monovalent) of less than 20 nM, more preferably less than 10 nM, more preferably less than 5 nM, where the binding affinity ranges are provided for binding to both LILRB1 and LILRB2, preferably measured using BLI under the conditions described in the Examples. Thus, the ABP of this embodiment is preferably an ABP of (targeting) LILRB1 and LILRB2. In another preferred embodiment of the invention, such antibodies are further characterized by significantly lower binding affinity to the counter-targets LILRA1 and LILRA3, where the binding is of lower affinity compared to the binding to LILRB1 and / or LILRB2. Preferably, the binding affinity, measured by KD (monovalent), of such ABPs of this embodiment for LILRA1 and LILRA3 is greater than 50 nM, more preferably greater than 100 nM, more preferably greater than 200 nM, and most preferably greater than 400 nM, when measured using BLI under the conditions described in the Examples section herein.

[0107] In a particularly preferred embodiment, the ABP of the present invention comprises a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences, a combination represented by antibody A-046, e.g., shown in column CDR-A-046 of Table B (e.g., heavy chain CDR1, CDR2 and CDR2 having the sequences represented by SEQ ID NOs: 361, 362, and 363, respectively, and light chain CDR1, CDR2 and CDR2 having the sequences represented by SEQ ID NOs: 365, 366, and 367, respectively), with each CDR independently and selectively having no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences. In another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1-CDR3 sequence, respectively, in the combination CDR-A-046, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1-CDR3 sequence, respectively, in the combination shown in the row of Table B marked by CDR-A-046, with each CDR independently and optionally having no more than one amino acid substitution, insertion or deletion compared to these sequences. In yet another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise the variable region sequences in the heavy and light chain variable domain combinations shown in the row of Table B marked by chain A-046. In each of these particularly preferred embodiments of the ABP, optionally, the ABP can inhibit the binding of an HLA-G protein or a variant thereof to a LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of 20 nM or less, or 10 nM or less, for example, 5 nM or less. Such IC50s can be determined using methods described elsewhere herein (see Example 3). Preferably, the ABP of this embodiment has a binding affinity for LILRB1 and / or LILRB2 measured by KD (monovalent) of less than 100 nM, more preferably less than 50 nM, more preferably less than 15 nM, where the binding affinity ranges are provided for binding to both LILRB1 and LILRB2, preferably measured using BLI under the conditions described in the Examples. Thus, the ABP of this embodiment is preferably an ABP of LILRB1 and LILRB2. In another preferred embodiment of the invention, such antibodies are further characterized by significantly lower binding affinity to the counter-targets LILRA1 and LILRA3, where the binding is of lower affinity compared to the binding to LILRB1 and / or LILRB2. Preferably, the binding affinity, measured by KD (monovalent), of such ABPs of this embodiment for LILRA1 and LILRA3 is greater than 100 nM, more preferably greater than 500 nM, more preferably greater than 1000 nM, and most preferably greater than 2000 nM, when measured using BLI under the conditions described in the Examples section herein.

[0108] In a particularly preferred embodiment, the ABP of the present invention may comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences, as represented by antibody A-047, e.g., as shown in column CDR-A-047 of Table B (e.g., heavy chain CDR1, CDR2 and CDR2 having the sequences set forth in SEQ ID NOs: 369, 370, and 371, respectively, and light chain CDR1, CDR2 and CDR2 having the sequences set forth in SEQ ID NOs: 373, 374, and 375, respectively), with each CDR independently and selectively having no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences. In another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1-CDR3 sequence, respectively, in the combination CDR-A-047, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1-CDR3 sequence, respectively, in the combination shown in the row of Table B marked by CDR-A-047, with each CDR independently and optionally having no more than one amino acid substitution, insertion or deletion compared to these sequences. In yet another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise the variable region sequences in the heavy and light chain variable domain combinations shown in the row of Table B marked by chain A-047. In each of these particularly preferred embodiments of the ABP, optionally, the ABP can inhibit the binding of an HLA-G protein, or a variant thereof, to a LILRB1 and / or LILRB2 protein, or a variant thereof, with an IC50 of 20 nM or less, or 10 nM or less, for example, 5 nM or less. Such IC50s can be determined using methods described elsewhere herein (see Example 3). Preferably, the ABP of this embodiment has a binding affinity for LILRB1 and LILRB2 measured by KD (monovalent) of less than 20 nM, more preferably less than 10 nM, more preferably less than 5 nM, where the binding affinity ranges are preferably provided for binding to both LILRB1 and LILRB2 measured using BLI under the conditions described in the Examples. Thus, the ABP of this embodiment is preferably an ABP of LILRB1 and LILRB2. In another preferred embodiment of the invention, such antibodies are further characterized by significantly lower binding affinity to the counter-targets LILRA1 and LILRA3, where the binding is of lower affinity compared to the binding to LILRB1 and / or LILRB2. Preferably, the binding affinity, measured by KD (monovalent), of such ABPs of this embodiment for LILRA1 and LILRA3 is greater than 20 nM, more preferably greater than 50 nM, and more preferably greater than 100 nM, when measured using BLI under the conditions described in the Examples section herein.

[0109] In a particularly preferred embodiment, the ABP of the present invention comprises a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences, as represented by antibody A-048, e.g., as shown in column CDR-A-048 of Table B (e.g., heavy chain CDR1, CDR2 and CDR2 having the sequences set forth in SEQ ID NOs: 377, 378, and 379, respectively, and light chain CDR1, CDR2 and CDR2 having the sequences set forth in SEQ ID NOs: 381, 382, ​​and 383, respectively), and may independently and selectively contain no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion in each CDR compared to these sequences. In another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1-CDR3 sequence, respectively, in the combination CDR-A-048, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1-CDR3 sequence, respectively, in the combination shown in the row of Table B marked by CDR-A-048, with each CDR independently and optionally having no more than one amino acid substitution, insertion or deletion compared to these sequences. In yet another particularly preferred embodiment, the ABP of the present invention may be an antibody or antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise the variable region sequences in the heavy and light chain variable domain combinations shown in the row of Table B marked by chain A-048. In each of these particularly preferred embodiments of the ABP, optionally, the ABP can inhibit the binding of an HLA-G protein or a variant thereof to a LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of 20 nM or less, or 10 nM or less, for example, 5 nM or less. Such IC50s can be determined using methods described elsewhere herein (see Example 3). Preferably, the ABP of this embodiment has a binding affinity for LILRB1 and LILRB2 measured by KD (monovalent) of less than 100 nM, more preferably less than 50 nM, more preferably less than 10 nM, where the binding affinity ranges are provided for binding to both LILRB1 and LILRB2, preferably measured using BLI under the conditions described in the Examples. Thus, the ABP of this embodiment is preferably an ABP of LILRB1 and LILRB2. In another preferred embodiment of the invention, such antibodies are further characterized by significantly lower binding affinity to the counter-targets LILRA1 and LILRA3, where the binding is of lower affinity compared to the binding to LILRB1 and / or LILRB2. Preferably, the binding affinity, measured by KD (monovalent), of such ABPs of this embodiment for LILRA1 and LILRA3 is greater than 50 nM, more preferably greater than 100 nM, more preferably greater than 500 nM, and most preferably greater than 1000 nM, when measured using BLI under the conditions described in the Examples section herein.

[0110] The ABPs of the present invention are preferably ABPs that exhibit an advantageous on-target / off-target profile, as shown in the Examples section. In particular, the present invention relates to ABPs that are cross-specific for binding to LILRB1 and LILRB2, but do not bind to or bind significantly less to LILRA1 and / or LILRA2. Thus, the ABPs of the present invention are characterized by maintaining cross-specificity, but are unable to bind to their off-targets in the LILRA receptor family, specifically LILRA1 and LILRA3, or are unable to significantly bind to LILRA1 and LILRA3.

[0111] Certain embodiments of the ABPs of the present invention also relate to ABPs that bind to the ECDs of LILRB1 and LILRB2 with a binding affinity that is at least 5-fold higher, or preferably 10-fold higher, compared to the binding of the ABP to the ECDs of LILRA1 and / or LILRA3, and preferably the ABP binding affinity is determined by bio-layer interferometry (BLI).

[0112] Furthermore, in some additional or alternative embodiments, the ABP of the present invention binds to the ECD of LILRB1 and / or LILRB2 with a binding dissociation constant (KD) that is at least 2-fold lower, preferably 3-fold lower, more preferably 5-fold lower, and most preferably 10-fold lower, compared to the binding KD of the ABP to the ECD of LILRA1 and / or LILRA3, the KD being determined by biolayer interferometry (BLI), preferably under the conditions described in Example 3 herein below.

[0113] Yet another embodiment of the present invention relates to an ABP that binds to the ECDs of LILRB1 and LILRB2 with a dissociation constant (KD) that is at least 2-fold lower, preferably 3-fold lower, more preferably 5-fold lower, and most preferably 10-fold lower than the KD of the ABP for the ECDs of LILRA1 and LILRA3, the KD being determined by biolayer interferometry (BLI), preferably under the conditions described in Example 3.

[0114] A specific embodiment of the present invention relates to antibodies having the above-characterized binding profiles for LILRB1 and LILRB2 and LILRA1 and LILRA3 (non-binding or lower binding thereof), wherein such ABPs of the present invention comprise one, preferably two, antibody heavy chain sequences and one, preferably two, antibody light chain sequences, which are derived from one or a combination of antibody parent clones A-001 to A-003, preferably A-001 and / or A-003. More preferably, the ABP comprises one, preferably two, antibody heavy chain variable sequences and one, preferably two, antibody light chain variable sequences, wherein the antibody heavy and light chain variable sequences comprise sequences that are at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identical to the antibody heavy or light chain variable sequences, respectively, shown for any of the parent antibody sequences A-001 to A-003, preferably A-001 and / or A-003 (see Table 1 below).

[0115] In a further embodiment of the present invention, the ABP of the present invention is preferably an ABP that competes for binding between the ECD of LILRB1 and / or LILRB2, or the ECD of a variant of LILRB1 and / or LILRB2, and a ligand or receptor of endogenous LILRB1 and / or LILRB2, and preferably the ligand or receptor of endogenous LILRB1 and / or LILRB2 is an HLA-G protein (or a variant of HLA-G).

[0116] Specifically, such an ABP of the present invention is characterized in that the lowest affinity selected from the group consisting of the binding affinity of the ABP to the ECD of LILRB1 and the binding affinity of the ABP to the ECD of LILRB2 is still at least two-fold higher, or preferably at least three-fold higher (in some embodiments, at least five-fold higher, more preferably at least ten-fold higher) than the highest binding affinity of the ABP to the ECD of an off-target such as LILRA1 or LILRA3. In other words, preferred ABPs are characterized in that the highest KD of such ABP for LILRB1 and LILRB2 is still two-fold lower, preferably three-fold lower (in some embodiments, at least five-fold lower, more preferably at least ten-fold lower) than the lowest KD of the two KDs of such ABP for LILRA1 and LILRA3.

[0117] Further Aspects and Embodiments of the ABP of the Invention

[0118] In a second aspect, the present invention relates to an ABP that competes with the ABP of the first aspect for binding to LILRB1 and / or LILRB2 proteins (e.g., the ECD of LILRB1 and / or LILRB2 proteins) or variants thereof, and in particular to an ABP that competes with one of the above-mentioned particularly preferred ABPs for binding to LILRB1 and / or LILRB2 proteins or variants thereof.

[0119] The term "competition," when used in the context of ABPs (e.g., modulator ABPs) that compete for the same antigen (or epitope presented by such antigen), refers to competition between ABPs, which can be determined by an assay in which the ABP being tested (e.g., an antibody or binding fragment thereof) blocks or inhibits (e.g., reduces) the binding of a reference ABP (e.g., a ligand or reference antibody) to a common antigen (e.g., LILRB1 and / or LILRB2, or a fragment thereof, such as the ECD of LILRB1 and / or LILRB2).

[0120] In a related aspect, the invention relates to an ABP that binds to the same epitope as the ABP of the first aspect.

[0121] The ABP of the second aspect of the present invention may comprise one or more features (or specific combinations thereof) of the ABPs described above. In particular, the ABP of the second aspect of the present invention may inhibit (e.g., inhibit) the binding of LILRB1 and / or LILRB2 proteins to their natural ligands, and / or the ABP of the second aspect of the present invention may modulate the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or variants of LILRB1 and / or LILRB2 (e.g., as described anywhere herein).

[0122] In certain embodiments of the invention, the ABPs of the invention have (or alternatively have) the ability to inhibit (e.g., block) binding to, interaction with, LILRB1 and / or LILRB2 proteins, or variants thereof, and natural ligands of LILRB1 and / or LILRB2 proteins; as well as the ABPs of the invention (including those of the first or second aspect above) may exhibit, exhibit, or otherwise possess other functional characteristics, in particular functional characteristics related to their usefulness in sensitizing cells to cell-mediated immune responses.

[0123] In further such specific embodiments, the ABPs of the present invention can antagonize immunosuppressive macrophage polarization, such as polarization toward M2 macrophages, thereby enhancing cell-mediated immune responses. Such enhancement can be assessed using a suitable assay, such as, for example, that described in Example 6 herein.

[0124] The term "immune cell" is art-recognized to describe any cell of an organism that participates in the immune system of that organism, particularly a mammal such as a human. White blood cells (leukocytes) are immune cells involved in the innate immune system, while cells of the adaptive immune system are a specialized type of white blood cell known as lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, while T cells are involved in cellular immune responses. In preferred embodiments of the present invention, the immune cell may be a myeloid cell, such as a T cell, and in particular (e.g., when an increased cell-mediated immune response is required, such as to treat cancer), the T cell may be a cytotoxic T cell (also known as a cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell, or killer T cell). CTLs are T cells that participate in killing cancer cells, infected cells (especially viral), or otherwise damaged cells. Other preferred immune cells for such embodiments include tumor-infiltrating lymphocytes (TILs). TILs are white blood cells that have left the bloodstream and migrated to tumors.Typically, TILs are a mixture of different types of cells (i.e., T cells, B cells, NK cells) in various proportions, with T cells being the most abundant cells.TILs can often be found in the stroma and within the tumor itself, and are involved in killing tumor cells.The presence of lymphocytes in tumors is often associated with better clinical outcomes.

[0125] Other specific functional characteristics of the ABPs of the present invention are (i) enhancing cell-mediated immune responses against mammalian cells expressing LILRB1 and / or LILRB2, or variants of LILRB1 and / or LILRB2, such as those mediated by activated cytotoxic T cells (CTLs); and / or (ii) increasing the activity and / or survival (and / or proliferation) of immune cells, such as T cells, in the presence of mammalian cells expressing LILRB1 and / or LILRB2, or variants of LILRB1 and / or LILRB2. In some embodiments, the mammalian cells expressing LILRB1 and / or LILRB2 can be cells associated with a disease, disorder, or condition, such as cancer cells (directly) associated with cancer. In other examples, the mammalian cells expressing LILRB1 and / or LILRB2 can be immune cells, such as T cells (see below), e.g., immune cells directly or indirectly associated with a disease, disorder, or condition.

[0126] Other specific functional characteristics of the ABPs of the present invention that are inhibitors or antagonists of LILRB1 and / or LILRB2 expression, function, activity and / or stability are any one, or combination or at least one functional characteristic of the inhibitor or antagonist modulators described herein, particularly those described above in the section "Modulators of LILRB1 and / or LILRB2 expression, function, activity and / or stability."

[0127] These particular functional characteristics of the ABPs of the present invention, which are activators or agonists of LILRB1 and / or LILRB2 expression, function, activity and / or stability, may be any one, or combination or at least one functional characteristic of the activator or agonist modulators described herein, particularly those described above in the section "Modulators of LILRB1 and / or LILRB2 expression, function, activity and / or stability."

[0128] In preferred embodiments of all ABPs of the present invention, the ABPs are isolated and / or substantially pure.

[0129] The term "isolated," as used herein in the context of a protein, such as an ABP (an example of which may be an antibody), refers to a protein that has been purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, preventative, research, or other use. An isolated ABP according to the present invention may be a recombinant, synthetic, or modified (non-natural) ABP. In the context of a nucleic acid or cell, the term "isolated," as used herein, refers to a nucleic acid or cell that has been purified from DNA, RNA, proteins or polypeptides or other contaminants (such as other cells) that would interfere with its therapeutic, diagnostic, preventative, research, or other use, or refers to a recombinant, synthetic, or modified (non-natural) nucleic acid. Preferably, an isolated ABP or nucleic acid or cell is substantially pure. In this context, a "recombinant" protein or nucleic acid is one that is produced using recombinant techniques. Methods and techniques for the production of recombinant nucleic acids and proteins are well known in the art.

[0130] The term "isolated," as used herein in the context of a protein, such as an ABP (an example of which may be an antibody), refers to a protein that has been purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, preventative, research, or other use. An isolated ABP according to the present invention may be a recombinant, synthetic, or modified (non-natural) ABP. The term "isolated," as used herein in the context of a nucleic acid or cell, refers to a nucleic acid or cell that has been purified from DNA, RNA, proteins or polypeptides or other contaminants (such as other cells) that would interfere with its therapeutic, diagnostic, preventative, research, or other use, or refers to a recombinant, synthetic, or modified (non-natural) nucleic acid. Preferably, an isolated ABP or nucleic acid or cell is substantially pure. In this context, a "recombinant" protein or nucleic acid is one that is produced using recombinant techniques. Methods and techniques for the production of recombinant nucleic acids and proteins are well known in the art.

[0131] In some embodiments, the ABP of the present invention can bind to LILRB1 and / or LILRB2, or a paralog, ortholog, or other variant thereof (e.g., any LILRB1 and / or LILRB2 or variant thereof described herein, etc.) (e.g., via one or more epitopes presented by one or more EC domains) with a KD of less than 20 nM, such as less than about 10 nM, 5 nM, or 2 nM (particularly less than about 1 nM). In preferred embodiments, the ABP of the present invention binds to said LILRB1 and / or LILRB2, or a variant thereof (e.g., said epitope of LILRB1 and / or LILRB2), with a KD of less than 100 pM. In more preferred embodiments, the ABP of the present invention binds to said LILRB1 and / or LILRB2, or a variant thereof, with a KD of less than 10 pM. In a most preferred embodiment, the ABP of the present invention binds to the LILRB1 and / or LILRB2 or variants thereof with a KD of less than 2 pM. Binding of the ABP of the present invention, such as the antibody of the present invention, to a human cell line expressing the LILRB1 and / or LILRB2 or variants thereof may, in some embodiments, occur with an EC50 of less than about 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, or 0.2 μg / mL, preferably an EC50 of less than 2 μg / mL. The ABPs of the present invention, such as the antibodies of the present invention, may in some embodiments bind to cynomolgus monkey cell lines expressing LILRB1 and / or LILRB2, or orthologs of their mutants, with an EC50 of less than about 10 μg / mL, less than 5 μg / mL, less than 2 μg / mL, 1 μg / mL, 0.5 μg / mL, or 0.2 μg / mL, preferably with an EC50 of less than 2 μg / mL.

[0132] In other embodiments, the ABP of the present invention (i) can bind to LILRB1 and / or LILRB2, or a variant of LILRB1 and / or LILRB2 thereof, with a KD of less than 20 nM, e.g., less than about 10 nM, 5 nM, or 2 nM (particularly less than about 1 nM), less than 100 pM, or less than 10 pM; and / or (ii) can bind to a human cell line expressing LILRB1 and / or LILRB2, or a variant of LILRB1 and / or LILRB2 thereof, with an EC50 of less than 2 μg / mL.

[0133] In some embodiments, the ABP of the invention may not bind to LILRA counters that target LILRA, such as not specifically binding to LILRA1 and LILRA3, or their orthologs or other variants. In alternative embodiments, the ABP of the invention binds to one or more, or any, of the LILRA proteins (preferably their ECDs) with a KD that is at least 2-fold higher, more preferably at least 3-fold, 4-fold, 5-fold, or 10-fold higher than its KD for binding to LILRB1 and / or LILRB2. In other such embodiments, the ABP of the invention binds to LILRA proteins with a KD of greater than 100 nM, preferably greater than 50 nM, and even more preferably greater than 20 nM.

[0134] The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined by plasmon resonance (BIAcore) or other methods. (登録商標) ), ELISA, and KINEXA. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably BIAcore. (登録商標) Another preferred method of the present invention for determining the KD values ​​of the ABPs of the present invention is by using a biosensor system such as the Octet Red system or by ELISA. (登録商標)The term "K" (or "K-assoc"), as used herein, broadly refers to the association rate of a particular antibody-antigen interaction, while the term "Kd" (or "K-diss"), as used herein, refers to the dissociation rate of a particular antibody-antigen interaction.

[0135] In yet another embodiment, the ABP of the present invention can compete with a ligand or receptor of endogenous LILRB1 and / or LILRB2 for binding to LILRB1 and / or LILRB2, or a variant thereof of LILRB1 and / or LILRB2, preferably wherein the ligand or receptor of endogenous LILRB1 and / or LILRB2 is HLA-G (or a variant of HLA-G). For example, in certain such embodiments, the ABP of the present invention (e.g., one that binds to (one or more epitopes presented by) the extracellular domain(s) of LILRB1 and / or LILRB2, or a paralog, ortholog, or other variant thereof) can inhibit (e.g., inhibit) the binding of HLA-G protein or a variant thereof to LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of 100 nM or less, 50 nM or less, or preferably 20 nM or less, e.g., 15 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less. In particular, in such embodiments, the ABP of the present invention can inhibit (e.g., inhibit) the binding of HLA-G protein or a variant thereof to LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of 10 nM or less, e.g., 5 nM or less, preferably 2 nM or less.

[0136] In one embodiment, the ABP of the present invention is a polyclonal antibody (mixture) or the antigen-binding fragment is a fragment of a polyclonal antibody (mixture).

[0137] In an alternative and preferred embodiment of all ABPs of the present invention, the ABP is an antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody, or the antigen-binding fragment is a fragment of a monoclonal antibody.

[0138] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of antibodies that are substantially identical based on their amino acid sequence. Monoclonal antibodies are typically highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (e.g., epitopes) on an antigen, each mAb is typically directed against a single determinant on the antigen. In addition to their specificity, mAbs are advantageous in that they can be synthesized by cell culture (e.g., hybridomas, recombinant cells, etc.) uncontaminated by other immunoglobulins. mAbs, as used herein, include, for example, chimeric, humanized, or human antibodies or antibody fragments.

[0139] Monoclonal antibodies according to the present invention can be prepared by methods well known to those skilled in the art. For example, mice, rats, or rabbits can be immunized with an antigen of interest together with an adjuvant. Splenocytes are collected as a pool from animals receiving several immunizations at regular intervals, with test bleeds performed to assess serum antibody titers. Splenocytes are either used immediately in fusion experiments or stored in liquid nitrogen for use in future fusions. Fusion experiments are then performed according to the procedure of Stewart & Fuller, J. Immunol Methods 1989, 123:45-53. Supernatants from wells containing growing hybrids are screened for mAb secretion, for example, by enzyme-linked immunosorbent assay (ELISA). ELISA-positive cultures are cloned by either limiting dilution or fluorescence-activated cell sorting, typically resulting in established hybridomas from single colonies. The ability of antibodies, including antibody fragments or subfragments, to bind to a specific antigen can be determined by binding assays known in the art, for example, using the antigen of interest as a binding partner.

[0140] In a further preferred embodiment, the ABP of the present invention is an antibody or an antigen-binding fragment thereof, wherein the antibody is a human antibody, a humanized antibody or a chimeric human antibody, or the antigen-binding fragment is a fragment of a human antibody, a humanized antibody or a chimeric human antibody.

[0141] Human antibodies can also be derived by in vitro methods. Suitable examples include, but are not limited to, phage display (CAT, MorphoSys, Dyax, Biosite / Medarex, Xoma, Yumab, Symphogen, Alexion, Affimed, etc.). In phage display, polynucleotides encoding single Fab or Fv antibody fragments are expressed on the surface of phage particles (see, e.g., Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581(1991); U.S. Patent No. 5,885,793). Phages are "screened" to identify antibody fragments with affinity for the target. Thus, certain such processes mimic immune selection through the display of antibody fragment repertoires on the surface of filamentous bacteriophage and subsequent selection of phage by their binding to the target. Certain such procedures result in the isolation of high-affinity, functional, neutralizing antibody fragments. Thus, a complete repertoire of human antibody genes can be generated by cloning naturally rearranged human V genes from peripheral blood lymphocytes (see, e.g., Mullinax et al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990)), or by generating fully synthetic or semi-synthetic phage display libraries containing human antibody sequences (see, Knappik et al. 2000; J Mol Biol 296:57; de Kruif et al., 1995; J Mol Biol 248:97).

[0142] Alternatively, the antibodies described herein may be derived from XenoMouse (登録商標)Such mice can be prepared using techniques that allow for the production of human immunoglobulin molecules and antibodies, and are deficient in the production of mouse immunoglobulin molecules and antibodies. In particular, preferred embodiments of transgenic production of mice and antibodies are disclosed in U.S. Patent Application No. 08 / 759,620, filed December 3, 1996, WO 98 / 24893, published June 11, 1998, and WO 00 / 76310, published December 21, 2000. See also Mendez et al., Nature Genetics, 15:146-156 (1997). Using such techniques, fully human monoclonal antibodies against a variety of antigens have been generated. Essentially, the XenoMouse (登録商標) Mice of a certain strain are immunized with an antigen of interest, such as LILRB1 and / or LILRB2, lymphocytes (e.g., B cells) are harvested from the hyperimmunized mice, and the harvested lymphocytes are fused with a myeloid cell line to prepare immortalized hybridoma cell lines. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. Other "humanized" mice are also commercially available: for example, Medarex mice, Kymab-Kymouse, Regeneron-Velocimmune mice, Kirin-TC mice, Trianni-Trianni mice, Omniab-Omni mice, Harbour Antibodies-H2L2 mice, and Merus-MeMo mice. Other "humanized" species are also available: rats: OmniAb-Omni rats, OMT-Uni rats; chickens: OmniAb-Omni chickens.

[0143] The term "humanized antibody" according to the present invention refers to an immunoglobulin chain or fragment thereof (e.g., Fab, Fab', F(ab')2, Fv, or other antigen-binding subsequence of an antibody) that contains minimal sequence (but typically still at least a portion) derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human immunoglobulin species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. Thus, at least a portion of the framework sequences of the antibody or fragment thereof may be human consensus framework sequences. In some instances, Fv framework residues of the human immunoglobulin may need to be replaced by corresponding non-human residues to increase specificity or affinity. Furthermore, humanized antibodies may contain residues found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, and typically at least two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which may be modified (e.g., by mutation or glycoengineering) to optimize one or more properties of such region and / or to improve the function of the (e.g., therapeutic) antibody, e.g., to increase or decrease Fc effector function or increase serum half-life. Exemplary such Fc modifications (e.g., Fc engineering or Fc enhancement) are described elsewhere herein.

[0144] The term "chimeric antibody" according to the present invention refers to an antibody whose light and / or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions that are identical or homologous to corresponding sequences from different species, such as mouse and human. Alternatively, the variable region genes may be derived from a particular antibody class or subclass, while the remainder of the chains are derived from another antibody class or subclass, either from the same or a different species. It also encompasses fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domains from a mouse antibody and the constant or effector domains from a human antibody, although other mammalian species may also be used.

[0145] In particular such embodiments, the ABP of the invention comprises an antigen-binding domain of an antibody, wherein the antigen-binding domain is that of a human antibody. Preferably, the ABP comprises an antigen-binding domain of an antibody or antigen-binding fragment thereof that is a human antigen-binding domain; (ii) the antibody is a monoclonal antibody or the antigen-binding fragment is a fragment of a monoclonal antibody; (iii) the antibody is a human antibody or a humanized antibody or the antigen-binding fragment is a fragment of a human antibody, a humanized antibody or a chimeric human antibody.

[0146] Human antibody light chains are generally classified as kappa and lambda light chains, each of which contains one variable region and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Human IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. Human IgM subtypes include IgM and IgM2. Human IgA subtypes include IgA1 and IgA2. In humans, IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and IgM isotypes contain 10 or 12 heavy chains and 10 or 12 light chains. The antibodies according to the invention may be IgG, IgE, IgD, IgA, or IgM immunoglobulins.

[0147] In some embodiments, the ABP of the present invention is an IgG antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgE antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgD antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgA antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgM antibody or a fragment thereof. Preferably, the ABP of the present invention is, comprises, or is derived from an IgG immunoglobulin or a fragment thereof; for example, a human, human-derived IgG immunoglobulin, or a rabbit- or rat-derived IgG and / or IgG2 immunoglobulin, or a fragment thereof. When the ABP of the present invention is, comprises, or is derived from a rat-derived IgG, preferably the ABP is, comprises, or is derived from a rat IgG2a or IgG2b immunoglobulin. When the ABP of the present invention is, comprises or is derived from human-derived IgG, more preferably the ABP of the present invention is, comprises or is derived from human IgG1, IgG2 or IgG4, and most preferably the ABP of the present invention is, comprises or is derived from human IgG1 or IgG2.

[0148] Thus, in a particular embodiment of the invention, the ABP is an antibody, wherein the antibody is an IgG, IgE, IgD, IgA, or IgM immunoglobulin, preferably an IgG immunoglobulin.

[0149] When the ABP of the invention comprises at least a portion of an immunoglobulin constant region (typically that of a human immunoglobulin), it may have such (e.g., human) immunoglobulin constant region modified, for example by glycoengineering or mutation, to optimize one or more properties of such region and / or to improve the function of the (e.g., therapeutic) antibody, for example to increase or decrease Fc effector function or to increase serum half-life.

[0150] The ABPs of the present invention, particularly those useful in the present methods, include antibodies that induce antibody-dependent cellular cytotoxicity (ADCC) of cells expressing LILRB1 and / or LILRB2. The ADCC of anti-LILRB1 and / or LILRB2 antibodies can be improved by using antibodies with low levels of fucose or lacking fucose. Antibodies lacking fucose have been correlated with enhanced ADCC (antibody-dependent cellular cytotoxicity) activity, particularly at low antibody doses (Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466).

[0151] A method for preparing fucoseless antibodies or antibodies with reduced fucose levels involves growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes an enzyme required for the fucosylation of polypeptides (alpha 1,6-fucosyltransferase).

[0152] Alternatively, during the expression of such antibodies, inhibitors of enzymes involved in sugar chain modification may be used, including tunicamycin, which selectively inhibits the formation of GlcNAc-PP-Dol, the first step in the formation of core oligosaccharides, the precursors of N-glycoside-linked sugar chains; castanospermine and W-methyl-l-deoxynojirimycin, which are inhibitors of glycosidase I; kifunensine, which is an inhibitor of mannosidase I; bromoconduritol, which is an inhibitor of glycosidase II; 1,4-deoxy-1,4-imino-D-mannitol, which is an inhibitor of mannosidase I; and swainsonine, which is an inhibitor of mannosidase II. Examples of glycosyltransferase-specific inhibitors include deoxy derivatives of substrates for N-acetylglucosamine transferase V (GnTV). In addition, 1-deoxynojirimycin is known to inhibit the synthesis of complex glycans and increase the proportion of high-mannose and hybrid glycans (Glycobiology series 2 -Destiny of Sugar Chain Cell, Katsutaka Nagai, SeniChiro Hakomori and Akira Kobata, 1993).

[0153] Based on these data, several cell lines have been genetically engineered to produce antibodies that are fucose-free or contain low levels of fucose, in order to manipulate the glycosylation pattern of IgG in order to select therapeutic monoclonal antibodies that exhibit specific profiles of Fc-γ-R binding that can be used in various pathologies (Mori et al., 2004; Yamane-Ohnuki et al., 2004).

[0154] Umana et al. and Davis et al. showed that IgG1 antibodies engineered to contain increasing amounts of bisected complex oligosaccharides (bisected A / -acetylglucosamine, GIcNAC) were able to induce potent ADCC compared to their parental counterparts (Umana et al., 1999; Davies et al., 2001). Second, the absence of fucose on human IgG1 N-linked oligosaccharides has been shown to improve FCGRIII binding and ADCC.

[0155] GLYCART BIOTECHNOLOGY AG (Zurich, CH) expressed N-acetylglucosaminyltransferase III (GnTIII), which catalyzes the addition of bisecting GIcNac residues to N-linked oligosaccharides, in a Chinese hamster ovary (CHO) cell line and demonstrated greater ADCC of the produced IgG1 antibody (WO 99 / 54342; WO 03 / 01 1878; WO 2005 / 044859).

[0156] WO2007 / 0166306 relates to the modification of the antibody anti-CD19, which contains 60% N-acetylglucosamine bisecting oligosaccharides and 10% non-fucosylated N-acetylglucosamine bisecting oligosaccharides, produced in mammalian human 293T embryonic kidney cells transfected with (i) the cDNA of the anti-CD19 antibody and (ii) the cDNA of the GnTIII enzyme.

[0157] Recombinant human IgG1 produced in YB2 / 0 cells (Shinkawa et al., 2003; Siberil et al., 2006) or CHO-Lecl3 (Shields et al., 2002) exhibited low fucose content or were deficient in fucose and showed enhanced ability to induce cell-mediated cytotoxicity compared to the same IgG1 produced in wild-type CHO cells. Conversely, no correlation between galactose and ADCC was observed, and bisecting GIcNAC content had only a slight effect on ADCC (Shinkawa et al., 2003).

[0158] By removing or replacing fucose from the Fc portion of antibodies, Kyowa Hakko Kogyo (Tokyo, Japan) enhanced Fc binding, improved ADCC, and therefore improved MAb efficacy (US 6,946,292). This improved Fc-γ-RIIIA-dependent effector function of hypofucosylated IgG has been shown to be independent of the Fc-γ-RIII allelic form (Niwa et al., 2005). Furthermore, it has recently been shown that the antigen density required to induce efficient ADCC is lower when IgG has a low fucose content compared to highly fucosylated IgG (Niwa et al., 2005).

[0159] Laboratoire Francais du Fractionnement et des Biotechnologies (LFB) (France) has shown that the Fuc / Gal ratio in MAb oligosaccharides should be 0.6 or less to obtain antibodies with high ADCC (FR 2 861 080).

[0160] Cardarelli et al., 2019, produced an anti-CD19 antibody in Ms-704PF CHO cells lacking the FUT8 gene, which encodes α-1,6-fucosyltransferase. The defucosylation of the antibody in this paper required engineering an enzyme-deficient cell line. Amino acid mutations were not considered in this report.

[0161] Herbst et al. generated a humanized IgG1 MAb, MEDI-551, expressed in a fucosyltransferase-deficient producer CHO cell line. This paper does not consider amino acid mutations (Herbst et al., 2010). S. Siberil et al. produced a MAb, anti-RhD, with a low fucose content using the rat myeloma YB2 / 0 cell line. The MAb produced in wild-type CHO cells exhibited a high fucose content (81%), whereas the same MAb produced in YB2 / 0 cells exhibited a lower fucose content (32%). This paper considers amino acid mutations (Siberil et al., 2006).

[0162] Thus, the ABPs of the present invention can be prepared with and / or have one or more of such glycoengineering (eg, afucosylation) approaches / antibody features described above.

[0163] An alternative method for increasing the ADDC activity of the ABPs of the present invention involves mutations in the Fc portion of such ABPs, particularly mutations that increase antibody affinity for the Fc-γ-R receptors.

[0164] Therefore, any of the above-described ABPs of the present invention can be produced using different antibody isotypes or mutant isotypes to control the degree of binding to different Fc-γ receptors. Antibodies lacking the Fc region (e.g., Fab fragments) lack binding to different Fc-γ receptors. The choice of isotype also affects binding to different Fc-γ receptors. The affinities of various human IgG isotypes for three different Fc-γ receptors, Fc-γ-RI, Fc-γ-RII, and Fc-γ-RIII, have been determined (Ravetch & Kinet, Annu. Rev. Immunol. 9, 457 (1991)). Fc-γ-RI is a high-affinity receptor that binds IgG in its monomeric form, while the latter two are low-affinity receptors that bind IgG only in its multimeric form. In general, both IgG1 and IgG3 have significant binding activity to three receptors, IgG4 to Fc-γ-RI, and IgG2, with only one type of Fc-γ-RII, called IIaLR (see Parren et al., J. Immunol. 148, 695 (1992)). Thus, the human isotype IgG1 is usually selected for stronger binding to Fc-γ receptors, while IgG2 or IgG4 are usually selected for weaker binding.

[0165] The correlation of increased Fc-γ-R binding with mutant Fc has been demonstrated using target cytotoxicity cell-based assays (Shields et al., 2001, J. Biol. Chem. 276:6591-6604; Presta et al., 2002, Biochem Soc. Trans. 30:487-490). Methods for increasing ADCC activity through specific Fc region mutations include Fc variants comprising at least one amino acid substitution at a position selected from the group consisting of 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, and 332, where the numbering of residues in the Fc region is that of the EU index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest) (National Institutes of Health, Bethesda, Md. 1987).

[0166] In a specific embodiment, said Fc variants comprise at least one substitution selected from the group consisting of the following, wherein the numbering of residues in the Fc region is that of the EU index as in Kabat: L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239D, S239E, S239N, S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T, V240M, F241W, F241L, F241Y, F241E, F241R, F243W, F243L, F243Y, F243R, F243Q, P244H, P245A, P247V,P247G, V262I, V262A, V262T, V262E, V263I, V263A, V263T, V263M, V264L, V264I, V264W, V264T, V264R, V264F,V264M, V264Y, V264E, D265G, D265N, D265Q, D265Y, D265F, D265V, D265I, D265L, D265H, D265T, V266I, V266A, V266T, V266M, S267Q, S267L, E269H, E269Y, E269F, E269R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T, Y296L,Y296I, Y296H, N297S, N297D, N297E, A298H, T299I, T299L, T299A, T299S, T299V, T299H, T299F, T299E, W313F,N325Q, N325L, N325I, N325D, N325E, N325A, N325T, N325V, N325H, A327N, A327L, L328M1328D, L328E, L328N, L328Q, L328F, L328I, L328V, L328T, L328H, L328A, P329F, A330L, A330Y, A330V, A330I, A330F, A330R, A330H, I332D, I332E, I332N, I332Q, I332T, I332H, I332Y and I332A.

[0167] The Fc variant may also be selected from the group consisting of the following, where the Fc region is of the EU index as in Kabat, see also WO2004029207, which is incorporated herein by reference: V264L, V264I, F241W, F241L, F243W, F243L, F241L / F243L / V262I / V264I, F241W / F243W, F241W / F243W / V262A / V264A, F241L / V262I, F243L / V264I, F243L / V262I / V264W, F241Y / F243Y / V262T / V264T, F241E / F243R / V262E / V264R, F241E / F243Q / V262T / V264E, F241R / F243Q / V262T / V264R, F241E / F243Y / V262T / V264R, L328M1328E, L328F, I332E, L3238M / I332E, P244H, P245A, P247V, W313F, P244H / P245A / P247V, P247G, V264I / I332E, F241E / F243R / V262E / V264R / I332E, F241E / F243Q / V262T / V264E / I332E, F241R / F243Q / V262T / V264R / I332E, F241E / F243Y / V262T / V264R / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, D265G, D265N, S239E / D265G, S239E / D265N, S239E / D265Q, Y296E, Y296Q, T299I, A327N, S267Q / A327S, S267L7A327S, A327L, P329F, A330L, A330Y, I332D, N297S, N297D, N297S / I332E, N297D / I332E, N297E / I332E, D265Y / N297D / I332E D265Y / N297D / T299L7I332E, D265F / N297E / I332E, L328I / I332E, L328Q / I332E, I332N, I332Q, V264T, V264F, V240I, V263I, V266I, T299A, T299S, T299V, N325Q, N325L, N325I, S239D, S239N, S239F, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q,S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239N / I332N, S239N / I332Q, S239Q / I332D, S239Q / I332N, S239Q / I332Q, Y296D, Y296N, F241Y / F243Y / V262T / V264T / N297D / I332E, A330Y / I332E, V264I / A330Y / I332E, A330L7I332E, V264I / A330L / I332E, L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239T, S239H, S239Y, V240A, V240T, V240M, V263A, V263T, V263M, V264M, V264Y, V266A, V266T, V266M, E269H, E269Y, E269F, E269R, Y296S, Y296T, Y296L, Y296I, A298H, T299H A330V, A330I, A330F, A330R, A330H, N325D, N325E, N325A, N325T, N325V, N325H, L328D / I332E, L328E / I332E, L328N / I332E, L328Q / I332E, L328V / I332E, L328T / I332E, L328H / I332E, L328I / I332E, L328A, I332T, I332H, I332Y, I332A, S239E / V264I / I332E, S239Q / V264I / I332E S239E / V264I / A330Y / I332E, S239E / V264I / S298A / A330Y / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D265V / N297D / I332E, S239D / D265I / N297D / I332E, S239D / D265L / N297D / I332E, S239D / D265F / N297D / I332E,S239D / D265Y / N297D / I332E, S239D / D265H / N297D / I332E, S239D / D265T / N297D / I332E, V264E / N297D / I332E, Y296D / N297D / I332E, Y296E / N297D / I332E, Y296N / N297D / I332E, Y296Q / N297D / I332E, Y296H / N297D / I332E, Y296T / N297D / I332E, N297D / T299V / I332E, N297D / T299I / I332E, N297D / T299L / I332E, N297D / T299F / I332E, N297D / T299H / I332E, N297D / T299E / I332E, N297D / A330Y / I332E, N297D / S298A / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L7I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, and S239D / 264I / A330L / I332E.

[0168] In certain embodiments, mutations at, adjacent to, or nearby sites in the hinge link region (e.g., substitution of residues 234, 235, 236, and / or 237 with other residues) can reduce affinity for Fc-γ receptors, particularly Fc-γ-RI receptors, in all isotypes (see, e.g., US6624821). Alternatively, positions 234, 236, and / or 237 are substituted with alanine, and position 235 is substituted with glutamic acid (see, e.g., US5624821). In the human IgG2 isotype, position 236 is deleted. Exemplary segments of amino acids at positions 234, 235, and 237 of human IgG2 are AlaAlaGly, VaiAlaAla, AlaAlaAla, VaiGluAla, and AlaGluAla. A preferred combination of mutations is L234A, L235E, and G237A, or for human isotype IgG1, L234A, L235A, and G237A. Particularly preferred ABPs of the present invention are antibodies with human isotype IgG1 and one of these three mutations in the Fc region. Other substitutions that reduce binding to Fc-γ receptors are the E233P mutation (particularly in mouse IgG1) and D265A (particularly in mouse IgG2a). Other examples of mutations and combinations of mutations that reduce Fc and / or C1q binding are E318A / K320A / R322A (particularly in mouse IgG1), L235A / E318A / K320A / K322A (particularly in mouse IgG2a). Similarly, residue 241 (Ser) in human IgG4 can be substituted, for example, with proline to disrupt Fc binding.

[0169] Additional mutations can be made in the constant region to modulate effector activity. For example, mutations can be made to the IgG1 or IgG2 constant region at A330S, P331S, or both. For IgG4, mutations can be made at E233P, F234V, and L235A, with a G236 deletion, or any combination thereof. IgG4 can also have one or both of the mutations S228P and L235E. The use of disrupted constant region sequences to modulate effector function is further described, for example, in WO2006 / 118,959 and WO2006 / 036291.

[0170] Additional mutations can be made in the constant region of human IgG to modulate effector activity (see, for example, WO2006 / 03291). These include the following substitutions: to human IgG1 (i) A327G, A330S, P331S; (ii) E233P, L234V, L235A, G236 deletion; (iii) E233P, L234V, L235A; (iv) E233P, L234V, L235A, G236 deletion, A327G, A330S, P331S; and (v) E233P, L234V, L235A, A327G, A330S, P331S; or particularly (vi) L234A, L235E, G237A, A330S, and P331S (e.g., to human IgG1), where the numbering of the residues in the Fc region is that of the EU index in Kabat. See also WO2004 / 029207, which is incorporated herein by reference.

[0171] The affinity of an antibody for Fc-γ-R can be altered by mutating specific residues in the heavy chain constant region. For example, disruption of the glycosylation site of human IgG1 can reduce the Fc-γ-R binding and therefore the effector function of the antibody (see, for example, WO2006 / 036291). The tripeptide sequences NXS and NXT (where X is any amino acid except proline) are enzyme recognition sites for glycosylation of N residues. Disruption of any of the tripeptide amino acids, particularly in the CH2 region of IgG, prevents glycosylation at that site. For example, mutation of N297 of human IgG1 prevents glycosylation and reduces Fc-γ-R binding to the antibody.

[0172] While ADCC and CDC activation is often desirable for therapeutic antibodies, there are situations in which an ABP of the present invention that cannot activate effector function is preferred (e.g., an ABP of the present invention that is an agnostic modulator). For these purposes, IgG4 has commonly been used, but this has recently fallen out of favor due to this subclass's unique ability to undergo Fab arm exchange, which allows heavy chain exchange between IgG4s in vivo, as well as residual ADCC activity. Therefore, an Fc engineering approach can be used to determine the key interaction sites of the Fc domain with Fc-γ receptors and C1q, and then these positions in the Fc of an ABP of the present invention can be mutated to reduce or eliminate binding. Alanine scanning by Duncan and Winter (1998; Nature 332:738) initially isolated the C1q binding site to the region surrounding the hinge and upper CH2 of the Fc domain. Genmab researchers identified the mutations K322A, L234A, and L235A and found that, in combination, they were sufficient to almost completely abolish Fc-γ-R and C1q binding (Hezareh et al., 2001; J Virol 75:12161). Using a similar approach, MedImmune later identified a set of three mutations, L234F / L235E / P331S (DUBED™), with very similar effects (Oganesyan et al., 2008; Acta Crystallographica 64:700). Another approach is to modify the glycosylation on asparagine 297 of the Fc domain, known to be required for optimal FcR interaction.Loss of binding to Fc-γR has been observed with N297 point mutations (Tao et al, 1989; J Immunol 143:2595), enzymatically deglycosylated Fc domains (Mimura et al, 2001; J Biol Chem 276:45539), antibodies recombinantly expressed in the presence of glycosylation inhibitors (Walker et al, 1989; Biochem J 259:347), and expression of the Fc domain in bacteria (Mazor et al 2007; Nat Biotechnol 25:563). Accordingly, the present invention also includes embodiments of ABPs in which such techniques or mutations have been used to reduce effector function.

[0173] IgG naturally persists for extended periods in (e.g., human) serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Despite this, numerous efforts have been made to manipulate the pH-dependent interaction between the Fc domain and FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. Researchers at PDL BioPharma have resulted in an approximately two-fold increase in IgG half-life in rhesus monkeys (Hinto et al., 2004; J Biol Chem 279:6213), and researchers at MedImmune have identified the mutation M252Y / S254T / T256E (termed YTE), which resulted in an approximately four-fold increase in IgG half-life in cynomolgus monkeys (Dall'Acqua et al., 2006; J Biol Chem 281:23514). The combination of the M252Y / S254T / T256E mutations with the point mutations H433K / N434F produces a similar effect (Vaccaro et al., 2005, Nat Biotechnol. Oct;23(10):1283-8). The ABP of the present invention may be PEGylated. PEGylation, i.e., chemical coupling with the synthetic polymer polyethylene glycol (PEG), has emerged as an accepted technique for developing long-acting biological agents, with approximately 10 clinically approved protein and peptide drugs to date (Jevsevar et al., 2010; Biotechnol J 5:113). The ABP of the present invention may also be subjected to PASylation, a biological alternative to PEGylation, to extend the plasma half-life of pharmaceutically active proteins (Schlapschy et al., 2013; Protein Eng Des Sei 26:489; XL-protein GmbH, Germany). Similarly, XTEN half-life extension technology from Amunix offers another biological alternative to PEGylation (Schellenberger, 2009, Nat Biotechnol;27(12):1186-90;doi:10.1038 / nbt.l588).Thus, the present invention also includes embodiments of ABPs in which such techniques or mutations are used to extend serum half-life, particularly in human serum.

[0174] Antibody fragments include "Fab fragments," which consist of one constant domain and one variable domain from each of the heavy and light chains, held together by the adjacent constant region of the light chain and the first constant domain (CH1) of the heavy chain. These can be formed from conventional antibodies by protease digestion, for example with papain, although similar Fab fragments can also be produced by genetic engineering. Fab fragments include Fab', Fab, and "Fab-SH" (Fab fragments containing at least one free sulfhydryl group).

[0175] Fab' fragments differ from Fab fragments in that they contain additional residues at the carboxy terminus of the first constant domain of the heavy chain including one or more cysteines from the antibody hinge region. Fab' fragments include "Fab'-SH" (Fab' fragments containing at least one free sulfhydryl group).

[0176] Further, antibody fragments include F(ab')2 fragments, which contain two light chains and two heavy chains containing a portion of the constant region ("hinge region") between the CH1 and CH2 domains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains. F(ab')2 fragments can be prepared from conventional antibodies by proteolytic cleavage with an enzyme that cleaves below the hinge region, for example, using pepsin, or by genetic engineering.

[0177] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions. A "single-chain antibody" or "scFv" is an Fv molecule in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding region.

[0178] The "Fc region" comprises two heavy chain fragments containing the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0179] Thus, in some embodiments, the ABP of the present invention is an antibody fragment selected from the list consisting of Fab', Fab, Fab'-SH, Fab-SH, Fv, scFv, and F(ab')2.

[0180] In those embodiments of ABPs that are fragments of immunoglobulins, such as antibody fragments, preferred are fragments that are capable of binding to the extracellular domain(s) of LILRB1 and / or LILRB2, or paralogs, orthologs, or other variants thereof (e.g., epitopes presented thereby), e.g., have any of the epitopes or other binding properties described herein, and more preferably, the fragments are modulators (e.g., inhibitors or antagonists) of the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or paralogues, orthologs, or other variants of LILRB1 and / or LILRB2.

[0181] In a preferred embodiment, the ABP of the present invention is an antibody in which at least a portion of the framework sequence of the antibody or fragment thereof is a human consensus framework sequence, for example, comprises a human germline-encoded framework sequence.

[0182] In some embodiments, the ABPs of the present invention are modified or engineered to increase antibody-dependent cellular cytotoxicity (ADCC). As will be appreciated by those skilled in the art, such ABPs of the present invention are particularly useful in treating diseases or disorders associated with cellular resistance to immune cells such as CTLs (e.g., LILRB1 and / or LILRB2 positive cancers), in that the ADCC mechanism (cell-mediated immune defense in which effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound by specific antibodies) is enhanced for cells that have resistance to immune cells such as CTLs, thus resulting in increased adhesion and / or lysis of such cells by effector cells of the immune system.

[0183] As used herein, "treatment" is synonymous with treating a disease, disorder or condition, including reducing the symptoms of the disease, disorder or condition, inhibiting the disease, disorder or condition, causing regression of the disease, disorder or condition, and / or curing the disease, disorder or condition.

[0184] Various techniques are known for modifying or engineering the ABPs of the invention to increase ADCC (Satoh et al., 2006; Expert Opin Biol Ther 6:1161; WO2009 / 135181), and such embodiments accordingly include those in which the ABPs of the invention can be afucosylated (GlycArt Biotechnology), e.g., the antibody is produced in CHO cells in which the endogenous FUT8 gene has been knocked out; or those in which the ABP can be a "glycoengineered antibody" (Seattle Genetics), e.g., in which a fucose analog is added to the antibody-expressing CHO cells, resulting in a significant reduction in fucosylation. Other afucosylation approaches that can be applied to the ABPs of the invention are described elsewhere herein.

[0185] Other techniques for modifying or engineering the ABPs of the invention to increase ADCC include mutations in the Fc portion of the ABP (as described in more detail elsewhere herein), in particular, one or more of human Fc residues 234, 235, 236 and / or 237, and / or residues 330, 331, are so mutated; such numbering of residues in the Fc region is according to the EU index in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest) (National Institutes of Health, Bethesda, Md. 1987).

[0186] Thus, in certain embodiments, the ABPs of the invention are modified or engineered to increase antibody-dependent cell-mediated cytotoxicity (ADCC), preferably wherein the ABP is afucosylated and / or the Fc of the ABP is mutated. In alternative embodiments, the ABPs of the invention are modified or engineered to decrease ADCC (e.g., when the Fc is mutated using one or more of the following residue changes: L234A, L235E, G237A, A330S, and / or P331S).

[0187] In other specific embodiments, the ABPs of the invention are modified to extend their serum half-life, particularly in human serum. For example, the ABPs of the invention may be PEGylated and / or PASylated, or may have an Fc region with T250Q / M428L, H433K / N434F / Y436, or M252Y / S254T / T256E / H433K / N434F modifications.

[0188] The ABPs of the present invention may be monospecific (i.e., have an antigen-binding domain that binds only one antigen) or multispecific (i.e., have two or more different antigen-binding domains that bind different antigens). For example, a "bispecific," "dual-specific," or "bifunctional" ABP or antibody is a hybrid ABP or antibody, respectively, that has two different antigen-binding sites. Bispecific antigen-binding proteins and antibodies are species of multispecific antigen-binding protein antibodies and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments (see, e.g., Songsivilai and Lachmann, 1990; Kostelny et al., 1992). The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which may be present on the same or different protein targets.

[0189] In certain such embodiments, the ABP may be a bispecific, trispecific, or tetraspecific antibody, and in particular the bispecific antibody is selected from the following: a T cell engager (BiTE) antibody, a dual affinity retargeting molecule (DART), a CrossMAb antibody, a DutaMab (商標) Antibodies, DuoBody antibodies; Triomab, TandAb, Bispecific NanoBody, Tandem scFv, Diabody, Single Chain Diabody, HSA Body, (scFv)2 HSA Antibody, scFv-IgG Antibody, Dock and Lock Bispecific Antibody, DVD-IgG Antibody, TBTI DVD-IgG, IgG-fynomer, Tetravalent Bispecific Tandem IgG Antibody, Dual Targeting Domain Antibody, Chemically Linked Bispecific (Fab')2 Molecules, Crosslinked mAb, Dual Acting Fab IgG (DAF-IgG), Ortho Fab-IgG, Bispecific CovX-Body, Bispecific Hexavalent Trimer, and ART-Ig.

[0190] Thus, in certain embodiments, the ABP of the invention is a multispecific antibody comprising at least two antigen-binding domains, each antigen-binding domain specifically binding to a different antigen epitope.

[0191] In some such embodiments of such an ABP, at least two of the different antigenic epitopes are epitopes presented by the ECD of the LILRB1 and / or LILRB2 protein, or at least one of the different antigenic epitopes is an epitope presented by the ECD of the LILRB1 and / or LILRB2 protein, and at least one of the different antigenic epitopes is an epitope presented by a protein other than LILRB1 and / or LILRB2, preferably an epitope other than an epitope presented by a protein other than HLA-G.

[0192] Thus, in some embodiments, the ABP of the present invention, when expressed on the surface of a mammalian cell, binds to the extracellular domain of LILRB1 and / or LILRB2, paralogs, orthologs, or other variants (e.g., via one or more first antigen-binding domains) and further comprises one or more additional antigen-binding domains that bind to an antigen(s) other than LILRB1 and / or LILRB2 or their variants. In certain embodiments of the ABP of the present invention, such other antigens may be another immunoglobulin superfamily gene (preferably not LILRA); and / or such other antigens may be antigens present on mammalian T cells. Antigens present on mammalian T cells that can be bound by such additional antigen-binding domains include CD3, CD40, OX-40, ICOS, and 4-1BB. In certain embodiments of the ABP of the present invention, such other antigens may be albumin, e.g., human albumin. It may also be another component of blood or serum, the binding of which by the ABP confers on the ABP an extended serum half-life, e.g., a half-life similar to that when bound to albumin.

[0193] In other embodiments, the ABP of the present invention may comprise two or more antigen binding regions, preferably two, three, or four antigen binding regions.

[0194] In yet other embodiments, the ABP of the present invention may comprise a chimeric antigen receptor (CAR), preferably comprising an extracellular antigen-binding region, a membrane anchor such as a transmembrane domain, and an intracellular region, e.g., an intracellular signaling region.

[0195] In a preferred embodiment, the ABP of the present invention may comprise at least one antibody constant domain, in particular, the at least one antibody constant domain is a CH1, CH2, or CH3 domain, or a combination thereof.

[0196] In further such embodiments, the ABPs of the invention having antibody constant domains comprise mutated Fc regions, e.g., to increase interaction of the Fc region with an Fc receptor (e.g., Saxena & Wu, 2016; Front Immunol 7:580), examples and embodiments of which are described elsewhere herein.

[0197] In other embodiments, the ABPs of the present invention may comprise an effector group and / or a labeling group.

[0198] The term "effector group" refers to any group, particularly a group attached to another molecule such as an antigen-binding protein, that acts as a cytotoxic agent. An example of a suitable effector group is a radioisotope or radionuclide. Other suitable effector groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable effector groups include calicheamicin, auristatin, geldanamycin, alpha-amanitin, pyrrolobenzodiazepines, and maytansine.

[0199] The term "label" or "label group" refers to any detectable label. Generally, labels are divided into various classes depending on the assay in which they are detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).

[0200] In some embodiments, the effector group or labeling group is attached to another molecule (such as ABP) via spacer arms of various lengths to reduce potential steric hindrance.

[0201] In another aspect, the present invention relates to an antigen-binding domain (ABD) of an ABP of the present invention, such as any ABP described above or elsewhere herein. In certain embodiments, the ABD of the present invention, when contained in an applicable scaffold, can bind to the ECD of LILRB1 and / or LILRB2 (or variants thereof). The ABD of the present invention may, in certain embodiments, be isolated and / or substantially pure.

[0202] Nucleic acids, nucleic acid constructs and (host) cells

[0203] In a third aspect, the present invention relates to nucleic acids encoding the ABP (or ABD) of the invention (such as those described above) or a component thereof. For example, the component encoded by the nucleic acid of the invention may be all or part of one chain of an antibody of the invention; or the component may be an scFV of said ABP. The component encoded by such a nucleic acid may be all or part of one or the other chain of an antibody of the invention, e.g., the component encoded by such a nucleic acid may be an ABD of the invention. The nucleic acid of the invention may also encode a fragment, derivative, mutant, or variant of the ABP of the invention, and / or may represent a "component which is a polynucleotide suitable and / or sufficient for use as a hybridization probe, a polymerase chain reaction (PCR) primer or sequence primer for identifying, analyzing, mutating, or amplifying polynucleotides encoding the polypeptide, an antisense or inhibitory nucleic acid (such as an RNAi / siRNA / shRNA or gRNA molecule) for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing."

[0204] In certain embodiments of the invention, the nucleic acids of the invention comprise nucleic acids having sequences encoding heavy or light chain CDRs, combinations of heavy and / or light chain CDR1, CDR2 and CDR3, or heavy or light chain variable domains (in each case as shown in Table 1), or functional fragments thereof. In other embodiments, the nucleic acid of the invention has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%; or 95% (preferably at least 75%) sequence identity to a nucleic acid sequence selected from the list consisting of (or has no more than 50, 40, 30, 20, 15, 10, or 5, preferably no more than 3, 2, or 1 base substitutions, insertions, or deletions, preferably at the third base, of a codon of the nucleic acid sequence); SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140, 149, 150, 159 160, 169, 170, 179, 180, 189, 190, 199, 200, 209, 210, 219, 220, 229, 230, 239, 240, 249, 250, 259, 260, 269, 270, 279, 280, 289, 290, 299, 300, 309, 310, 319, 320, 329, 330, 339, 340, 349, 350, 359, 360, 369 and 370; Preferably, such nucleic acids encode the heavy or light chain variable domains of the ABPs of the invention, for example the corresponding heavy or light chain variable domains having the amino acid sequences set out in Table 1, optionally with no more than 10, 9, 8, 7, 6, 5, 4, preferably 3, 2 or 1 amino acid substitutions, insertions or deletions compared to these sequences.

[0205] Nucleic acids according to the invention may be DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, and may optionally be linked to polynucleotides that are not naturally linked. In some embodiments, such nucleic acids may contain one or more non-natural (e.g., synthetic) nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more, particularly 1 to about 5, or preferably all instances of a particular nucleotide in the sequence); and / or such nucleic acids may include (e.g., be conjugated to) another chemical moiety, such as a label or effector group; e.g., a label or effector group described elsewhere herein.

[0206] In one embodiment, the nucleic acids of the invention can be isolated or substantially pure. In another embodiment, the nucleic acids of the invention can be recombinant, synthetic, and / or modified, or in any other way non-naturally occurring. For example, the nucleic acids of the invention can contain at least one nucleic acid substitution (or deletion) modification (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 such modifications, particularly 1 to about 5 such modifications, preferably 2 or 3 such modifications) compared to a naturally occurring product (e.g., a human nucleic acid).

[0207] The nucleic acid can be of any suitable length, such as about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000, or more nucleotides. For example, an siRNA nucleic acid can be preferably about 15 to about 25 base pairs in length (preferably about 19 to about 21 base pairs in length); an shRNA nucleic acid can preferably include a 20 to 30 base pair stem, a loop of at least 4 nucleotides, and a nicotinamide adenine dinucleotide overhang at the 3' end; a microRNA can be preferably about 22 base pairs in length; and an mRNA or DNA sequence encoding an ABP of the present invention or a component thereof (such as a heavy chain, light chain, or IgG antibody) can be preferably about 500 to 1,500 nucleotides in length. More preferably, the nucleic acid encoding the mammalian light chain of the antibody can be about 630 to about 650 nucleotides, and the nucleic acid encoding the mammalian heavy chain of the antibody can be about 1,300 to about 1,650 nucleotides. The nucleic acid can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid. The nucleic acid can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0208] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full length) can be isolated from B cells of mice, rats, llamas, alpaca, chickens, or rabbits immunized with LILRB1 and / or LILRB2 antigens, or fragments thereof, such as one or more EC domains (or polynucleotides encoding and capable of expressing LILRB1 and / or LILRB2 antigens, or fragments thereof). Nucleic acids can be isolated by conventional procedures, such as PCR.

[0209] Changes can be introduced by mutation into the sequence of the nucleic acid of the present invention. Such changes, depending on their nature and position in the codon, can result in a change in the amino acid sequence of the polypeptide (e.g., antigen-binding protein) that they encode. Mutations can be introduced using any technique known in the art.

[0210] In one embodiment, one or more specific amino acid residues can be altered, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues can be altered, for example, using a random mutagenesis protocol. However, once created, the mutant polypeptide can be expressed and screened for desired properties. Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues.

[0211] Other changes that can be made to the sequences of the nucleic acids of the present invention (e.g., by mutation) may not alter the amino acid sequence of the encoded polypeptide, but may result in changes in the stability and / or expression efficiency of the encoded polypeptide. For example, by codon optimization, the expression of a given polypeptide sequence can be improved by utilizing more common codons for a given amino acid found in the species in which the nucleotide is expressed. Methods of codon optimization, and alternative methods (such as optimizing CpG and G / C content), are described, for example, in Hass et al., 1996 (Current Biology 6:315); WO1996 / 09378; WO2006 / 015789 and WO 2002 / 098443.

[0212] In a related aspect, the present invention relates to a nucleic acid construct (NAC) comprising at least one nucleic acid of the invention (as described above). Such a NAC can include one or more additional features that enable expression of the encoded ABP or a component of the ABP (e.g., an ABD) in a cell (e.g., a host cell). Examples of NACs of the invention include, but are not limited to, plasmid vectors, viral vectors, mRNA, non-episomal mammalian vectors, and expression vectors, e.g., recombinant expression vectors. Nucleic acid constructs of the invention can include a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a cell, such as a host cell (see below). Nucleic acid constructs of the invention are typically recombinant nucleic acids and / or can be isolated and / or substantially pure. Recombinant nucleic acids are typically non-naturally occurring; particularly if they contain portions derived from different species and / or synthetic, in vitro, or mutagenesis methods.

[0213] In some embodiments, the NAC of the present invention comprises one or more constructs containing nucleic acids encoding either a heavy chain or a light chain. In some embodiments, the NAC of the present invention comprises two constructs, one containing nucleic acids encoding a heavy antibody chain and the other containing nucleic acids encoding a light antibody chain, such that expression from both constructs can produce a complete antibody molecule. In some embodiments, the NAC of the present invention comprises a construct containing nucleic acids encoding both a heavy and a light antibody chain, such that a complete antibody molecule can be expressed from a single construct. In other embodiments, the NAC of the present invention can comprise a single construct encoding a single chain sufficient to form an ABP of the present invention; for example, the encoded ABP may be an scFv or a single endothelial antibody (such as a camelid antibody).

[0214] In some embodiments, the NAC of the present invention comprises sequences encoding all or part of a constant region, allowing all or part of a heavy and / or light chain to be expressed.

[0215] The NAC according to the present invention can comprise (or consist of) an mRNA molecule comprising an open reading frame encoding the ABP of the present invention, together with upstream and downstream elements (e.g., 5' and / or 3' UTR and / or poly-A stretch) that allow, for example, expression of the ABP, preferably mRNA stability and / or expression of the ABP. For the use of mRNA as a NAC for introducing and expressing polynucleotides in cells, see, for example, Zangi et al. (Nat. Biotechnol. vol. 31, 898-907(2013)), Sahin et al. ((2014) Nature Reviews Drug Discovery 13:759), and Thess et al. (Mol. Ther. vol. 23 no.9, 1456-1464(2015)). Specific UTRs that may be included in the mRNA NACs of the present invention include the 5'UTR of the TOP gene (WO 2013 / 143699) and / or histone stem loops (WO 2013 / 120629). The mRNA NACs of the present invention may further include one or more chemical modifications (EP 1 685 844) including a 5'-cap such as m7G(5')ppp, (5'(A, G(5')ppp(5')A, or G(5')ppp(5')G, and / or at least one nucleotide that is an analogue of a naturally occurring nucleotide, such as phosphorothioate, phosphoramidate, peptide nucleotide, methylphosphonate, 7-deaza-guanosine, 5-methylcytosine, or inosine.

[0216] NACs, such as DNA-, retroviral- and mRNA-based NACs of the invention, can be used in gene therapy methods to treat or prevent diseases of the immune system (see therapeutic methods below), whereby a NAC comprising an expressible sequence encoding an ABP of the invention is administered to a cell or organism (e.g., by transfection). In particular, the use of mRNA therapeutics for the expression of antibodies is known from WO2008 / 083949.

[0217] In another related aspect, the present invention relates to cells (such as host cells and / or recombinant host cells) comprising one or more nucleic acids or NACs of the present invention. Preferably, such cells are capable of expressing the ABP (or a component thereof) encoded by said NAC. For example, if an ABP of the present invention comprises two distinct polypeptide chains (e.g., the heavy and light chains of IgG), a cell of the present invention can comprise a first NAC encoding (and capable of expressing) the heavy chain of such ABP and a second NAC encoding (and capable of expressing) the light chain of such ABP; or the cell can comprise a single NAC encoding both chains of such ABP. In these methods, such a cell of the present invention can express a functional (e.g., binding and / or inhibitory) ABP of the present invention. The (host) cell of the present invention can be one of the mammalian, prokaryotic, or eukaryotic host cells described elsewhere herein, particularly when the cell is a Chinese hamster ovary (CHO) cell.

[0218] In certain embodiments of such aspects, the (host) cells are human cells; in particular, they may be human cells sampled from a particular individual (e.g., autologous human cells). In such embodiments, such human cells can be grown and / or engineered in vitro to introduce the NAC of the present invention. A utility of engineered human cells from a particular individual may be to produce the ABP of the present invention, including reintroducing a population of such engineered human cells into a human subject, such as for use in therapy. In certain such uses, the engineered human cells may be introduced into the same human individual from which they were originally sampled, e.g., as autologous human cells.

[0219] The human cells subjected to such manipulation can be any germ cell or somatic cell type in the body. For example, the donor cells can be germ cells or somatic cells selected from the group consisting of fibroblasts, B cells, T cells, dendritic cells, keratinocytes, adipocytes, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neurons, glial cells, astrocytes, cardiac cells, esophageal cells, muscle cells, melanocytes, hematopoietic cells, macrophages, monocytes, and mononuclear cells. The donor cells can be obtained from any organ or tissue in the body, for example, cells from an organ selected from the group consisting of the liver, stomach, intestine, lung, pancreas, cornea, skin, gallbladder, ovaries, testes, kidneys, heart, bladder, and urethra.

[0220] Pharmaceutical Composition

[0221] For therapeutic use, the ABP, nucleic acid or NAC of the invention (or cells such as host cells) can be formulated into a pharmaceutical composition suitable for facilitating administration to an animal or human. The term "pharmaceutical composition" refers to a mixture of substances comprising a therapeutically active substance (such as an ABP of the invention) for pharmaceutical use.

[0222] Thus, in a fourth aspect, the present invention relates to a pharmaceutical composition comprising a compound that is a modulator of the expression, function, activity, and / or stability of immunoglobulin superfamily member 11 (LILRB1 and / or LILRB2), or a variant of LILRB1 and / or LILRB2, and a pharmaceutically acceptable carrier, stabilizer, and / or excipient. For example, the modulator of LILRB1 and / or LILRB2 is an ABP of the present invention, and / or at least one NAC of the present invention, and / or a (host) cell of the present invention. Thus, in a related aspect, a pharmaceutical composition is provided herein that comprises an ABP of the present invention, and / or at least one NAC of the present invention, and / or a (host) cell of the present invention, and a pharmaceutically acceptable excipient or carrier.

[0223] In a preferred embodiment, the pharmaceutical composition comprises an ABP of the present invention, e.g., in such an embodiment, the modulator of LILRB1 and / or LILRB2 is an ABP of the present invention (e.g., a LILRB1 and / or LILRB2 inhibitory ABP of the present invention).

[0224] For example, pharmaceutical compositions of the present invention may contain 0.1% to 100% (w / w) of active ingredient (e.g., a modulator of LILRB1 and / or LILRB2), for example, about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably between about 1% to about 20%, about 10% to 50%, or about 40% to 90%.

[0225] As used herein, the term "pharmaceutically acceptable" excipient, stabilizer, or carrier is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffers, lubricants, release-controlling vehicles, diluents, emulsifiers, wetting agents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Auxiliary agents can also be incorporated into the compositions.

[0226] A pharmaceutical composition of the invention (or for use with the invention) is typically formulated to be compatible with its intended route of administration, including oral, parenteral, e.g., intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical), and transmucosal administration.

[0227] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application and containing (or for use with) a compound of the invention (e.g., a modulator of LILRB1 and / or LILRB2) can include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin; propylene glycol or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfate; a chelating agent such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0228] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Kolliphor (登録商標) EL (formerly Cremophor EL (商標)BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, an injectable composition should typically be sterile and fluid to the extent that easy syringability exists. It should typically be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0229] Sterile injectable solutions can be prepared by incorporating a compound of the present invention (e.g., a modulator of LILRB1 and / or LILRB2) in the required amount in an appropriate solvent, optionally with one or a combination of ingredients described herein, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those described herein. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0230] Oral compositions and compositions comprising (or for use with) a compound of the present invention (e.g., a LILRB1 and / or LILRB2 inhibitor) generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, where the compound in the liquid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as MG stearate or Stealth; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavor.

[0231] Additionally, compounds of the present invention (or those for use with the present invention) (e.g., modulators of LILRB1 and / or LILRB2) can be administered rectally. Rectal compositions can be in any rectally acceptable dosage form, including, but not limited to, creams, gels, emulsions, enemas, suspensions, suppositories, and tablets. One preferred dosage form is a suppository, having a shape and size designed for introduction into the rectal opening of the human body. Suppositories typically soften, melt, or dissolve at body temperature. Suppository excipients include, but are not limited to, theobroma oil (cocoa butter), glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

[0232] For administration by inhalation, the compounds of the invention (or for use with the invention) (e.g., modulators of LILRB1 and / or LILRB2) are typically delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0233] Cells, such as immune cells (e.g., CAR T cells), for use in the present invention can be included in pharmaceutical formulations suitable for administration into the bloodstream or directly into a tissue or organ. The appropriate format is determined by a skilled artisan (e.g., a physician) for each patient, tissue, and organ according to standard procedures. Suitable pharmaceutically acceptable carriers and their formulations are known in the art (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980). When formed into a pharmaceutical composition, such cells are preferably formulated in a solution at a pH of about 6.5 to about 8.5. Excipients for rendering the solution isotonic can also be added, such as buffer solutions known in the art, e.g., pH buffered with sodium phosphate, e.g., 4.5% mannitol or 0.9% sodium chloride. Other pharmaceutically acceptable agents can also be used to make the solution isotonic, including, but not limited to, dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. In one embodiment, the media formulation is adjusted to preserve cells while maintaining their health and identity. For example, a premix containing an anticoagulant (ACD-A) aqueous solution, an equal volume of dextrose (50%), and phosphate-buffered saline (PBS) is typically premixed and aliquoted in a volume that matches or approximates the cellular matrix or environment in which the cells are extracted from the tissue or organ.

[0234] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. For transdermal administration, pharmaceutical compositions can be formulated into ointments, salves, gels, or creams, as generally known in the art.

[0235] In certain embodiments, pharmaceutical compositions are formulated for sustained or controlled release of the compounds of the present invention (e.g., modulators of LILRB1 and / or LILRB2). Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are readily apparent to those skilled in the art. Materials can also be commercially available (e.g., liposomes containing monoclonal antibodies against viral antigens, targeted to infected cells) and used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0236] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral, rectal or parenteral compositions in dosage unit form.As used herein, dosage unit form comprises physically discrete units suitable as unit dosages for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with the required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined and directly depends on the unique characteristics of active compound and the specific therapeutic effect to be achieved, and the limitations inherent in the skilled person who formulates such active compound for the treatment of individuals.

[0237] In some embodiments, the pharmaceutical composition comprising a modulator of LILRB1 and / or LILRB2 is in a unit dosage form of 10-1000 mg of the modulator of LILRB1 and / or LILRB2. In some embodiments, the pharmaceutical composition comprising a modulator of LILRB1 and / or LILRB2 is in a unit dosage form of 10-200 mg of the modulator of LILRB1 and / or LILRB2. In some embodiments, the pharmaceutical composition comprising an ABP is in a unit dosage form of 200-400 mg of the modulator of LILRB1 and / or LILRB2. In some embodiments, the pharmaceutical composition comprising a modulator of LILRB1 and / or LILRB2 is in a unit dosage form of 400-600 mg of the modulator of LILRB1 and / or LILRB2. In some embodiments, the pharmaceutical composition comprising a modulator of LILRB1 and / or LILRB2 is in a unit dosage form of 600-800 mg of the modulator of LILRB1 and / or LILRB2. In some embodiments, the pharmaceutical composition comprising a modulator of LILRB1 and / or LILRB2 is in a unit dose form of 800-100 mg of the modulator of LILRB1 and / or LILRB2.

[0238] Exemplary unit dosage forms for pharmaceutical compositions comprising modulators of LILRB1 and / or LILRB2 are provided as tablets, capsules (e.g., as powders, granules, microtablets, or micropellets), suspensions, or single-use pre-filled syringes. In certain embodiments, kits for producing single-dose administration units are provided. The kits can include both a first container containing a dried active ingredient and a second container containing an aqueous formulation. Alternatively, the kits can include single-chamber and multi-chamber pre-filled syringes.

[0239] The toxicity and therapeutic effectiveness (e.g., efficacy) of such active ingredients can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as procedures for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Active agents that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0240] Data obtained from cell culture assays and animal studies can be used in formulating a dosage range of the active ingredient (e.g., a modulator of LILRB1 and / or LILRB2), such as for use in humans. The dosage of such active ingredients lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any active ingredient used in the therapeutic approaches of the invention, a (therapeutically) effective dose can be initially estimated from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active ingredient that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used, for example, to more accurately determine useful (e.g., effective) amounts or doses for administration to humans. The pharmaceutical compositions can be included in a container, pack, or dispenser along with instructions for administration.

[0241] In the context of the present invention, an effective amount of a modulator of LILRB1 and / or LILRB2 or pharmaceutical composition can be one that elicits the biological, physiological, pharmacological, therapeutic, or medical response in a cell, tissue, system, body, animal, individual, patient, or human that is sought by a researcher, scientist, pharmacologist, pharmaceutical scientist, veterinarian, medical doctor, or other clinician, such as alleviating the effects / symptoms of a disorder, disease, or condition, such as a proliferative disorder, e.g., cancer or tumor, or killing or inhibiting the proliferation of cells involved in a proliferative disorder, e.g., tumor cells. Effective amounts can be determined by standard procedures, including those described below.

[0242] According to all aspects and embodiments of the medical uses and methods of treatment provided herein, an effective amount administered at least once to a subject in need of treatment with a modulator of LILRB1 and / or LILRB2 is typically about 0.01 mg / kg to about 100 mg / kg per administration, e.g., about 1 mg / kg to about 10 mg / kg per administration. In some embodiments, an effective amount of a modulator of LILRB1 and / or LILRB2 administered at least once to said subject is about 0.01 mg / kg to about 0.1 mg / kg per administration, about 0.1 mg / kg to about 1 mg / kg per administration, about 1 mg / kg to about 5 mg / kg per administration, about 5 mg / kg to about 10 mg / kg per administration, about 10 mg / kg to about 50 mg / kg per administration, or about 50 mg / kg to about 100 mg / kg per administration.

[0243] For the prevention or treatment of disease, the appropriate dosage of a LILRB1 and / or LILRB2 modulator (or pharmaceutical composition comprising same) depends on the type of disease being treated, the severity and course of the disease, whether the LILRB1 and / or LILRB2 modulator and / or pharmaceutical composition is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history, age, age, size / weight and response to the LILRB1 and / or LILRB2 modulator and / or pharmaceutical composition, and the discretion of the attending physician. The LILRB1 and / or LILRB2 modulator and / or pharmaceutical composition is suitably administered to the patient at one time or over a series of treatments. When such LILRB1 and / or LILRB2 inhibitors and / or pharmaceutical compositions are administered over a series of treatments, the total number of administrations for a given course of treatment may consist of a total of about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than about 10 treatments. For example, treatment may be given once a day (or 2, 3, or 4 times a day) for a week, a month, or even several months. In certain embodiments, the course of treatment can be continued indefinitely.

[0244] The amount of LILRB1 and / or LILRB2 modulator and / or pharmaceutical composition administered depends on variables such as the type and severity of the disease or indication being treated, the overall health, age, size / weight of the patient, the in vivo efficacy of the LILRB1 and / or LILRB2 modulator and / or pharmaceutical composition, and the route of administration. The initial dose can be increased above the upper level to rapidly achieve the desired blood or tissue level. Alternatively, the initial dose can be lower than the optimal dose, and the daily dose can be gradually increased during the course of treatment. Human doses can be optimized, for example, in known Phase I dose-escalation studies designed to consist of a relatively low initial dose, e.g., about 0.01 mg / kg to about 20 mg / kg of the active ingredient. Dosing frequency can vary depending on factors such as the route of administration, dosage, and the disease being treated. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks. Formulations of (or for use with) the LILRB1 and / or LILRB2 modulators of the present invention are within the skill of those in the art. In some embodiments of the present invention, such LILRB1 and / or LILRB2 modulators are lyophilized and reconstituted in buffered saline at the time of administration. The LILRB1 and / or LILRB2 modulators and / or pharmaceutical compositions may further result in a reduced recurrence of the disease being treated, or may reduce the incidence of drug resistance or increase the time until drug resistance develops; in the case of cancer, may result in an increase in progression-free survival and / or overall survival.

[0245] "MODULATION USES, MEDICAL USES AND THERAPEUTIC METHODS FOR MODULATING LILRB1 AND / OR LILRB2"

[0246] Modulating compounds of LILRB1 and / or LILRB2, in particular modulating compounds that inhibit the interaction of LILRB1 and / or LILRB2 with their, or their variants of LILRB1 and / or LILRB2, natural ligands (such as HLA-G), and / or ABPs, NACs, (host) cells and pharmaceutical compositions of the present invention can be used in a variety of ways to modulate the expression, function, activity and / or stability of LILRB1 and / or LILRB2 (or their variants), including their use for treatment or prevention.

[0247] Thus, in a further aspect, provided herein is a method for modulating the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or variants of LILRB1 and / or LILRB2, comprising contacting cells expressing the LILRB1 and / or LILRB2 or variants thereof with the above-described regulatory compound, particularly an ABP of the present invention or a NAC encoding the ABP. When such an ABP is a modulator of the expression, function, activity, and / or stability of the LILRB1 and / or LILRB2 or variants thereof, it thereby modulates the expression, function, activity, and / or stability of the LILRB1 and / or LILRB2 or variants thereof. Such a method can be performed on cells present ex vivo, i.e., cells contained in a container or vessel, such as those used in a research facility. Accordingly, in such embodiments, such methods of the present invention can be described as in vitro methods of modulating the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or LILRB1 and / or LILRB2 variants thereof. However, in alternative embodiments, the methods may be performed using cells in the body, e.g., are in vivo methods of modulating the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or LILRB1 and / or LILRB2 variants thereof.

[0248] In such embodiments, particularly, such in vitro (or in vivo) methods include inhibiting the function and / or activity of LILRB1 and / or LILRB2, or variants thereof, when such a modulatory compound (e.g., ABP) is an inhibitor and / or antagonist of such function and / or activity. In some embodiments of such methods, it further comprises contacting the cells with immune cells, such as CTLs or TILs. Preferably, the ABP is an antibody or antibody fragment and is an inhibitor or antagonist of the function and / or activity of LILRB1 and / or LILRB2, or variants thereof.

[0249] In some embodiments, such in vitro (or in vivo) methods include activating the function and / or activity of LILRB1 and / or LILRB2, or variants thereof, when such a modulatory compound (e.g., an ABP) is an activator and / or agonist of such function and / or activity. In some embodiments of such methods, it further includes contacting the cells with immune cells, such as CTLs or TILs. Preferably, the ABP is an antibody or antibody fragment and is an activator and / or agonist of the function and / or activity of LILRB1 and / or LILRB2, or variants thereof. In certain embodiments of these aspects, the modulating method comprises contacting a cell expressing LILRB1 and / or LILRB2, or a variant thereof, with a modulating compound as described above that is an activator and / or agonist of the function and / or activity of LILRB1 and / or LILRB2, or a variant thereof, wherein the method mediates any one or combination of at least one of the functional characteristics or effects of the activating or agonist modulators described herein, particularly those described above in the section "Modulators of LILRB1 and / or LILRB2 expression, function, activity and / or stability."

[0250] In certain other embodiments of these aspects, the modulating method comprises contacting a cell expressing said LILRB1 and / or LILRB2, or a variant thereof, with an above-described modulating compound that is an inhibitor and / or antagonist of the function and / or activity of LILRB1 and / or LILRB2, or a variant thereof, wherein the method mediates any one or combination of at least one of the functional characteristics or effects of the inhibitor or antagonist modulator described herein, particularly as described above in the section "Modulators of LILRB1 and / or LILRB2 Expression, Function, Activity and / or Stability."

[0251] In certain embodiments, the modulatory compound (particularly an ABP) is an inhibitor and / or antagonist of the function and / or activity of LILRB1 and / or LILRB2 or a variant thereof, and inhibits the interaction between the natural ligand (such as HLA-G) of the LILRB1 and / or LILRB2 protein or a variant thereof and the LILRB1 and / or LILRB2 protein or a variant thereof; i.e., such a compound inhibits the binding function and / or activity of the LILRB1 and / or LILRB2 protein or a variant thereof.

[0252] In a preferred embodiment of the therapeutic aspect, a modulatory compound (e.g., an inhibitor or antagonist of the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or a variant thereof), such as an ABP, or a NAC encoding said ABP, can (i) modulate the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or a variant thereof, and / or (ii) enhance a cell-mediated immune response against mammalian cells and reduce or decrease the resistance of cells (e.g., tumor cells expressing natural ligands of LILRB1 and / or LILRB2 (e.g., HLA-G or variants)) to the immune response. In other particularly preferred embodiments of the present invention, an ABP (such as an inhibitor or antagonist of the expression, function, activity and / or stability of LILRB1 and / or LILRB2 or a variant thereof, in particular one that inhibits the binding function and / or activity of LILRB1 and / or LILRB2 proteins to a ligand of LILRB1 and / or LILRB2), or a NAC encoding said ABP, enhances or increases the susceptibility of cells (such as tumor cells expressing a natural ligand of LILRB1 and / or LILRB2 (e.g., HLA-G or a variant)) to an immune response.

[0253] The term "resistance" refers to the acquired or natural resistance of cells (e.g., of a proliferative disorder) involved in a disease (e.g., a proliferative disorder), e.g., tumor or cancer cells, to a patient's own immune response (e.g., a cell-mediated immune response) or to an immune response assisted by immunotherapy, such as adoptive T cell transfer or treatment with checkpoint blockers. Thus, resistant cells (e.g., resistant tumor or cancer cells) are more likely to evade humoral and / or cellular immune defense mechanisms and survive in a subject with a disorder (e.g., a tumor or cancer). Treatment of a resistant proliferative disorder, such as tumor / cancer resistance, is considered effective in the context of the present invention when cells involved in the proliferative disorder (e.g., cells of a cancer tumor) are more susceptible or rendered susceptible to an immune response (e.g., a cell-mediated immune response), i.e., more likely to be recognized and / or neutralized (e.g., by a cytotoxic process such as apoptosis) by the subject's immune response, compared to non-treatment.

[0254] Thus, in certain embodiments of the invention, cells involved in the disease may be resistant to a cell-mediated immune response; and / or such cells may have or exhibit a resistant phenotype.

[0255] In a preferred embodiment of the present invention, the terms "cellular resistance," "cell resistance," and the like refer to the resistance of target cells (such as tumor or cancer cells) to a cell-mediated immune response, e.g., a cytotoxic T lymphocyte (CTL) response (e.g., the tumor or tumor cells are unresponsive to CTLs targeting the tumor cells, or have a reduced or limited response to CTLs). Tumor cells may exhibit a reduced or limited response when contacted with CTLs specific for an antigen expressed on the tumor cells. A reduced or limited response is a reduction to 90% cytotoxic T cell response, preferably 80%, 70%, 60%, 50%, or more preferably 40%, 30%, 20%, or less. In this case, 100% refers to a state in which CTLs are able to kill all target cells involved in the proliferative disorder in a sample. Whether subject cells (e.g., tumor cells) are resistant to a patient's (cell-mediated) immune response can be tested in vitro by contacting a sample of subject cells (e.g., autologous tumor cells) with (e.g., autologous) T cells and then quantifying (e.g.,) tumor cell survival / proliferation rates. Alternatively, a decrease in the (cell-mediated) immune response can be determined by comparing cancer samples from the same cancer before and after tolerance has been acquired (e.g., induced by treatment), or by comparing cancer samples from a different cancer known not to be resistant to CTLs. In contrast, treatments of the present invention involve sensitizing cells involved in a proliferative disorder to CTLs, thereby reducing the resistance of such cells. A decrease in (e.g., tumor) cell resistance to CTLs is preferably a significant increase in CTL toxicity, preferably a 10% increase, more preferably a 20%, 30%, 40%, 50%, 60%, 70%, 80% or more increase, and even more preferably a 2-fold, 3-fold, 4-fold, 5-fold or more increase.

[0256] In certain embodiments, the resistant phenotype of cells involved in a proliferative disorder is exhibited by cells when a subject suffering from a proliferative disorder (e.g., cancer or tumor) has previously been treated with an (immuno)therapy, e.g., such proliferative disorder progresses despite such prior (immuno)therapy. For example, a class of subjects suitable for the various treatment methods of the present invention may be those whose tumor (or cancer) has progressed (e.g., relapsed or recurred, or failed to respond) after prior treatment with a cancer immunotherapy. In certain embodiments, such prior treatment may be any immunotherapy as described elsewhere herein, including adoptive immune cell transfer (e.g., TCR or CART cell therapy), anti-tumor vaccines, or antibodies binding to immune checkpoint molecules (e.g., CTLA-4, PD-1, or PD-L1). In other embodiments, the subject may be suffering from a tumor or cancer, and such cancer may have progressed (e.g., relapsed or recurred, or failed to respond) after prior radiation therapy.

[0257] The immune response is a cell-mediated immune response, such as that mediated by T cells, including cytotoxic T cells and / or TILs, particularly in such embodiments; and / or the immune response is lysis and / or killing of cells, such as those mediated by cytotoxic T cells and / or TILs, particularly those expressing LILRB1 and / or LILRB2, or variants thereof. In other particular such embodiments, the immune response is a cytotoxic immune response against cells (such as tumor cells), particularly a cell-mediated cytotoxic immune response, such as that mediated by T cells, including cytotoxic T cells and / or TILs. LILRB1 and / or LILRB2-expressing cells are found in the myeloid compartment and regulate cell-mediated immune responses.

[0258] Specifically, in certain preferred embodiments of such treatment aspects, modulatory compounds as disclosed herein, particularly ABPs (e.g., those that are inhibitors or antagonists of the expression, function, activity, and / or stability of LILRB1 and / or LILRB2 or their variants), or NACs encoding such ABPs, enhance or increase the killing and / or lysis of cells associated with proliferative disorders, such as tumor cells. Such effects are brought about through modulation of cells expressing LILRB1 and / or LILRB2 or their variants (e.g., macrophage cells); such immune responses are mediated by cytotoxic T cells and / or TILs, and / or by enhancing or increasing the sensitivity of cells of the immune system and / or by reducing or diminishing the immunosuppressive effect of cells expressing LILRB1 and / or LILRB2 or their variants on the (cytotoxic) immune response. Thus, LILRB1- and / or LILRB2-expressing cells contacted with the agents of the present invention are less immunoinhibitory and support the subject's immune response against the proliferative disorder being treated.

[0259] In certain such preferred embodiments, cells expressing LILRB1 and / or LILRB2, or variants thereof, are cells of the myeloid compartment, such as macrophages, or in some instances may be cancer cells or cells derived from tumor cells. Exemplary cancer or tumor cells may be those described or exemplified elsewhere herein.

[0260] In another preferred embodiment of such a treatment aspect, a modulatory compound, in particular an ABP (such as an inhibitor or antagonist of the expression, function, activity and / or stability of LILRB1 and / or LILRB2 or a variant thereof, in particular an inhibitor of the HLA-G binding function of LILRB1 and / or LILRB2 or a variant thereof), or a NAC encoding said ABP, may increase the activity and / or survival of T cells (and / or increase T cell proliferation), and in one embodiment, may result in an enhancement of the (cytotoxic) immune response mediated by such T cells.

[0261] Thus, in a fifth aspect, the present invention relates to a method for treating a disease, disorder, or condition in a mammalian subject by administering to the subject a product that is a modulator of the expression, function, activity, and / or stability of immunoglobulin superfamily member 11 (LILRB1 and / or LILRB2) or variants of LILRB1 and / or LILRB2 thereof. In a related aspect, the present invention relates to a product for use in medicine, the product being a compound that is a modulator of the expression, function, activity, and / or stability of immunoglobulin superfamily member 11 (LILRB1 and / or LILRB2) or variants of LILRB1 and / or LILRB2 thereof. In particular embodiments of these medical / therapeutic inventions, the modulatory compound (such as an ABP) is an inhibitor of the binding-HLA-G binding function of LILRB1 and / or LILRB2 or variants thereof; and / or the product is selected from the list consisting of an ABP, an ABD, a nucleic acid, a NAC, or a recombinant host cell of the invention, in particular an ABP of the invention.

[0262] In a related aspect, the present invention also relates to a method of treating or preventing a disease, disorder or condition in a mammalian subject in need thereof, the method comprising administering to the subject at least once an effective amount of a modulating compound as described above, or, in particular, administering to the subject at least once an effective amount of an ABP, NAC, (host) cell or pharmaceutical composition as described above.

[0263] In another related aspect, the present invention also relates to the use of an above-mentioned product of the invention or an above-mentioned modulatory compound (especially an ABP of the invention) for the manufacture of a medicament, particularly for the treatment of a disease, disorder or condition in a mammalian subject, particularly where the disease, disorder or condition is one described herein.

[0264] The term "treatment" as used herein is meant to include therapy, e.g., therapeutic treatment, as well as prophylactic or suppressive measures for a disease (or disorder or condition). Thus, for example, successful administration of a LILRB1 and / or LILRB2 inhibitor before the onset of a disease results in treatment of the disease. "Treatment" also encompasses administration of a LILRB1 and / or LILRB2 inhibitor after the onset of a disease to ameliorate or eradicate the disease (or its symptoms). Administration of a LILRB1 and / or LILRB2 inhibitor after the onset of clinical symptoms and subsequent clinical symptoms also includes treatment of the disease, with possible alleviation of clinical symptoms and possibly amelioration of the disease. "Subjects in need of treatment" include subjects (e.g., human subjects) already with a disease, disorder, or condition, as well as subjects prone to or suspected of having a disease, disorder, or condition, including subjects in which the disease, disorder, or condition is to be prevented.

[0265] In particular embodiments of these aspects, the modulatory compound is as described above and / or is an ABP, a NAC, a (host) cell or a pharmaceutical composition of the invention; in particular an ABP of the invention and / or an inhibitory nucleic acid of the invention.

[0266] Such compounds may, for example, in preferred embodiments, be inhibitors or antagonists of the expression, function, activity, and / or stability of LILRB1 and / or LILRB2, or variants of LILRB1 and / or LILRB2. In particular, the compounds inhibit the binding of a natural ligand of the LILRB1 and / or LILRB2 protein (e.g., HLA-G or a variant thereof) to the LILRB1 and / or LILRB2 protein (or a variant thereof), and in particular inhibit the binding of the HLA-G protein (or a variant thereof) to human LILRB1 and / or LILRB2 protein (or a variant thereof), for example, inhibiting the binding between the ECDs of such proteins; preferably, such proteins (or variants) and the inhibitors are as described above.

[0267] Such compounds may be, for example, compounds (such as ABPs or inhibitor nucleic acids) that have any one or any combination of the following characteristics: * specific binding to human LILRB1 and / or (preferably) LILRB2 (e.g., specific binding to one comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) or SEQ ID NO: 358 (LILRB2), e.g., with a KD of 50 nM or less, or 20 nM or less, more preferably 10 nM or less, or 5 nM or less); * lack of specific binding to LILRA proteins, such as preferably LILRA1 and / or LILRA3; * Stimulates T cell activation, e.g., as measured by increased T cell proliferation or IFN-γ secretion, e.g., in a mixed lymphocyte reaction (MLR) assay; * stimulating the differentiation or activation of monocytes into macrophages, for example stimulating the differentiation of monocytes into pro-inflammatory macrophages, as shown in the assays described in the Examples; * inhibiting the binding of LILRB1 and / or (preferably and) LILRB2 to HLA-A and HLA-B, preferably to HLA-G; *Has a binding profile as shown in Table 2; * Promoting pro-inflammatory polarization of macrophages towards M1 macrophages; and * Does not induce (or trigger) basophil activation.

[0268] In other aspects described elsewhere herein, methods are provided for detecting and / or diagnosing a disease, disorder or condition in a mammalian subject.

[0269] In one particular embodiment, the disease, disorder or condition is characterized by a pathological immune response.

[0270] In further particular embodiments, the disease, disorder, or condition is characterized by cells associated with the disease, disorder, or condition, such as cancer cells, by expression of a natural ligand of LILRB1 and / or LILRB2, particularly by expression of HLA-G. For example, the disease, disorder, or condition may be associated with the unwanted presence of a natural ligand of LILRB1 and / or LILRB2, such as, for example, a disease or condition characterized by HLA-G-positive cells or cells positive for such a ligand, particularly LILRB1- and / or LILRB2-positive monocytes and / or macrophages (particularly TAMs).

[0271] In yet another specific embodiment, a subject suffering from or suspected of suffering from a disease, disorder, or condition is characterized by having (i) cancer positive for a natural ligand of LILRB1 and / or LILRB2, such as HLA-G, and / or (ii) LILRB1 and / or LILRB2 positive immune cells, particularly LILRB1 and / or LILRB2 positive monocytes and / or macrophages; and / or (iii) LILRB1 and / or LILRB2 positive immune cells, particularly LILRB1 and / or LILRB2 positive monocytes (or macrophages); preferably, such LILRB1 and / or LILRB2 positive immune cells are present in or associated with the site of the cancer or tumor (such as being present in the tumor bed or tumor microenvironment (TME) of such cancer or tumor, particularly with the presence of TAMs and / or MDSCs).

[0272] Diseases, disorders or conditions treatable by the subject matter of the present invention are, in certain modified embodiments, characterized by the expression of ligands of LILRB1 and / or LILRB2; in particular, characterized by abnormal, e.g., over- (or under-)expression or expression or activity, of ligands of LILRB1 and / or LILRB2 (in particular HLA-G) in a given cell or tissue (such as a cell or tissue involved in a proliferative disorder of a subject) compared to that in a healthy subject or normal cell.

[0273] In still further particular embodiments, the disease, disorder or condition is characterized by LILRB1 and / or LILRB2 expression and / or activity in immune cells, e.g., macrophages, and in particular, such cells express LILRB1 and / or LILRB2 mRNA and / or protein and / or are positive for such LILRB1 and / or LILRB2 expression and / or activity.

[0274] In another particular embodiment, the disease, disorder or condition is a proliferative disorder (or a condition associated with such a disorder or disease), particularly when the product or regulatory compound (such as an ABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, particularly an ABP of the invention) is an inhibitor and / or antagonist of the expression, function, activity and / or stability of LILRB1 and / or LILRB2, or variants thereof of LILRB1 and / or LILRB2.

[0275] "Proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. A proliferative disorder does not imply any limitation on the rate of cell proliferation, but merely indicates a loss of normal controls affecting growth and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same cell division rate as normal cells, but do not respond to signals that limit such proliferation. Within the scope of "proliferative disorder" is a neoplasm or tumor, which is an abnormal growth of tissue or cells. Cancer is understood in the art and includes any of a variety of malignant neoplasms characterized by the proliferation of cells that have the ability to invade surrounding tissues and / or metastasize to new colonization sites. Proliferative disorders include carcinoma, atherosclerosis, rheumatoid arthritis, idiopathic pulmonary fibrosis, and cirrhosis of the liver. Non-cancerous proliferative disorders also include psoriasis and its various clinical forms, Reiter's syndrome, erythrohidrosis pilaris, and repeated hyperproliferative cell proliferation in the skin, such as hyperproliferative variants of keratinization disorders (e.g., actinic keratosis, senile keratosis), and scleroderma.

[0276] In more particular embodiments, the proliferative disorder is a cancer or tumor, particularly a solid tumor (or a condition associated with such a cancer or tumor), including, but not limited to, head and neck cancer, squamous cell carcinoma, multiple myeloma, solitary plasmacytoma, renal cell carcinoma, retinoblastoma, germ cell tumor, and hepatoblastoma. Hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, any hematological malignancy (e.g., chronic lymphoblastic leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, chronic myeloblastic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, hairy cell leukemia, mast cell leukemia, mast cell neoplasm, follicular lymphoma) lymphoma), follicular large cell lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt's lymphoma, mycosis fungoides, Sézary syndrome, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, chronic myeloproliferative disorder, myelofibrosis, myeloid metaplasia, systemic mastocytosis), and tumors of the central nervous system (e.g., brain tumor, glioblastoma, non-glioblastoma brain tumor, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, and choroid plexus papilloma), myeloproliferative disorders (e.g., polycythemia vera, thrombocythemia, idiopathic myelofibrosis), soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, or liver cancer.

[0277] In a preferred embodiment, various aspects of the present invention relate to the ABP of the present invention being used to detect, prevent, and / or treat a proliferative disorder, including, but not limited to, carcinoma (including breast cancer, prostate cancer, gastric cancer, lung cancer, colorectal and / or colon cancer, hepatocellular carcinoma, melanoma), lymphoma (including non-Hodgkin's lymphoma and mycosis fungoides), leukemia, sarcoma, mesothelioma, brain cancer (including glioma), germ cell tumor (including testicular cancer and ovarian cancer), choriocarcinoma, renal cancer, pancreatic cancer, thyroid cancer, head and neck cancer, endometrial cancer, cervical cancer, bladder cancer, or gastric cancer.

[0278] Thus, in a preferred embodiment, the proliferative disorder is cancer, such as lung cancer, breast cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, melanoma, myeloma, kidney cancer, head and neck cancer, Hodgkin's lymphoma, bladder cancer or prostate cancer, in particular one selected from the list consisting of melanoma, lung cancer (such as non-small cell lung cancer), bladder cancer (such as urothelial carcinoma), kidney cancer (such as renal cell carcinoma), head and neck cancer (such as squamous cell carcinoma of the head and neck) and Hodgkin's lymphoma. Preferably, the proliferative disorder is melanoma or lung cancer (such as non-small cell lung cancer).

[0279] In particularly preferred embodiments, the disease, disorder, or condition is a cancer that is positive for a ligand of LILRB1 and / or LILRB2 (e.g., HLA-G) and / or is characterized by the presence of LILRB1 and / or LILRB2-positive immune cells, particularly LILRB1 and / or LILRB2-positive monocytes and / or macrophages, and / or is a cancer (or other proliferative disorder) characterized as being resistant and / or refractory to blockade of an immune checkpoint molecule (e.g., resistant and / or refractory to treatment for blockade of an immune checkpoint molecule), e.g., blockade using a ligand for an immune checkpoint molecule (e.g., blockade of PD1 / CTLA4, further described below; similar to Gao et al., 2017). For example, in one such embodiment, the disease, disorder, or condition can be a proliferative disorder (e.g., cancer) that is resistant and / or refractory to PD1 / CTLA4 blockade therapy.

[0280] In further particular embodiments, the disease, disorder or condition is an infectious disease (or a condition associated with such a disorder or disease), particularly when the product or regulatory compound (such as the ABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, particularly the ABP of the invention) is an inhibitor and / or antagonist of the expression, function, activity and / or stability of LILRB1 and / or LILRB2, or variants thereof of LILRB1 and / or LILRB2.

[0281] The term "infectious disease" is art-recognized and, as used herein, includes any pathogen or disease, disorder, or condition associated with (e.g., resulting from or caused by) a pathogen that infects mammalian cells, preferably human cells. Examples of such pathogens include bacteria, yeast, fungi, protozoa, mycoplasma, viruses, prions, and parasites.Examples of infectious diseases include: (a) viral diseases, such as diseases resulting from infection with the following viruses: adenovirus, herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), poxvirus (e.g., orthopoxviruses such as smallpox or vaccinia, or molluscum contagiosum virus), picornavirus (e.g., rhinovirus or enterovirus), orthomyxovirus (e.g., influenza virus), paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), cononavirus (e.g., SARS), papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), hepadnavirus (e.g., hepatitis B virus), flavivirus (e.g., hepatitis C virus or dengue virus), or retrovirus (e.g., lentiviruses such as HIV); (b) bacterial diseases, such as infection with bacteria. For example, diseases resulting from infection with the following bacteria: Escherichia, Enterobacteria, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus or Bordetella; (c) other infectious diseases, for example Chlamydia, fungal diseases (including but not limited to candidiasis), aspergillosis, histoplasmosis, cryptococcal meningitis, parasitic diseases (including but not limited to malaria), Pneumocystis pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis infections, and prions that cause human diseases such as Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, familial insomnia, and kuru (a virus endemic to New Guinea).

[0282] In yet another particular embodiment, the disease, disorder or condition is one associated with an overactive or undesirably active immune system, such as an autoimmune, allergic or inflammatory condition, particularly an allergy, autoimmunity, transplant rejection, inflammation, graft-versus-host disease or sepsis (or a condition associated with such a disease, disorder or condition), particularly when the product or modulatory compound (e.g., an ABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, particularly an ABP of the invention) is an activator and / or agonist of the expression, function, active ingredient and / or stability of LILRB1 and / or LILRB2, or variants thereof of LILRB1 and / or LILRB2.

[0283] In accordance with the medical uses and methods of treatment disclosed herein, the subject is a mammal and may include mice, rats, rabbits, monkeys, and humans. In a preferred embodiment, the mammalian subject is a human patient.

[0284] In one embodiment, the cells involved in the proliferative disorder are resistant to a humoral or cell-mediated immune response. For example, the cells involved in the proliferative disorder (e.g., cancer or tumor cells) are resistant and / or refractory to blockade of an immune checkpoint molecule, such as blockade using a ligand for the immune checkpoint molecule, in an exemplary case, blockade of PD1 / CTLA4 (similar to Gao et al., 2017).

[0285] In particular, the therapeutic methods may be applied to proliferative disorders that have been subjected to known immunotherapy (such as therapy for blockade of immune checkpoint molecules, e.g., blockade of PD1 / CTLA4), particularly known immunotherapy with ligands to immune checkpoint molecules. For example, in certain embodiments, a modulator (e.g., antagonist) of LILRB1 and / or LILRB2, e.g., an ABP of the present invention, may be for use in treating a proliferative disorder in a subject in need thereof, and the subject has been subjected to a known immunotherapy, particularly a known administration of a ligand to an immune checkpoint molecule.

[0286] In other methods, a modulatory (e.g., inhibitory) compound (e.g., an ABP such as one of the present invention) may be used in combination with a different anti-proliferative therapy, particularly a different anti-cancer therapy, particularly when the different anti-proliferative therapy is an immunotherapy, particularly an immunotherapy using a ligand against an immune checkpoint molecule. Thus, the composition can be used to treat a proliferative disorder in a subject in need thereof, wherein the subject is subjected to co-treatment (e.g., combination treatment) with an immunotherapy, particularly a co-treatment with a ligand against an immune checkpoint molecule.

[0287] In such embodiments, the ligand binds to an immune (inhibitory) checkpoint molecule. For example, such a checkpoint molecule may be selected from the group consisting of A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1 (or one of its ligands PD-L1 and PD-L2), TIM-3 (or its ligand galectin-9), TIGIT, and VISTA. In particular, in such embodiments, the ligand binds to a checkpoint molecule selected from CTLA-4, PD-1, and PD-L1. In other more particular embodiments, the ligand is an antibody selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, BGB-A317, atezolizumab, avelumab, and durvaluma; in particular, an antibody selected from the group consisting of ipilimumab (Yervoy), nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), and atezolizumab (TECENTRIQ).

[0288] When a therapeutic method or use of the present invention (e.g., involving an ABP of the present invention) is used in combination with any such other procedure (e.g., another drug or cancer immunotherapy, e.g., a ligand that binds to an immune (inhibitory) checkpoint molecule), such method or use being a combination treatment regimen may include embodiments in which such exposure / administration is simultaneous. In alternative embodiments, such administration may be sequential, particularly embodiments in which the LILRB1 and / or LILRB2 modulator (e.g., an ABP of the present invention) is administered before such other procedure. For example, such a LILRB1 and / or LILRB2 modulator may be administered continuously within about 14 days (e.g., before) of the other procedure, for example, within about 10 days, 7 days, 5 days, 2 days, or 1 day (e.g., before) of the other procedure; further, the LILRB1 and / or LILRB2 modulator may be administered continuously within about 48 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 5 minutes (e.g., before) of the other procedure.

[0289] In certain embodiments, the medical use or composition is for use in enhancing an immune response in a subject, preferably for use in supporting a cell-mediated immune response in a subject, e.g., a T-cell mediated immune response in a subject, e.g., for treating a proliferative disease such as a cancer disease.

[0290] In certain embodiments, treatment can involve the transfer of cells to a subject, preferably the transfer of immune cells to a subject, more preferably adoptive T cell transfer. For example, such cells can be the subject's own cells, e.g., autoimmune cells such as the subject's T cells, dendritic cells, or natural killer (NK) cells.

[0291] In a preferred embodiment of the medical use or composition, the modulatory compound (e.g., the ABP of the present invention) is an inhibitor or antagonist of the expression, function, activity and / or stability of LILRB1 and / or LILRB2, or a variant thereof of LILRB1 and / or LILRB2, and inhibition of the expression, function, activity and / or stability of LILRB1 and / or LILRB2, or a variant thereof of LILRB1 and / or LILRB2 enhances the immune response, preferably enhances a cell-mediated immune response in a subject, such as a T-cell-mediated immune response in a subject, for example, to treat an infectious disease or a proliferative disease, such as a cancer disease, particularly when the composition is an ABP of the present invention.

[0292] In such embodiments, the immune response can be enhanced by increasing the activity, proliferation, and / or survival of T cells, and in particular, increasing the activity of T cells includes increasing the production of one or more proinflammatory cytokines by such T cells (such as TILs). Preferably, in such embodiments, the cytokine is selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-12, IL-17, and IL-18, tumor necrosis factor (TNF)[α], interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor such as IL-2 (and / or IL-17 or IFN-γ).

[0293] Such an increase in T cell activity, proliferation and / or survival may be associated with, in particular, inhibition of the interaction between LILRB1 and / or LILRB2 and their ligands, mediated by LILRB1 and / or LILRB2-mediated signaling.

[0294] Administration of a modulatory (e.g., inhibitory) compound (e.g., an ABP of the present invention) is, in certain embodiments, associated with inhibition of the interaction between LILRB1 and / or LILRB2 and their ligand(s), particularly mediated by LILRB1 and / or LILRB2-mediated signal transduction.

[0295] In other specific embodiments, administration of a modulatory compound (e.g., an ABP of the present invention) reduces or decreases the resistance of cells (e.g., tumor cells and / or cells expressing LILRB1 and / or LILRB2, or mutants of LILRB1 and / or LILRB2 thereof) to an immune response, preferably the compound enhances or increases the susceptibility of cells (e.g., tumor cells and / or cells expressing LILRB1 and / or LILRB2, or mutants of LILRB1 and / or LILRB2 thereof) to an immune response.

[0296] In preferred embodiments, the medical use is for the treatment of a proliferative disorder (such as cancer as described herein) in a mammalian subject in need thereof, in some such embodiments, the subject is a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, e.g., a human patient.

[0297] "Cells and methods for producing the ABPs / NACs of the present invention"

[0298] As noted above, in one aspect, the present invention provides cells, such as (recombinant) host cells or hybridomas, capable of expressing the ABPs described above. In another aspect, the present invention provides cells comprising at least one NAC encoding the ABP or a component of an ABP described above. The cells of the present invention can be used in the methods provided herein to produce the ABPs and / or NACs of the present invention.

[0299] In certain embodiments, the cells are isolated or substantially pure, and / or are recombinant cells, and / or are non-naturally occurring cells (i.e., not found in nature or a product of nature), such as hybridomas.

[0300] Therefore, in another aspect, the present invention provides a method for producing a recombinant cell line capable of expressing an ABP specific for LILRB1 and / or LILRB2, or an ABP specific for a variant of LILRB1 and / or LILRB2, comprising: * Provision of suitable host cells; * providing at least one genetic construct comprising a coding sequence encoding the ABP of the present invention; * introducing said genetic construct into said suitable host cell; and * Optionally, expressing said genetic construct by said suitable host cell under conditions that allow expression of said ABP.

[0301] In yet another aspect, the present invention provides a method for producing the ABPs described above, comprising, for example, culturing one or more cells of the invention under conditions that allow expression of said ABPs.

[0302] Therefore, in another aspect, the present invention provides a method for producing an ABP specific to LILRB1 and / or LILRB2, or an ABP specific to a variant of LILRB1 and / or LILRB2, comprising: * providing a hybridoma or (host) cell capable of expressing an ABP according to the invention, such as a recombinant cell line comprising at least one genetic construct comprising a coding sequence encoding said compound or ABP; and * Culturing said hybridoma or host cells under conditions that allow expression of the ABP.

[0303] To produce a recombinant ABP of the present invention, a DNA molecule encoding the protein (e.g., an antibody, light chain and / or heavy chain, or fragment thereof) is inserted into an expression vector (or NAC) such that the sequence is operably linked to transcriptional and translational control sequences. Alternatively, a DNA molecule encoding an ABP can be chemically synthesized. The synthetic DNA molecule can be ligated to other appropriate nucleotide sequences, including, for example, constant region-encoding sequences and expression control sequences, to generate a conventional gene expression construct encoding the desired ABP. To produce an ABP of the present invention, one skilled in the art can select from a wide variety of expression systems known in the art, such as those reviewed by Kipriyanow and Le Gall, 2004. Expression vectors include, but are not limited to, plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. The term "expression vector" or "NAC" includes any vector suitable for expressing foreign DNA. Examples of such expression vectors are viral vectors, such as adenovirus, vaccinia virus, baculovirus and adeno-associated virus vectors. In this context, the expression "viral vector" is understood to mean both DNA and viral particles. Examples of phage or cosmid vectors include pWE15, M13, λEMB13, λEMB14, λFIXII, λDASHII, λZAPII, λgT10, λgt11, Charon4A, and Charon21A. Examples of plasmid vectors include pBR, pUC, pB1uescriptII, pGEM, pTZ, and pET families. Various shuttle vectors can be used, for example, vectors capable of autonomous replication in multiple host microorganisms, such as E. coli and Pseudomonas. Artificial chromosome vectors are also considered expression vectors. Expression vectors and expression control sequences are selected to be compatible with cells, such as host cells. Examples of mammalian expression vectors include pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRepS, D H26S, D HBB, pNMT1, pNMT41, and pNMT81, which are available from Invitrogen. TM pCI is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV are available from Agilent Technologies, and pTRES is available from Clontech and its affiliates.

[0304] To produce antibodies, antibody light chain genes and antibody heavy chain genes can be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector. A convenient vector encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered to allow for easy insertion and expression of any VH or VL sequence, as described above, and the CH1 and / or upper hinge region contains at least one amino acid modification of the present invention. The constant chain is typically a kappa or lambda antibody light chain. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the (host) cell. DNA encoding the antibody chain can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature antibody chain DNA. The signal peptide may be an immunoglobulin signal peptide or a heterologous peptide derived from a non-immunoglobulin protein. Alternatively, the DNA sequence encoding the antibody chain may already contain a signal peptide sequence.

[0305] In addition to the DNA sequence encoding the ABP (antibody) chain, the recombinant expression vector carries regulatory sequences including promoters, enhancers, termination and polyadenylation signals, and other expression control elements that control the expression of the antibody chain in (host) cells. Examples of promoter sequences (exemplified for expression in mammalian cells) are promoters and / or enhancers derived from CMV (such as the CMV Simian Virus 40 (SV40) promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdM1P)), polyoma, and strong mammalian promoters such as the native immunoglobulin promoter and actin promoter. Examples of polyadenylation signals include BGH polyA, SV40 late or early polyA; or the 3'UTR of an immunoglobulin gene, etc., can be used.

[0306] The recombinant expression vector may also have sequences that regulate replication of the vector in (host) cells (e.g., an origin of replication) and a selectable marker gene. Nucleic acid molecules encoding the heavy chain or antigen-binding portion thereof and / or the light chain or antigen-binding portion thereof of the antibody of the present invention, and vectors containing these DNA molecules, can be introduced into (host) cells, such as bacterial cells or higher eukaryotic cells, such as mammalian cells, according to transfection methods well known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer with viral vectors.

[0307] It is within the skill of the art to express the heavy and light chains of an antibody or fragment thereof from a single expression vector or from two separate expression vectors. Preferably, the DNA molecules encoding the heavy and light chains are present on two vectors that are co-transfected into a (host) cell, preferably a mammalian cell.

[0308] Mammalian cell lines available as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44, BI-HEX-CHO) cells, NSO, SP2 / 0 cells, HeLa cells, HEK293 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, or derivatives / progeny of any such cell line. Other mammalian cells, including, but not limited to, human, mouse, rat, monkey, and rodent cell lines, or other eukaryotic cells, including, but not limited to, yeast, insect, and plant cells, or prokaryotic cells, such as bacteria, can be used. The antibody molecules of the invention are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody molecule in the host cells.

[0309] According to some embodiments of the methods for producing ABPs, after expression, the intact antibody (or antigen-binding fragment of the antibody) can be recovered and isolated using purification techniques well known in the art, such as protein A, protein G, affinity tags, e.g., glutathione-S-transferase (GST), and histidine tags.

[0310] ABPs are preferably recovered from the culture medium as secreted polypeptides or, for example, when expressed without a secretory signal, can be recovered from host cell lysates. To obtain substantially homogeneous preparations of ABPs, it is necessary to purify the ABP molecules using standard protein purification methods used for recombinant and host cell proteins. For example, state-of-the-art purification methods useful for obtaining the ABP molecules of the present invention include, as a first step, the removal of cells and / or particulate cell debris from the culture medium or lysate. The ABP is then purified from contaminating soluble proteins, polypeptides, and nucleic acids by, for example, fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse-phase HPLC, Sephadex chromatography, or chromatography on silica or cation exchange resins. Preferably, the ABP is purified by standard Protein A chromatography, for example, using a Protein A spin column (GE Healthcare). Protein purity can be verified by reducing SDS-PAGE. ABP concentration can be determined by measuring absorbance at 280 nm and utilizing the protein-specific extinction coefficient. As a final step in the process for obtaining an ABP molecule preparation, the purified ABP molecule may be dried, eg, lyophilized, for therapeutic use.

[0311] Thus, in certain embodiments of such aspects, the methods of the invention comprise the further step of isolation and / or purification of the ABP.

[0312] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising the ABP, which comprises formulating the ABP isolated by the above method into a pharmaceutically acceptable form.

[0313] In another aspect, provided herein is a method for producing a pharmaceutical composition comprising the above-described NAC, comprising formulating the NAC prepared by the above-described method into a pharmaceutically acceptable form.

[0314] According to some embodiments, the method of making a pharmaceutical composition comprises the further step of combining said ABP and / or NAC with a pharmaceutically acceptable excipient or carrier.

[0315] In some embodiments of the method for producing a pharmaceutical composition comprising an ABP, the ABP is typically labeled with a detectable labeling group before being formulated into a pharmaceutically acceptable form. Various methods for labeling proteins are known in the art and can be used. Suitable labeling groups include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is attached to the ABP via spacer arms of various lengths to reduce potential steric hindrance.

[0316] Thus, in certain embodiments of such aspects, the ABP is a modified antibody and the method comprises the further step of addition of a functional moiety selected from a detectable labeling group or a cytotoxic moiety.

[0317] "Detection / Diagnostic / Monitoring Methods"

[0318] LILRB1 and / or LILRB2 can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and / or conditions associated with the undesirable presence of LILRB1 and / or LILRB2-positive cells, or cells positive for variants of LILRB1 and / or LILRB2, and / or cellular resistance to cell-mediated immune responses; in particular, abnormal and / or localized expression / activity of LILRB1 and / or LILRB2 (in particular phosphorylated LILRB1 and / or LILRB2) can be used for such purposes. The diseases, disorders, and / or conditions so detected, diagnosed, or monitored may be those described elsewhere herein.

[0319] In a related aspect, the invention provides a method for determining the presence or amount of LILRB1 and / or LILRB2 (or variants thereof) in a biological sample from a subject, comprising: * contacting the sample with an ABP capable of binding to LILRB1 and / or LILRB2 (or a variant thereof); and * Detecting binding between LILRB1 and / or LILRB2 (or variants thereof) and ABP in a biological sample.

[0320] In a preferred embodiment, the proteins LILRB1 and / or LILRB2 (or variants thereof) are detected with the ABP of the present invention.

[0321] In certain embodiments, the biological sample comprises (preferably) cells or tissues of the subject, or extracts of such cells or tissues, particularly such cells are those involved in a proliferative disorder (e.g., cells of the tumor microenvironment, or immune cells present at the site of the tumor). The tumor or cells thereof may be or be derived from one of the tumors described elsewhere herein.

[0322] In certain embodiments of such aspects, the method comprises: * Providing (eg, by obtaining a biological sample from a subject), in particular where such a step occurs before the detecting step.

[0323] In certain embodiments, such detection and / or determination methods can be carried out as diagnostic tools, such as a method for diagnosing whether a mammalian subject (such as a human subject or patient) has a disease, disorder, or condition (such as those described above), in particular a proliferative disorder such as a cancer or a tumor (or is at risk of developing such a disease, disorder, or condition), or is associated with the undesirable presence of LILRB1 and / or LILRB2 positive cells, or cells positive for variants of LILRB1 and / or LILRB2, and / or is associated with cellular resistance to a cell-mediated immune response, and / or is associated with (e.g., aberrant) expression or activity of LILRB1 and / or LILRB2 (or variants thereof), or has cellular resistance to a cell-mediated immune response.

[0324] In certain embodiments of these detection, determination and / or diagnosis methods, the cellular resistance to a cell-mediated immune response is cellular resistance to a T cell-mediated immune response.

[0325] In certain embodiments, a biological sample is obtained from a mammalian subject, such as a human patient. The term "biological sample" is used in its broadest sense and can refer to a bodily sample obtained from a subject (e.g., a human patient). For example, a biological sample can include a clinical sample, i.e., a sample derived from a subject. Such samples can include, but are not limited to, peripheral body fluids that may or may not contain cells, such as blood, urine, plasma, mucus, bile, pancreatic juice, supernatant, and serum; tissue or fine needle biopsy samples; tumor biopsy samples or sections (or cells thereof), and archived samples with known diagnostic, treatment, and / or outcome history. Biological samples can also include sections of tissue, such as frozen sections taken for histological purposes. The term "biological sample" can also encompass any material derived by processing a sample. Derived substances can include, but are not limited to, cells (or their progeny) isolated from the biological sample, nucleic acids and / or proteins extracted from the sample. Processing of a biological sample may include one or more of filtration, distillation, extraction, amplification, concentration, fixation, inactivation of interfering components, addition of reagents, and the like.

[0326] The detection, determination and / or diagnosis methods of the invention can be carried out as in vitro methods, for example, using a kit (or components thereof) of the invention.

[0327] In some embodiments of these detection, determination, and / or diagnosis methods, the biological sample is a tissue sample from a subject, e.g., a tumor or cancer sample from a subject. Such a sample may include tumor cells and / or blood cells (e.g., monocytes and T cells). As noted above, such a tissue sample may be a biopsy sample of a tumor or cancer, such as a needle biopsy sample, or a tumor biopsy section or archived sample thereof. Such a tissue sample may include live, dead, or fixed cells, such as from a tumor or cancer, which may be suspected of expressing (e.g., abnormally or localized) the applicable biomarker to be determined.

[0328] In other embodiments of these detection, determination and / or diagnosis methods, the biological sample is a blood sample from the subject, such as a sample of immune cells (e.g., monocytes and T cells) present in the blood.

[0329] In some embodiments, the determination and / or diagnosis methods of the present invention can further comprise comparing the detected amount (or activity) (of protein or mRNA) of the applicable biomarker (i.e., LILRB1 and / or LILRB2, or variants thereof) (i.e., LILRB1 and / or LILRB2, or variants thereof) with a standard value or cut-off value, wherein a detected amount greater than the standard value or cut-off value indicates a phenotype (or a risk of developing a phenotype) associated with the undesirable presence of LILRB1 and / or LILRB2-positive cells (or cells positive for LILRB1 and / or LILRB2 variants thereof), and / or associated with cellular resistance to a cell-mediated immune response in the subject, and / or associated with (e.g., abnormal) expression or activity of LILRB1 and / or LILRB2 (or variants thereof) in the subject. Such a standard value or cut-off value may be determined from the use of an assay, or may be predetermined from one or more values ​​obtained from a study or multiple samples with known phenotypes. For example, a cut-off value for a diagnostic test may be determined by analysis of samples taken from patients in the context of a clinical trial and determining the cut-off according to the desired (or obtained) sensitivity and / or specificity of the test.

[0330] Examples of methods useful for detecting (e.g., the presence, absence, or amount) of applicable biomarkers (i.e., LILRB1 and / or LILRB2, or variants thereof) include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) that use an ABP (e.g., of the present invention), such as an antibody or antigen-binding fragment thereof, that specifically binds to such applicable biomarkers.

[0331] Such methods can use monoclonal or polyclonal antibodies. Examples of monoclonal antibodies are described elsewhere herein. As used herein, the term "polyclonal antibody" refers to a mixture of antibodies that are genetically distinct because they are produced by plasma cells derived from multiple somatic recombination and clonal selection events, and typically recognize different epitopes of the same antigen.

[0332] Alternatively, the presence of an applicable biomarker (i.e., LILRB1 and / or LILRB2, or variants thereof) can be detected by detecting the presence of mRNA encoding such applicable biomarker or a fragment of such mRNA. Methods for detecting the presence of such mRNA (or fragment) can include PCR (such as quantitative RT-PCR), hybridization (such as Illumina chip), nucleic acid sequencing, etc. Such methods can include or include using one or more nucleic acids described herein, such as PCR primers or probes, or hybridization probes, that bind (e.g., specifically) to such mRNA.

[0333] For such detection, determination, or diagnostic applications, ABPs or nucleic acids are typically labeled with a detectable labeling group. Generally, labeling groups are divided into various classes depending on the assay in which they are detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags, etc.). Suitable labeling groups include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y,99 Tc, 111 In, 125 I, 131 I), fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the ABP or nucleic acid via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used. For example, the ABP or nucleic acid can be labeled with a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags, etc.).

[0334] Thus, in certain embodiments of the detection / diagnostic method (or kit therefor), the means (e.g., ABP or nucleic acid) for detecting (e.g., detector) the protein or mRNA of the applicable biomarker (e.g., LILRB1 and / or LILRB2) is, for example, conjugated to a detectable label. The term "label" or "labeling group" refers to any detectable label, including those described herein.

[0335] In certain embodiments, the detection / diagnostic methods of the present invention include immunohistochemistry (IHC) or immunocytochemistry (ICC) assays. The terms "IHC" and "ICC" are art-recognized and include the meaning of techniques used to localize antigen expression that relies on specific epitope-antibody interactions. IHC typically refers to the use of tissue sections, while ICC typically describes the use of cultured cells or cell suspensions. In both methods, positive staining is typically visualized using a molecular label (e.g., which may be fluorescent or chromogenic). Briefly, the sample is typically fixed to preserve cellular integrity and then subjected to incubation with a blocking reagent to prevent nonspecific binding of the antibody. The sample is then typically incubated with a primary (and sometimes secondary) antibody, and the signal is visualized for microscopic analysis.

[0336] "Detection / Genomic / Monitoring Kit"

[0337] In a seventh aspect, the present invention provides a kit for determining the presence, absence, amount, function, activity, and / or expression of an applicable biomarker (i.e., LILRB1 and / or LILRB2, or a variant thereof) in a sample (e.g., a biological sample), e.g., on cells in the sample, e.g., a kit for carrying out a diagnostic or determination method or a detection method (or monitoring or prognostic method) of the present invention. The kit comprises an ABP and / or nucleic acid as described above, and optionally one or more additional components.

[0338] In certain embodiments of the kit, additional components may include an ABP or nucleic acid or instructions describing how to use the kit, e.g., to detect the presence of an applicable biomarker in a sample by detecting binding between the ABP and a protein of such applicable biomarker and / or by detecting binding between a nucleic acid and the mRNA of such applicable biomarker. Such instructions may consist of a printed manual or computer-readable memory containing such instructions, or may include instructions for identifying, obtaining, and / or using one or more other components to be used with the kit.

[0339] In other particular embodiments of the kit, the additional components may include one or more other requirements, components, reagents, or other means useful for using the kit or practicing the detection methods of the invention (including any such requirements, components, reagents, or means disclosed herein useful for such practice). For example, the kit may further include reaction and / or binding buffers, labels, enzyme substrates, secondary antibodies, and samples, materials, or moieties, etc.

[0340] The present invention, in the context of the full disclosure, also relates in certain embodiments to the following itemized embodiments: Item 1: An isolated antigen binding protein (ABP) that specifically binds to the extracellular domain (ECD) of leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and / or LILRB2 proteins, wherein the isolated ABP comprises at least one complementarity determining region (CDR) and is capable of inhibiting the binding of LILRB1 and / or LILRB2 to its natural ligand (a natural ligand of LILRB1 and / or LILRB2, such as HLA-G), and preferably the ABP does not bind to, or binds with lower affinity to, leukocyte immunoglobulin-like receptor subfamily A (LILRA) proteins, such as LILRA1 and / or LILRA3. Item 2: The isolated ABP according to Item 1, comprising at least one complementarity-determining region 3 (CDR3) having 90% sequence identity or having three or less, preferably one or less, amino acid substitutions compared to a sequence selected from the following: SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, and 351. Item 3: The isolated ABP according to Item 1 or 2, wherein the ABP is an antibody or an antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, and at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the combinations in the table below for heavy chain and / or light chain CDRs, CDR-A-001 to CDR-A-044: [Table I-1] [Table I-2] [Table I-3] Each independently optionally has no more than three or no more than two, preferably no more than one amino acid substitution, insertion or deletion compared to these sequences. Item 4: The isolated ABP according to any one of Items 1 to 3, wherein the ABP is an antibody or an antigen-binding fragment thereof, which is composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences. (i) wherein at least one, preferably both, of the antibody heavy chain sequences comprise the heavy chain CDR1 to CDR3 sequences in the combination CDR-A-010, respectively, and at least one, preferably both, of the antibody light chain sequences comprise the light chain CDR1 to CDR3 sequences in the combination CDR-A-010, respectively, each independently optionally comprising no more than one amino acid substitution, insertion or deletion compared to these sequences; and preferably, the ABP specifically binds to LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of 10 nM or less, preferably about 5 nM or less; or (ii) wherein at least one, preferably both, of the antibody heavy chain sequences comprises the heavy chain CDR1 to CDR3 sequences in the combination CDR-A-026, respectively, and at least one, preferably both, of the antibody light chain sequences comprises the light chain CDR1 to CDR3 sequences in the combination CDR-A-026, respectively, each independently optionally comprising one or less amino acid substitutions, insertions or deletions compared to these sequences, and preferably the ABP specifically binds to LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of 10 nM or less, preferably about 5 nM or less. Item 5: An isolated ABP, preferably an isolated ABP, that competes with the ABP described in any one of items 1 to 4 for binding to the ECD of the LILRB1 and / or LILRB2 protein and is capable of inhibiting the binding of the LILRB1 and / or LILRB2 protein or a mutant thereof to a natural ligand of LILRB1 and / or LILRB2. Item 6: An isolated ABP according to any one of items 1 to 5, which specifically binds to LILRB1 and / or LILRB2 with a KD of 100 nM or less (preferably 50 nM or less, more preferably 10 nM or less); preferably, an isolated ABP that binds to LILRA such as LILRA1 and / or LILRA3 with a KD of 50 nM or more (preferably 100 nM or more, more preferably 200 nM or more). Item 7: (i) an isolated ABP according to any one of items 1 to 6, which inhibits the binding of a natural ligand of LILRB1 and / or LILRB2 to LILRB1 and / or LILRB2, wherein such a ligand is preferably HLA-G, and / or (ii) an isolated ABP, for example, which reduces the occurrence of an immunosuppressive phenotype in the tumor microenvironment. Item 8: The isolated ABP according to any one of items 1 to 7, which is an antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody or the antigen-binding fragment is a fragment of a monoclonal antibody. Item 9: The isolated ABP according to any one of items 1 to 8, which is an antibody or an antigen-binding fragment thereof, wherein the antibody is a human antibody, a humanized antibody, or a chimeric human antibody, or the antigen-binding fragment is a fragment of a human antibody, a humanized antibody, or a chimeric human antibody. Item 10: An isolated nucleic acid encoding an ABP, or an antigen-binding fragment or monomer of an ABP, wherein the ABP is one according to any one of items 1 to 9. Item 11: A recombinant host cell comprising the nucleic acid of Item 10. Item 12: A pharmaceutical composition comprising (i) the ABP according to any one of Items 1 to 9, or (ii) the nucleic acid according to Item 10, or (iii) the recombinant host cell according to Item 11, and a pharmaceutically acceptable carrier, stabilizer and / or excipient. Item 13: A product for use in medicine selected from the list consisting of (i) the isolated ABP according to any one of Items 1 to 9, and (ii) the isolated nucleic acid according to Item 10, (iii) the recombinant host cell according to Item 11, and (iv) the pharmaceutical composition according to Item 12. Item 14: The pharmaceutical composition according to Item 13, for use in treating an infectious or proliferative disorder associated with a suppressed cell-mediated immune response due to the expression or activity of LILRB1 and / or LILRB2, or a mutant thereof. Item 15: The product for use in medicine according to Item 14, wherein cells involved in the proliferative disorder express natural ligands of LILRB1 and / or LILRB2, and such cells induce an immunosuppressive phenotype in immune cells such as macrophages. Item 16: A product for use in medicine according to any one of items 13 to 15, wherein the product is for use in enhancing the immune response in a mammalian subject, preferably for use in promoting immunoactivation of a humoral or cell-mediated immune response in a subject, such as promoting pro-inflammatory polarization of macrophages towards an M1 phenotype, for treating an infectious disease, e.g. for treating a proliferative disease such as a cancer disease. Item 17: A product for use in the medicament according to any one of Items 13 to 16 for use in the treatment of a proliferative disorder resistant and / or refractory to PD1 / CTLA4 blockade therapy. Item 18: A product for use according to any one of items 13 to 17, having any one or any combination of the following characteristics: a. specific binding to human LILRB1 and / or (preferably) LILRB2 (e.g., comprising the amino acid sequences of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (LILRB2)), e.g., with a KD of 50 nM or less, or 20 nM or less; more preferably, 10 nM or less, or 5 nM or less; b. lack of specific binding to LILRA proteins, e.g., preferably LILRA1 and / or LILRA3; c. stimulates T cell activation, e.g., as determined by increased T cell proliferation or IFN-γ secretion, e.g., in a mixed lymphocyte reaction (MLR) assay; d. stimulating the differentiation or activation of monocytes into macrophages, e.g., stimulating the differentiation of monocytes into pro-inflammatory macrophages, as shown in the assays described in the Examples; e. inhibiting the binding of LILRB1 and / or (preferably and) LILRB2 to HLA-A and HLA-B, preferably HLA-G; f. has a binding profile as shown in Table 2; g. Promoting pro-inflammatory polarization of macrophages towards M1 macrophages; and h. Does not induce (or induce) basophil activation.

[0341] The present invention, in the context of a complete disclosure, also relates in certain embodiments to the embodiments of Section B set forth in the following sections: Item B1: An isolated antigen binding protein (ABP) that specifically binds to the extracellular domain (ECD) of leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and LILRB2 proteins, wherein the isolated ABP comprises at least one, preferably three or six complementarity determining regions (CDRs) and is capable of inhibiting the binding of LILRB1 and LILRB2 to their natural ligands (the natural ligands of LILRB1 and / or LILRB2, e.g., HLA-G), and wherein the ABP does not bind to LILRA1 and LILRA3 or binds with less affinity (at least two-fold less) to LILRA1 and LILRA3. Item B2: An isolated ABP of item B1, wherein the ABP binds to the ECD of LILRB1 and LILRB2 with at least a 5-fold higher, or preferably a 10-fold higher, binding affinity compared to the binding of the ABP to the ECD of LILRA1 and / or LILRA3, and preferably the ABP binding affinity is determined by biolayer interferometry (B1I). Item B3: An isolated ABP of item B1 or B2, wherein the ABP binds to the ECD of LILRB1 and / or LILRB2 with a binding dissociation constant (KD) that is at least 2-fold lower, preferably 3-fold lower, more preferably 5-fold lower, and most preferably 10-fold lower, compared to the binding KD of the ABP to the ECD of LILRA1 and / or LILRA3, the KD being determined by biolayer interferometry (B1I), preferably under the conditions described in Example 3. Item B4: The isolated ABP of item B3, wherein the ABP binds to the ECDs of LILRB1 and LILRB2 with a dissociation constant (KD) that is at least 2-fold lower, preferably 3-fold lower, more preferably 5-fold lower, and most preferably 10-fold lower than the KD of the ABP for the ECDs of LILRA1 and LILRA3, and the KD is determined by biolayer interferometry (B1I), preferably under the conditions described in Example 3. Item B5: The isolated ABP of any one of items B1 to B4, wherein the ABP comprises one, preferably two, antibody heavy chain sequences and one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence are derived from one or a combination of antibody parent clones A-001 to A-003, preferably A-001 and / or A-003. Item B6: The isolated ABP according to item B5, wherein the ABP comprises one, preferably two, antibody heavy chain variable sequences and one, preferably two, antibody light chain variable sequences, wherein the antibody heavy and light chain variable sequences comprise sequences that are at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identical to the antibody heavy or light chain variable sequences, respectively, set forth for any of the parent antibody sequences A-001 to A-003, preferably A-001 and / or A-003. Item B7: The isolated ABP according to any one of items B1 to B6, wherein the ABP competes with a ligand or receptor of endogenous LILRB1 and / or LILRB2 for binding to the ECD of LILRB1 and / or LILRB2, or to the ECD of a variant of LILRB1 and / or LILRB2, preferably wherein the endogenous ligand or receptor of LILRB1 and / or LILRB2 is an HLA-G protein (or a variant of HLA-G). Item B8: The isolated ABP according to item B7, wherein the ABP is capable of inhibiting the binding of an HLA-G protein or a variant thereof to a LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of less than 100 nM, less than 50 nM, or preferably less than 20 nM, for example less than 15 nM or less than 10 nM, as determined by ELISA. Item B9: SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, and 383, or having no more than three or two, preferably no more than one amino acid substitution, deletion or insertion. Item B10: The isolated ABP of any one of Items B1 to B9, wherein the ABP is an antibody or an antigen-binding fragment thereof and is composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the following combinations of heavy chain and / or light chain CDRs, CDRs-A-001 to CDRs-A-048: [Table II-1] [Table II-2] Each independently and optionally has no more than three or no more than two, preferably no more than one amino acid substitution, insertion or deletion compared to these sequences. Item B11: An isolated ABP, preferably an isolated ABP, which competes with the ABP described in any one of items B1 to B10 for binding to the ECD of LILRB1 and / or LILRB2 protein and is capable of inhibiting the binding of LILRB1 and / or LILRB2 protein or a mutant thereof to a natural ligand of LILRB1 and / or LILRB2. Item B12: An isolated ABP according to any one of items B1 to B11, which (i) inhibits the binding of a natural ligand of LILRB1 and / or LILRB2 to LILRB1 and / or LILRB2, such ligand being preferably HLA-G, and / or (ii) reduces the development of an immunosuppressive phenotype, for example, in the tumor microenvironment. Item B13: The isolated ABP according to any one of items B1 to B12, which is an antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody, or the antigen-binding fragment is a fragment of a monoclonal antibody. Item B14: An isolated nucleic acid encoding an ABP, or an antigen-binding fragment or monomer of an ABP, wherein the ABP is one according to any one of items B1 to B13. Item B15: A recombinant host cell comprising a nucleic acid according to item B14. Item B16: A pharmaceutical composition comprising (i) an ABP according to any one of items B1 to B13, or (ii) a nucleic acid according to item B14, or (iii) a recombinant host cell according to item B15, and a pharmaceutically acceptable carrier, stabilizer and / or excipient. Item B17: A product for use in medicine selected from the list consisting of: (i) an isolated ABP according to any one of items B1 to B13, and (ii) an isolated nucleic acid according to item B14; (iii) a recombinant host cell according to item B15, and (iv) a pharmaceutical composition according to item B16. Item B18: A product for use according to item B17, wherein the use in medicine is the treatment of a proliferative disorder, wherein cells involved in the proliferative disorder express natural ligands of LILRB1 and / or LILRB2, and such cells preferably induce an immunosuppressive phenotype in immune cells such as macrophages of the subject. Item B19: Product for use according to item B17 or B18 for use in enhancing the immune response in a treated mammalian subject, preferably for use in promoting immunoactivation of humoral or cell-mediated immune response in a subject, such as promoting pro-inflammatory polarization of macrophages towards M1 phenotype, for treating infectious diseases, for example for treating proliferative diseases such as cancer diseases. Item B20: A product for use according to any one of items B17 to B19, having one or any combination of the following characteristics: a. specific binding to human LILRB1 and / or (preferably) LILRB2 (e.g., comprising the amino acid sequences of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (LILRB2)), e.g., with a KD of 50 nM or less, or 20 nM or less; more preferably, 10 nM or less, or 5 nM or less; b. lack of specific binding to LILRA proteins, e.g., preferably LILRA1 and / or LILRA3; c. stimulation of T cell activation, as determined, for example, by increased T cell proliferation or IFN-γ secretion, e.g., in a mixed lymphocyte reaction (MLR) assay; d. stimulating the differentiation or activation of monocytes into macrophages, e.g., stimulating the differentiation of monocytes into pro-inflammatory macrophages, as shown in the assays described in the Examples; e. inhibiting the binding of LILRB1 and / or (preferably and) LILRB2 to HLA-A and HLA-B, preferably HLA-G; f. has a binding profile as shown in Table 2; g. Promoting pro-inflammatory polarization of macrophages into M1 macrophages; and h. Does not induce (or induce) basophil activation.

[0342] As used herein, the terms "of the present invention," "in accordance with the present invention," "based on the present invention," and the like are intended to refer to all aspects and embodiments of the present invention described and / or claimed herein.

[0343] As used herein, the term "comprising" is to be interpreted as encompassing both "including" and "consisting of," both meanings being specifically intended, and therefore each individually disclosed embodiment according to the present invention. As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. For example, "A and / or B" and "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, respectively, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" refer to an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. The terms typically indicate deviations of ±20%, ±15%, ±10%, and, for example, ±5% from the indicated numerical value. As will be understood by a person skilled in the art, such specific deviations from the numerical value for a given technical effect depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have such a deviation greater than 1, as compared to an artificial or engineered technical effect. As will be understood by a person skilled in the art, such specific deviations from the numerical value for a given technical effect depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have such a deviation relative to a man-made or engineered technical effect that is greater than 1. Where an indefinite or definite article is used when referring to a singular noun, e.g., "a", "an" or "the", this includes the plural of that noun unless something else is specifically stated.

[0344] It will be understood that application of the teachings of the present invention to a particular problem or environment, and modification of the present invention or the inclusion of additional features thereto (such as further aspects and embodiments), is within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.

[0345] Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0346] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0347] Certain aspects and embodiments of the present invention will now be described, by way of example, with reference to the description, figures, and tables set forth herein. Such examples of methods, uses, and other aspects of the present invention are representative only and should not be construed as limiting the scope of the invention solely to such representative examples. [Example]

[0348] The following is an example:

[0349] Example 1: "Generation and maturation of antibodies that bind to human LILRB1 and / or LILRB2"

[0350] Cross-specific antibodies against LILRB2 and LILRB1 with improved affinity were selected by phage display from an antibody gene library based on parental V gene sequences with diversified CDR-H1 / H2 and CDR-L3, respectively. For each parental sequence, two diversified libraries were constructed: (i) keeping the light chain constant and diversifying CDR-H1 and CDR-H2, and (ii) keeping the heavy chain constant and diversifying CDR-L3. The CDR-H1 / 2 diversified library contained between 8e8 and 4e9 derivatives, and the CDR-L3 diversified library contained more than 2e7 derivatives of each parental sequence. Diversification of the CDR sequences was based on rational design.

[0351] Optimized selection conditions were applied to select for higher affinity binders, improve cross-specificity to LILRB2 / LILRB1, and simultaneously reduce binding to LILRA1 / LILRA3. Briefly, the antibody phage library was blocked with 2x ChemiB1OCKER (Merck Millipore) and pre-adsorbed to streptavidin-coated magnetic beads loaded with biotinylated LILRA1 and LILRA3. In the first panning round, biotinylated recombinant target protein (LILRB1) was added at a concentration of 10 nM and incubated with a 20-fold molar excess of non-biotinylated recombinant LILRA1 and LILRA3 for 1 h at room temperature. Antibody phage bound to recombinant LILRB1 were isolated using streptavidin magnetic beads (Dynabeads M-280, ThermoFisher) and washed with DPBST, including an extended wash step, for 30 minutes in the presence of 100 nM non-biotinylated target protein and 500 nM non-biotinylated recombinant LILRA1 and LILRA3. Antibody phage particles were eluted with 10 μg / ml trypsin and used to infect mid-logarithmic E. coli TGI for phage amplification.

[0352] Panning rounds 2 and 3 were performed equivalent to panning round 1 with the following modifications to increase the selection pressure for higher affinity: the concentration of biotinylated recombinant target protein (LILRB2 in panning round 2 and LILRB1 in panning round 3) was further restricted (1 nM in panning round 2 and 0.5 nM in panning round 3). After capturing the biotinylated antigen with bound antibody phage on streptavidin magnetic beads, an initial washing step with DPBST was performed. To further increase the stringency of the wash and select for slower dissociation rates, beads were suspended in 1.5 ml of PBST containing 100 nM non-biotinylated recombinant target protein and 100 nM non-biotinylated LILRA1 and LILRA3 and incubated at room temperature for up to 20 hours.

[0353] Enrichment of higher affinity binders and optimal selection stringency was monitored by determining the selection output of each condition and phage titer in the panning round.

[0354] Parental and mature scFv antibodies of the present invention that selectively bind to human LILRB1 and / or LILRB2 proteins have been identified and are set forth in Table 1, which shows, for each such antibody, the heavy and light chain CDR sequences and variable region sequences contained in each such antibody.

[0355] Antibodies A-001, A-002, and A-003 constitute selected parent antibodies. Antibodies A-004 to A-048 constitute derivatives of one or two heavy and / or light chain sequences of the parent molecule. In particular, A-004 to A-021 are heavy chain (HC) variants of the parent antibody A-001. A-022 to A-033 are HC variants of the parent antibody A-003. A-034 to A-036 are light chain (LC) variants of the parent antibody A-001, and A-037 is an LC variant of A-002. A-038 to A-044 are LC variants of the parent antibody A-003.

[0356] Antibody A-045 is a derivative of antibody A-001 and contains the HC of A-010 and the LC of A-036. A-046 is a derivative of antibody A-001 and contains the HC of A-015 and the LC of A-036. A-047 is a combination of the HC and LC chains derived from A-001 and A-003 and contains the HC of A-025 and the LC of A-038. A-048 is a combination of the HC and LC chains derived from A-001 and A-003 and contains the HC of A-025 and the LC of A-039. Table 1: Amino acid sequences of the CDRs and variable regions of the ABPs of the invention, and nucleic acid sequences encoding the variable regions of the ABPs of the invention. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0357] Example 2 "Target and Non-Target Binding Affinities"

[0358] To identify affinity-improved binders specific for LILRB2 and LILRB1, but not for LILRA1 and LILRA3, monoclonal FaBs were expressed in E. coli after panning rounds 2 and 3. After bacterial lysis, recombinant LILRB2, LILRB1, LILRA1, and LILRA3 were tested for their binding properties and cross-reactivity profiles by standard ELISA at 1:100 or 1:25 dilutions for maturation of the heavy and light chains, respectively. Briefly, biotinylated recombinant LILRB2, LILRB1, LILRA1, and LILRA3 were immobilized at 1 μg / mL on streptavidin-coated 384-well Maxisorp plates. The surface was blocked with 2% (w / v) BSA (bovine serum albumin) in PBST. After three washing cycles with PBST, E. coli lysate in 2% (w / v) BSA was applied to the immobilized antigen and incubated for 1.5 hours. After three washing cycles with PBST to remove all unbound antibody, bound Fab antibody was detected with a goat anti-human Fab antibody conjugated with horseradish peroxidase. After three washing cycles with PBST, the ELISA was developed with TMB substrate.

[0359] ELISA-positive hits with the desired cross-reactivity profile and improved binding over the parental clones were then analyzed for their cell-binding properties by standard multiplexed flow cytometry. Briefly, ExpiCHO cells transiently transfected with constructs expressing the extracellular domains (ECDs) of LILRB2, LILRB1, and LILRA1, or LILRA3 fused to an intracellular GFP tag were incubated with different concentrations (50 nM and unstained) of CellTrace ELISA. (商標) Multiplexed flow cytometry analysis (LILRB2 vs. LILRA1 and LILRB1 vs. LILRA3) was performed by staining with Violet (Invitrogen). Differentially stained cell lines were mixed at a 1:1 ratio, and 30,000 cells were incubated with E. coli lysate containing monoclonal Fab in a 384-well format. Unbound antibody was removed by washing the cells three times with FACS buffer (DPBS + 3% (v / v) FCS). Bound antibody was detected with a mouse anti-human Fab antibody conjugated to AlexaFluor647. Dead cells were excluded by Zombie Yellow Dye (Biolegend) staining, and the median fluorescence intensity (MFI) of AlexaFluor647 was measured using two different CellTrace (商標) Violet (Invitrogen) stained cell populations were analyzed to determine specific binding to cells expressing LILRB2 and LILRB1, and reduced binding to LILRA1 and LILRA3.

[0360] The off-rates of the best cell binders were analyzed by biolayer interferometry (BLI) on an OctetRED96e system in standard kinetic experiments using recombinant LILRB2 and LILRB1 proteins. Briefly, biotinylated recombinant LILRB2 or LILRB1 was immobilized on a streptavidin-coated biosensor. The sensor was immersed in E. coli lysate containing a monoclonal Fab diluted 1:1 with 2x Kinetics Buffer supplemented with 20 μg / mL biocytin and 4% (w / v) BSA, and the antibody was allowed to bind for 420 seconds. The sensor was then immersed in 1x Kinetics Buffer supplemented with 10 μg / mL biocytin and 2% (w / v) BSA, and off-rates were measured over a 420-second period. All sensorgrams were double-referenced against 1x Kinetics Buffer supplemented with 10 μg / mL biocytin and 2% (w / v) BSA and an empty streptavidin sensor, and sensor drift and E. coli lysate binding potential were subtracted. Dissociation rates were fitted using a 1:1 binding model.

[0361] Affinity was determined by BLI on an Octet RED96e system using the ECDs of recombinant LILRB2, LILRB1, LILRA1, and LILRA3. Briefly, IgG was captured on an anti-human capture (AHC) biosensor (Sartorius). The sensor was immersed in a well containing 100 nM recombinant LILRB2 or LILRB1 ECD, or 900 nM LILRA1 ECD or LILRA3, and incubated for 300 seconds to measure association. The sensor was then immersed in 1x Kinetics Buffer, and dissociation was measured over 420 seconds. All sensorgrams were double-referenced against 1x Kinetics Buffer and an unloaded streptavidin sensor, and sensor drift and analyte potential binding were subtracted. Affinity was determined by applying a 1:1 binding model.

[0362] Binding curves of the antibody clones of the invention are shown in Figure 2. Dissociation constants (KD) are shown below in Table 2 for the antibodies tested. Surprisingly, all parental and mature antibody clones of the invention show cross-specificity between the "on" targets LILRB1 and LILRB2, but significantly lower binding to both the "off" targets LILRA1 and LILRA3.

[0363] Example 2.1: "ABP Binding to LILRB1 and LILRB2 in the Presence of Off-Target LILRA1 or LILRA3"

[0364] To examine the ability of ABPs to bind to LILRB1 and LILRB2 in the presence of LILRA1 or LILRA3, biolayer interferometry (BLI) experiments were performed using an Octet RED96e system. Briefly, biotinylated recombinant LILRB2 ECD (extracellular domain) or LILRB1 ECD was immobilized on a streptavidin-coated biosensor. After recording baseline signals in assay buffer (1:10, 10x kinetics buffer, Sartorius, diluted with DPBS), the sensors were immersed in wells containing 10 nM antibody in either assay buffer, assay buffer supplemented with 20 nM recombinant LILRA1 ECD or LILRA3 ECD, or assay buffer supplemented with 100 nM recombinant LILRA1 ECD or LILRA3 ECD, and incubated at 25°C for 600 seconds to measure association. To exclude signal changes caused by LILRA1 or LILRA3 ECD, samples without IgG were also measured. The sensor was then immersed in 1x kinetics buffer and dissociation was measured over 600 s. All samples for the same antibody were measured in one assay cycle (baseline association-dissociation), and the sensor was regenerated with 10 mM glycine pH 1.5 between cycles. All sensorgrams were double-referenced against 1x kinetics buffer and an empty streptavidin sensor, and sensor drift and analyte potential binding were subtracted. Binding was normalized to the binding response after 600 s of association in 1x kinetics buffer, which was set as 1 for each antibody.

[0365] Binding curves of the antibodies of the invention are shown for LILRB2 (Figure 11) and LILRB1 (Figure 12).

[0366] Surprisingly, A-045 showed no or only slight loss of binding capacity to LILRB1 or LILRB2 in the presence of LILRA1 or LILRA3, whereas a dramatic loss of binding capacity to detectable binding was observed for the cross-specific reference molecule (Ref062 [NGM]) due to the high affinity of Ref062 for LILRA1 and LILA3. Similarly, a dramatic loss of binding capacity to LILRB1 was observed for the reference molecule (Ref051 [Biond / Sanofi BND-22]) in the presence of LILRA1 or LILRA3.

[0367] Example 3: "Inhibition of LILRB1 and LILRB2 interaction with HLA-G"

[0368] HLA-G inhibition was tested by ELISA. 384-well Maxisorp plates were coated with 2 μg / mL B2M-HLA-G fused to mouse IgG2a-Fc, and the plates were blocked with 2% (w / v) BSA in PBST. 30 nM biotinylated LILRB2 ECD or LILRB1 ECD was mixed with a 1:1000 dilution of avidin-HRP (Biolegend, 405103) in a 1:1 volume ratio and incubated at room temperature for 1 h. 15 μl of the antibody dilution series (300 nM to 0.3 nM) was mixed with 30 μL of premixed LILRB / avidin-HRP solution (final antibody concentrations 100 nM to 0.1 nM) and incubated at room temperature for 1 h. After three washing cycles with PBST, 20 μL of the antibody / LILRB / avidin-HRP solution was transferred to the plate with immobilized HLA-G and incubated at room temperature for 15 minutes. After three washing cycles with PBST, residual LILRB binding to HLA-G was detected by developing an ELISA using TMB substrate. The respective binding curves are shown in Figures 3, 4, and 5. The IC50 values ​​for inhibiting LILRB1 / 2-HLA-G binding are shown in Table 2. Table 2: Biochemical assay results Dissociation constants (KD) for binding target or off-target proteins and IC50 for ligand binding inhibition are provided. [Table 2-1] [Table 2-2] nb=non-bonded

[0369] Example 4.1: "Cross-specific LILRB1 / 2 antibodies of the present invention reduce M2-like polarization of monocyte-derived macrophages"

[0370] To characterize the effects of cross-specific LILRB1 / 2 antibodies and the reference monospecific LILRB2 antibody on macrophage phenotype and function, a functional bone marrow assay was performed. M0 macrophages were generated by culturing isolated monocytes in medium (RPMI with 10% FBS + 1% P / S) containing 50 ng / ml recombinant human M-CSF (Peprotech) and the indicated concentrations of an in-house cross-specific LILRB1 / 2 antibody, a reference cross-specific LILRB1 / 2 antibody (Ref062 [NGM707]), a reference monospecific LILRB2 antibody (Ref047 [Merck MK-4830]), or the appropriate isotype control (Ref001) for 6 days at 37°C in six-well tissue culture plates. M0 macrophages were removed from the plate using a cell scraper and polarized to M2-like macrophages (RPMI) in 6-well tissue culture plates for 2 days at 37°C in medium (RPMI containing 10% FBS + 1% p / S) containing 20 ng / ml recombinant human IL-4 (Peprotech), 20 ng / ml recombinant human IL-10 (Peprotech), and 20 ng / ml recombinant human TGF-β (Peprotech), and the indicated concentrations of an in-house cross-specific LILRB1 / 2 antibody, a reference cross-specific LILRB1 / 2 antibody (Ref062 [NGM707]), a monospecific LILRB2 antibody (Ref047 [Merck MK-4830]), or the appropriate isotype control (Ref001). On day 8, supernatants were collected for analysis of cytokines via Luminex Assay (R&D Systems), and M2-like macrophages were analyzed by flow cytometry for various surface markers indicative of an M2-like phenotype.

[0371] As shown in Figure 6A, cross-specific LILRB1 / 2 antibodies and mono-specific LILRB2 antibodies induced a significant reduction in the M2-like macrophage phenotype marker CD163, consistent with a less suppressive M2-like polarization of macrophages. Consistent with this, cross-specific LILRB1 / 2 antibodies and mono-specific LILRB2 antibodies also significantly reduced the secretion of immunosuppressive CCL13 and CCL23 cytokines by macrophages following M2-like polarization (see Figures 6B and 6C). Surprisingly, all cross-specific LILRB1 / 2 antibodies reduced the M2 marker CD163 and immunosuppressive cytokines at concentrations much lower than the mono-specific LILRB2 antibody, demonstrating superior potency when both LILRB1 and LILRB2 are blocked. Furthermore, when comparing cross-specific LILRB1 / 2 antibodies, A-010 exhibited even greater potency than the reference molecule Ref062.

[0372] Such results further demonstrate the ability of the antibodies of the present invention to selectively bind to both LILRB1 and LILRB2, but not to their counter-targets LILRA1 and LILRA3. Furthermore, the cross-specific antibodies of the present invention effectively reduce M2-like macrophage polarization, thereby providing a means to reduce the immunosuppressive function of the LILRB-HLA-G axis, for example, in the tumor microenvironment.

[0373] Example 4.2: "Cross-specific LILRB1 / 2 antibodies of the present invention reduce M2-like polarization of monocyte-derived macrophages and rescue cytotoxic T cell activation"

[0374] Myelosuppression assays were performed as described above for functional bone marrow assays. However, on day 8, supernatants were collected for analysis of myeloid cytokines, after which isolated autologous T cells were added to macrophages at a 1:5 ratio and co-cultured for an additional 3 days. On day 11, supernatants were again collected for analysis of T cell cytokines via Luminex Assay (R&D Systems), and T cells were analyzed by flow cytometry for surface activation markers.

[0375] The cross-specific LILRB1 / 2 antibody A-045 and the reference molecule Ref-062, as well as the monospecific LILRB2 antibody Ref-047, induced a significant reduction in the M2-like macrophage phenotype marker CD163 (Figure 6D), consistent with a less suppressive M2-like polarization of macrophages. Consistent with this, all antibodies also significantly reduced the secretion of the immunosuppressive CCL18 following M2-like polarization (Figure 6E). Regarding the rescue of T cell activation, all antibodies upregulated the early activation marker CD69 on T cells and increased the secretion of the proinflammatory cytokines GM-CSF and IFNγ (Figure 6FH). Compared to the reference molecules, A-045 showed specifically superior potency in T cell rescue.

[0376] Example 4.3: "Cross-specific LILRB1 / 2 antibodies of the present invention induce repolarization of monocyte-derived M1- and M2-like macrophages"

[0377] To characterize the effects of cross-specific LILRB1 / 2 antibodies on macrophage phenotype and function, a functional bone marrow assay was performed. M0 macrophages were generated by culturing isolated monocytes in medium (RPMI with 10% FBS + 1% P / S) containing 50 ng / ml recombinant human M-CSF (Peprotech) and the indicated concentrations of an in-house cross-specific LILRB1 / 2 antibody, a reference cross-specific LILRB1 / 2 antibody (Ref062 [NGM707]), or the appropriate isotype control (Ref001) at 37°C for 6 days in six-well tissue culture plates. M0 macrophages were removed ...

Claims

1. An isolated antigen binding protein (ABP) that specifically binds to the extracellular domain (ECD) of leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and LILRB2 proteins, wherein the isolated ABP comprises at least one, preferably three or six complementarity determining regions (CDRs), and is capable of inhibiting the binding of LILRB1 and LILRB2 to their natural ligands (the natural ligands of LILRB1 and / or LILRB2, e.g., HLA-G), and wherein the ABP does not bind to LILRA1 and LILRA3 or binds to LILRA1 and LILRA3 with lower affinity (at least two-fold less).

2. The isolated ABP of claim 1, wherein the ABP binds to the ECD of LILRB1 and LILRB2 with a binding affinity that is at least 5-fold higher, or preferably 10-fold higher, compared to the binding of the ABP to the ECD of LILRA1 and / or LILRA3, and preferably the ABP binding affinity is determined by biolayer interferometry (BLI).

3. The isolated ABP of claim 1 or 2, wherein the ABP binds to the ECD of LILRB1 and / or LILRB2 with a binding dissociation constant (KD) that is at least 2-fold lower, preferably 3-fold lower, more preferably 5-fold lower, and most preferably 10-fold lower than the binding KD of the ABP to the ECD of LILRA1 and / or LILRA3, and the KD is determined by biolayer interferometry (BLI), preferably under the conditions described in Example 3.

4. The isolated ABP of claim 3, wherein the ABP binds to the ECD of LILRB1 and LILRB2 with a dissociation constant (KD) that is at least 2-fold lower, preferably 3-fold lower, more preferably 5-fold lower, and most preferably 10-fold lower than the KD of the ABP for the ECD of LILRA1 and LILRA3, and the KD is determined by biolayer interferometry (BLI), preferably under the conditions described in Example 3.

5. 5. The isolated ABP according to any one of claims 1 to 4, wherein the ABP comprises one, preferably two, antibody heavy chain sequences and one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequences and the antibody light chain sequences are derived from one or a combination of antibody parent clones A-001 to A-003, preferably A-001 and / or A-003.

6. 6. The isolated ABP of claim 5, wherein the ABP comprises one, preferably two, antibody heavy chain variable sequences and one, preferably two, antibody light chain variable sequences, wherein the antibody heavy and light chain variable sequences comprise sequences that are at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identical to the antibody heavy or light chain variable sequences, respectively, shown for any of the parent antibody sequences of antibodies A-001 to A-003, preferably A-001 and / or A-003.

7. The isolated ABP of any one of claims 1 to 6, wherein the ABP competes for binding between the ECD of LILRB1 and / or LILRB2, or the ECD of a variant of LILRB1 and / or LILRB2, and a ligand or receptor of endogenous LILRB1 and / or LILRB2, and preferably the ligand or receptor of endogenous LILRB1 and / or LILRB2 is an HLA-G protein (or a variant of HLA-G).

8. The isolated ABP of claim 7, wherein the ABP is capable of inhibiting the binding of the HLA-G protein or a variant thereof to the LILRB1 and / or LILRB2 protein or a variant thereof with an IC50 of less than 100 nM, less than 50 nM, or preferably less than 20 nM, for example less than 15 nM or less than 10 nM, as determined by ELISA.

9. SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379 and 9. The isolated ABP of any one of claims 1 to 8, comprising at least one complementarity determining region 3 (CDR3) having an amino acid sequence with at least 90% sequence identity compared to a sequence selected from 383, or having no more than three or two, preferably no more than one amino acid substitution, deletion or insertion.

10. The isolated ABP according to any one of claims 1 to 9, wherein the ABP is an antibody or an antigen-binding fragment thereof composed of at least one, preferably two antibody heavy chain sequences and at least one, preferably two antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the following combinations of heavy and / or light chain CDRs, CDR-A-001 to CDR-A-048: Table 1-1 Table 1-2 Table 1-3 wherein each independently and optionally has no more than three or no more than two, preferably no more than one amino acid substitution, insertion or deletion compared to these sequences.

11. A heavy chain variable region sequence comprising: - heavy chain CDR1 of SEQ ID NO: 353, optionally with no more than 3 or 2, preferably no more than 1 amino acid substitution, insertion or deletion compared to this sequence; - a heavy chain CDR2 of SEQ ID NO: 354, optionally having no more than 3 or 2, preferably no more than 1 amino acid substitution, insertion or deletion compared to this sequence; - a heavy chain CDR3 of SEQ ID NO: 355, optionally having no more than 3 or 2, preferably no more than 1 amino acid substitution, insertion or deletion compared to this sequence; and, A light chain variable region sequence comprising: a light chain CDR1 of SEQ ID NO: 357, optionally with no more than 3 or 2, preferably no more than 1 amino acid substitution, insertion or deletion compared to this sequence; - a light chain CDR2 of SEQ ID NO: 358, optionally having no more than 3 or 2, preferably no more than 1 amino acid substitution, insertion or deletion compared to this sequence; a light chain CDR3 of SEQ ID NO: 359, optionally with no more than three or two, preferably no more than one amino acid substitution, insertion or deletion compared to this sequence; 11. The isolated ABP of any one of claims 1 to 10, comprising:

12. the antibody heavy chain variable region sequence of SEQ ID NO: 356, optionally having no more than 10, 9, 8, 7, 6, 5, 4, 3 or 2, preferably no more than 1, amino acid substitutions, insertions or deletions compared to this sequence; and, the antibody light chain variable region sequence of SEQ ID NO: 360, optionally having no more than 10, 9, 8, 7, 6, 5, 4, 3 or 2, preferably no more than 1, amino acid substitutions, insertions or deletions compared to this sequence; 12. The isolated ABP of any one of claims 1 to 11, comprising:

13. The isolated ABP of any one of claims 1 to 12, wherein the ABP binds to the same epitope in the extracellular domain of LILRB1 and / or LILRB2 as antibody clone A-045.

14. 14. The isolated ABP according to any one of claims 1 to 13, characterized in that the ABP comprises: a. the ABP specifically binds to LILRB1 and LILRB2 and binds to the same epitope on LILRB1 and LILRB2 as an antibody of Table 1, preferably A-001, A-003, A-010 or A-045; b. the ABP does not bind to LILRA1 or binds to LILRA1 with lower affinity; c. the ABP does not bind to LILRA3 or binds to LILRA3 with lower affinity; d. When the ABP binds to LILRB1 and / or LILRB2 on human monocytes, it reduces the M2-like polarization of monocytes.

15. 15. The isolated ABP of claim 14, which does not bind to or binds with lower affinity to LILRA1 and LILRA2 compared to the binding affinity of the ABP to LILRB1 and LILRB2.

16. e. The isolated ABP of claim 14 or 15, further characterized in that the ABP, when bound to LILRB1 on human macrophages, induces phagocytosis of the cells.

17. f. The isolated ABP of any one of claims 14 to 16, wherein the ABP reduces the expression of a marker of M2-like macrophages (such as CD163, CD206, or CD209) when bound to human LILRB1 and / or LILRB2 on human monocytes or macrophages.

18. g. The isolated ABP of any one of claims 14 to 17, wherein the ABP increases the expression of a marker of M1-like macrophages (or CD86, etc.) when bound to human LILRB1 and / or LILRB2 on human monocytes or macrophages.

19. h. An isolated ABP described in any one of claims 14 to 18, wherein the ABP, upon binding to human LILRB1 and / or LILRB2 on human monocytes or macrophages, induces the secretion of pro-inflammatory cytokines (or increases the secretion of GM-CSF, IFNγ and IL-9, etc.), preferably independent of the presence of LILRA1 and / or LILRA3.

20. The isolated ABP according to any one of claims 1 to 19, wherein the ABP is an inhibitor or antagonist of LILRB1 and LILRB2, preferably an inhibitor or antagonist of the interaction of LILRB1 and LILRB2 with their natural ligands such as HLA-G.

21. 21. The isolated ABP of any one of claims 1 to 20, wherein the ABP, when bound to LILRB1 or LILRB2 on macrophages, reduces polarization to M2-like macrophages or repolarizes M2-like macrophages.

22. 22. The isolated ABP of any one of claims 1 to 21, wherein the ABP, when bound to LILRB1 or LILRB2 on macrophages, reduces polarization into M2-like macrophages or repolarizes M2-like macrophages into M1-like macrophages.

23. An isolated ABP, preferably an isolated ABP, that competes with the ABP described in any one of claims 1 to 22 for binding to the ECD of LILRB1 and / or LILRB2 protein and is capable of inhibiting the binding of LILRB1 and / or LILRB2 protein, or a variant thereof, to the natural ligand of LILRB1 and / or LILRB2.

24. (i) inhibiting the binding of a natural ligand of LILRB1 and / or LILRB2 to LILRB1 and / or LILRB2, wherein such a ligand is preferably HLA-G; and / or (ii) An isolated ABP according to any one of claims 1 to 23, which reduces the occurrence of an immunosuppressive phenotype, for example in a tumor microenvironment.

25. 25. The isolated ABP of any one of claims 1 to 24, which is an antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody or the antigen-binding fragment is a fragment of a monoclonal antibody.

26. 26. An isolated nucleic acid encoding an ABP, or an antigen-binding fragment or monomer of an ABP, wherein the ABP is according to any one of claims 1 to 25.

27. 27. The isolated nucleic acid of claim 26, comprising a nucleic acid having a sequence encoding, in each case, a heavy or light chain CDR, a combination of heavy and / or light chain CDR1, CDR2 and CDR3, or a heavy or light chain variable domain as shown in the following table (described as Table 1 but described below as Table 2): Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15

28. 28. A nucleic acid construct (NAC) comprising the nucleic acid of claim 26 or 27, optionally with one or more additional features that allow expression of the encoded ABP or component of the ABP in a cell, such as a host cell.

29. 29. The NAC of claim 28, comprising two constructs, one of which contains nucleic acid encoding a heavy antibody chain and the other of which contains nucleic acid encoding a light antibody chain, wherein expression from both constructs is capable of producing a complete antibody molecule.

30. 30. A recombinant host cell comprising a nucleic acid according to claim 26 or 27, or a NAC according to claim 28 or 29.

31. 31. A pharmaceutical composition comprising (i) an ABP according to any one of claims 1 to 25, or (ii) a nucleic acid according to claim 26 or 27, or (iii) a NAC according to claim 28 or 29, or (iv) a recombinant host cell according to claim 30, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

32. 32. A product for use in medicine selected from the list consisting of: (i) an ABP according to any one of claims 1 to 25, or (ii) a nucleic acid according to claim 26 or 27, or (iii) a NAC according to claim 28 or 29, or (iv) a recombinant host cell according to claim 30, and (iv) a pharmaceutical composition according to claim 31.

33. 33. A product for use in medicine according to claim 32, wherein the use in medicine is the treatment of a proliferative disorder, wherein cells involved in the proliferative disorder express natural ligands of LILRB1 and / or LILRB2, and wherein the cells preferably induce an immunosuppressive phenotype in immune cells such as macrophages of the subject.

34. 34. A product for use in medicine according to claim 32 or 33 for use in enhancing the immune response in a treated mammalian subject, preferably for use in promoting immunoactivation of a humoral or cell-mediated immune response in a subject, such as promoting pro-inflammatory polarization of macrophages towards an M1 phenotype, for treating an infectious disease, for treating a proliferative disease such as a cancer disease.

35. 35. A product for use in medicine according to any one of claims 32 to 34, which product has any one or combination of the following characteristics: a. specific binding to human LILRB1 and / or (preferably) LILRB2 (e.g., comprising the amino acid sequences of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (LILRB2)), e.g., with a KD of 50 nM or less, or 20 nM or less, more preferably 10 nM or less, or 5 nM or less; b. lack of specific binding to LILRA proteins, such as preferably LILRA1 and / or LILRA3; c. stimulation of T cell activation, as measured, for example, by increased T cell proliferation or IFN-γ secretion, e.g., in a mixed lymphocyte reaction (MLR) assay; d. stimulation of monocyte differentiation or activation into macrophages, e.g., stimulation of monocyte differentiation into pro-inflammatory macrophages, as demonstrated in the assays described in the Examples; e. Inhibition of binding of LILRB1 and / or (preferably and) LILRB2 to HLA-A and HLA-B, preferably HLA-g; f. Presence of a binding profile as shown in Table 3 (although written as Table 2, 3); g. Promoting pro-inflammatory polarization of macrophages into M1 macrophages, and h. does not induce (or trigger) basophil activation; and i. Induction of secretion of pro-inflammatory cytokines in macrophages, such as GM-CSF, IFNγ and IL-9; j. Induces phagocytosis. Table 3-1 Table 3-2

36. 36. A product for use in medicine according to any one of claims 32 to 35, wherein the ABP is characterized by binding to the epitope of the extracellular domain of LILRB1 and LILRB2 that is bound by the antibody clone A-045.

37. 26. A method for inducing phagocytosis in cells, preferably macrophages, comprising contacting the macrophages with an ABP according to any one of claims 1 to 25.

38. 17. A method of modulating macrophage polarization, comprising the step of contacting a target cell with an ABP according to any one of claims 1 to 16, thereby inducing or enhancing an M1-like macrophage phenotype in said target cell.

39. 39. The method of claim 38, wherein the target cell is a monocyte or macrophage.

40. 40. The method of claim 38 or 39, wherein macrophage polarization is regulated in the presence of cells expressing LILRA1 and / or LILRA3.

41. 41. The method of any one of claims 38 to 40, wherein said modulation is repolarization of an M2-like phenotype to an M1-like phenotype.

42. 26. A method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABP according to any one of claims 1 to 25.

43. 43. The method of claim 42, wherein the treatment comprises the use of any one of claims 32 to 36.

44. 44. The method according to claim 42 or 43, wherein the disease is a proliferative disease, preferably cancer.

45. 45. The method of any one of claims 42-44, wherein said treating comprises antagonizing immunosuppressive macrophage polarization in said subject, such as polarization towards M2 macrophages, thereby enhancing a cell-mediated immune response against cells involved in said disease.

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