Humanized anti-CD11B antibodies and methods of use thereof

Abstract

The present disclosure provides humanized anti-CD11b antibodies and methods of use thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 487,174, filed on February 27, 2024, which is expressly incorporated herein by reference in its entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence list (044546.00003SequenceListing.xml, size: 32kB; and creation date: February 27, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to the field of immunology. Specifically, this disclosure relates to humanized anti-CD11b antibodies and methods of using them. Background Technology

[0006] The human gene ITGAM encodes the CD11b strand of the CD11b / CD18 integrin (also known as Mac-1, αMβ2, CR3, or SLEB6; UniProt accession number P11215). Mac-1 is a member of the β2 integrin family and is non-covalently linked to the α strand (e.g., α...). L α M α X or α DMac-1 is composed of common β2 subunits (e.g., CD18). β2-integrins are expressed on the cell surface as transmembrane heterodimers with a large extracellular region (for ligand binding), a transmembrane domain, and a short cytoplasmic tail. Mac-1 is expressed on the cell surface of monocytes, macrophages, dendritic cells, neutrophils, and lymphocytes (such as NK cells and activated T cells). More than 100 proteins have been reported as ligands for Mac-1, including ICAM-1, ICAM-2, fibrinogen, iC3b, collagen, factor X, and NIF. Most ligands bind to the globular head of Mac-1 (αMI domain), while some bind to other parts of Mac-1 (such as the β-propeller domain). Like other integrins, Mac-1 remains inactive, bent, and closed in the absence of stimulation. In the presence of stimulation such as inflammatory cytokines, chemokines, or TLR agonists, the extracellular domain of Mac-1 extends, allowing the head to access its ligands. Mac-1 ligand recognition regulates various cellular functions associated with leukocyte recruitment and host defense. See Vandendriessche et al., Front Cell Dev Biol., Feb 11; 9:624025 (2021); Rica et al., Front Immunol., Apr 29; 12:662164 (2021).

[0007] Mac-1 is a major phagocytic receptor used to opsonize particles such as iC3b-coated bacteria. Mac-1 clears microbes and immune complexes through phagocytosis, making it an important regulator of host defense and tissue homeostasis. See Rica et al., (2021). Mac-1 is also highly expressed in microglia, where Mac-1-mediated phagocytosis at weakly signaling synapses is crucial for neurodevelopment. See Jensen et al., “Complement receptor 3forms a compact high-affinity complex with iC3b.” J. Immunol., June 15; 206(12):3032-3042 (2021). Meanwhile, iC3b / C3dg-induced binding to Mac-1 on macrophages and subsequent phagocytosis also play a role in the pathology of blood and CNS disorders observed with aberrant complement activation. In autoimmune hemolytic anemias (“AIHA”), such as warm autoimmune hemolytic anemia (“wAIHA”) and cold agglutinin disease (“CAD”), autoantibodies specific to erythrocyte membrane proteins bind to erythrocytes, leading to the clearance of erythrocytes by macrophages in the liver or spleen (i.e., extravascular hemolysis). Paroxysmal nocturnal hemoglobinuria (“PNH”) is another rare blood disorder primarily caused by a deficiency of complement regulators, leading to the formation of the erythrocyte membrane attack complex (“MAC”) and subsequent intravascular hemolysis, although extravascular hemolysis via macrophage phagocytosis is also thought to play a role in disease progression. See Zaninoni et al., “The immunomodulatory effect and clinical efficacy of daratumumab in a patient with cold agglutinin disease.” Front Immunol., 6 June 3; 11:946 (2020); Lin et al., “Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria.” Blood, J. American Society of Hematology 126.7:891-894 (2015). Immune thrombocytopenic purpura (“ITP”) is also an autoimmune blood disorder characterized by thrombocytopenia caused by platelets targeting autoantibodies. Autoantibody-coated platelets are mediated by F cγ-rays and complement receptors are disrupted by macrophages in the spleen and liver. Furthermore, recent studies have demonstrated the role of microglia Mac-1-mediated neuronal phagocytosis in neurodegenerative diseases such as Alzheimer's disease, dementia, and spinal muscular atrophy. See Jensen et al., (2021).

[0008] In addition to phagocytosis, Mac-1 plays a crucial role in cell-cell interactions and cytokine secretion, which are essential for the recruitment of immune cells to infected or damaged tissues and for inflammatory responses. This process makes a vital contribution to the immune defense system by eliminating pathogens and cellular debris. See Vandendriessche et al., (2021); Rica et al., (2021). However, Mac-1-mediated adhesion has been proposed as a mechanism for erythrocyte adhesion to endothelial cells in sickle cell disease. See Lombardi et al., “Factor H interferes with the adhesion of sicklered cells to vascular endothelium: a novel disease-modulating molecule,” Haematologica 104.5 (2019):919. Furthermore, fibrinogen binding to CR3 on leukocytes is thought to induce nephrotic syndrome in sickle cell disease by increasing the secretion of inflammatory cytokines and reactive oxygen species (“ROS”). See Nasimuzzaman et al., “Elimination of the fibrinogen integrinαMβ2-binding motif improves renalpathology in mice with sickle cell anemia,” Blood Advances, 3.9:1519-1532 (2019).

[0009] Brief description of exemplary aspects

[0010] The association of Mac-1 with immune-inflammatory dysregulation in many diseases has generated interest in therapeutic agents that disrupt the interaction between Mac-1 and its ligands. Several Mac-1 antagonists have been developed in the form of small molecules, peptides, or monoclonal antibodies (“mAbs”). See Vandendriessche et al., (2021); Rica et al., (2021). Although not yet tested in clinical trials, Mac-1 blocking mAbs, more specifically CD11b blocking mAbs, such as M1 / 70 (Springer et al., “Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novelleukocyte differentiation antigens.” European J. Immunology, 8.8:539-551 (1978); Rotshenker, “Microglia and macrophage activation and the regulation of complement-receptor-3 (CR3 / MAC-1)-mediated myelin phagocytosis in injury and disease.” J. Molecular Neuroscience 21:65-72 (2003)); OKM10 (Talle et al., “Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies.” Cellular Immunology, 78.1:83-99 (1983); Wright et al., “Identification of the C3bi receptor of human monocytes and macrophages by using monoclonalantibodies." PNAS 80.18:5699 - 5703(1983)); anti - Mo1 (Arnaout et al., "Inhibition of phagocytosis of complement C3 - or immunoglobulin G - coated particles and of C3bi binding by monoclonal antibodies to a monocyte - granulocyte membrane glycoprotein (Mol).", J. Clinical Investigation, 72.1:171 - 179(1983)); MN - 41 (Eddy et al., "The distribution of the CR3 receptor on human cells and tissue as revealed by a monoclonal antibody.", Clinical Immunology and Immunopathology 31.3:371 - 389(1984); 5C6 (Rotshenker (2003)); (Rosen et al., "Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo.", J. Experimental Medicine, 166.6:1685 - 1701(1987)); and CBRM 1 / 5 (Lin et al., (2015); Diamond et al., "A subpopulation of Mac - 1(CD11b / CD18) molecules mediates neutrophil adhesion to ICAM - 1 and fibrinogen.", J. Cell Biology, 120.Mac-1 blocking effects have been demonstrated in various in vitro settings (2:545-556 (1993)). M1 / 70 and 5C6 inhibited mouse myelin phagocytosis in mouse microglia in vitro. See Rotshenker (2003). CBRM 1 / 5 has been shown to bind only to activation-dependent novel epitopes of CR3 and block the interaction of Mac-1 with its ligands, such as ICAM-1 and fibrinogen. See Diamond et al. (1993). Thus, CBRM 1 / 5 recognizes activated neutrophils and monocytes but not resting neutrophils and monocytes, thereby specifically blocking Mac-1 in activated cells. In vitro, CBRM 1 / 5 blocks phagocytosis of C3dg-coated PNH erythrocytes by monocytes. See Lin et al. (2015). However, despite these encouraging results, there is a need in the field for humanized Mac-1 antibodies that can be used, for example, as therapeutic agents without inducing undesirable immune responses and corresponding negative side effects that would otherwise reduce clinical utility.

[0011] Therefore, this disclosure provides modified Mac-1mAbs (CD11b polypeptide chains targeting Mac-1) that can be administered as human therapeutic agents. Modifications include humanization, affinity maturation, and Fc mutation to reduce immunogenicity, increase binding affinity, and modulate Fc receptor-mediated effector function. Thus, this disclosure addresses the need for humanized antibodies capable of specifically binding to polypeptides encoded by the human gene ITGAM (e.g., the CD11b polypeptide, which forms part of the Mac-1 heterodimer as explained above).

[0012] In a first general aspect, this disclosure provides a binder comprising a humanized antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide expressed by the human gene ITGAM.

[0013] In some aspects, the polypeptide comprises the polypeptide or a fragment thereof represented by SEQ ID NO:1 or SEQ ID NO:2. The fragment may comprise any portion of SEQ ID NO:1 or SEQ ID NO:2 (e.g., any 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive amino acids of either sequence, or a range having endpoints defined by any pair of the aforementioned size values).

[0014] In some respects, the binder contains a full-length antibody.

[0015] In some respects, the binder contains human IgG antibodies.

[0016] In some respects, the binder contains an antibody with an Fc domain that binds less to FcγR than an antibody with a wild-type Fc domain.

[0017] In some respects, the binder contains an antibody having an antibody Fc domain that has increased effector functionality compared to an antibody having a wild-type Fc domain.

[0018] In some respects, the binder contains a humanized antibody or an antigen-binding fragment thereof that binds to a detectable marker.

[0019] In some respects, the conjugate comprises a humanized antibody or an antigen-binding fragment thereof bound to a conjugate containing a cytotoxic agent.

[0020] In some aspects, the antibody or fragment thereof comprises: a) a heavy chain variable domain comprising a CDR1 region having a polypeptide sequence represented by SEQ ID NO:3, a CDR2 region having a polypeptide sequence represented by SEQ ID NO:4, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO:5; and / or b) a light chain variable domain comprising: a CDR1 region having a polypeptide sequence represented by SEQ ID NO:6, a CDR2 region having a polypeptide sequence represented by SEQ ID NO:7, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO:8.

[0021] In some respects, a) the CDR1, CDR2, and / or CDR3 regions of the heavy chain variable domain are represented by polypeptide sequences having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:3 to SEQ ID NO:5; and / or b) the CDR1, CDR2, and / or CDR3 regions of the light chain variable domain are represented by polypeptide sequences having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:6 to SEQ ID NO:8.

[0022] In some respects, the binder comprises an antigen-binding fragment, wherein the fragment includes a Fab fragment, a Fab′ fragment, an F(ab′)2 fragment, a single-chain antibody (“scFv”), a dimerized V region fragment (“biantibody”), or a disulfide-stabilized V region fragment (“dsFv”).

[0023] In some aspects, the binder comprises: a) a heavy chain variable region (“VH”) selected from SEQ ID NO:21 to SEQ ID NO:24; and / or b) a light chain variable region (“VL”) selected from SEQ ID NO:25 to SEQ ID NO:28.

[0024] In some aspects, the binder comprises: a) one or more heavy chain variable regions (“VH”) having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NO:21 to SEQ ID NO:24; and / or b) one or more light chain variable regions (“VL”) having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NO:25 to SEQ ID NO:28.

[0025] In some aspects, the binder comprises: a) one or more heavy chain variable regions (“VH”) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO:21 to SEQ ID NO:24; and / or b) one or more light chain variable regions (“VL”) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO:25 to SEQ ID NO:28.

[0026] In some aspects, the binder comprises: a) a heavy chain variable region (“VH”) comprising the polypeptide sequence of SEQ ID NO:22 and a light chain variable region (“VL”) comprising the polypeptide sequence of SEQ ID NO:28; b) a VH comprising the polypeptide sequence of SEQ ID NO:23 and a VL comprising the polypeptide sequence of SEQ ID NO:26; c) a VH comprising the polypeptide sequence of SEQ ID NO:23 and a VL comprising the polypeptide sequence of SEQ ID NO:27; d) a VH comprising the polypeptide sequence of SEQ ID NO:23 and a VL comprising the polypeptide sequence of SEQ ID NO:28; or e) a VH comprising the polypeptide sequence of SEQ ID NO:24 and a VL comprising the polypeptide sequence of SEQ ID NO:28.

[0027] In some aspects, the binder comprises: a) a heavy chain variable region (“VH”) comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:22, and a light chain variable region (“VL”) comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:28; b) a heavy chain variable region (“VH”) comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:28; VH, which contains a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23, and VL, which contains a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23; c) containing a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:26; NO:23 VH comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23, and VL comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23; d) comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:27; NO:23 is compared to a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:28, and to a polypeptide sequence comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:28;Or e) a VH comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:24, and a VL comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:28.

[0028] In some aspects, the binder comprises: a) a heavy chain variable region (“VH”) comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:22, and a light chain variable region (“VL”) comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:28; b) a VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and a VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:26; c) a VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and a light chain variable region (“VL”) comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and a light chain variable region (“VL”) comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:26, and a light chain variable region (“VL”) comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23 ... NO:27 compared to a VL having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions; d) a VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and a VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:28; or e) a VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:24, and a VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:28.

[0029] In a second general aspect, this disclosure provides a method for treating a pathology associated with phagocytosis in a human subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of the binding agent described herein.

[0030] In some respects, treating a pathology involves reducing, eliminating, or modulating one or more symptoms of the pathology.

[0031] In some respects, the pathology is autoimmune hemolytic anemia (“AIHA”), immune thrombocytopenic purpura (“ITP”), sickle cell disease, or neurodegenerative diseases (e.g., Alzheimer’s disease, dementia, or spinal muscular atrophy).

[0032] In a third general aspect, this disclosure provides a composition comprising any of the binding agents described herein and a pharmaceutically acceptable carrier.

[0033] In a fourth general aspect, this disclosure provides a nucleic acid molecule encoding any of the binding agents described herein.

[0034] In some respects, nucleic acids include genomic DNA, recombinant vectors, or mRNA.

[0035] In a fifth general aspect, this disclosure provides a host cell comprising any of the nucleic acid molecules described herein.

[0036] In some respects, host cells include Escherichia coli, Sf9, COS, HEK293, or CHO cells.

[0037] In a sixth general aspect, this disclosure provides a pharmaceutical composition comprising any nucleic acid molecule described herein and a pharmaceutically acceptable carrier.

[0038] To achieve the foregoing and related objectives, the one or more aspects include the features fully described below and specifically pointed out in the claims. The following description elaborates on certain illustrative features of the one or more aspects. However, these features indicate only a few of the various ways in which the principles of the aspects may be employed, and this description is intended to include all such aspects and their equivalents. Attached Figure Description

[0039] The accompanying drawings described herein illustrate and illustrate exemplary aspects of this disclosure, but are not intended to limit the scope of the invention as defined by the claims.

[0040] Figure 1 This is a multiple sequence alignment (“MSA”) showing the alignment of five exemplary heavy chain variable regions (named “VH1” to “VH5”) used in the humanized anti-CD11b antibody construct prepared according to this disclosure. The sequence of each heavy chain represents a portion of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19.

[0041] Figure 2The image shows an alignment of six exemplary light chain variable regions (designated “VL1” to “VL6”) used in a humanized anti-CD11b antibody construct prepared according to this disclosure. The sequence of each heavy chain represents a portion of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20.

[0042] Figure 3 A graph illustrating the results of flow cytometry analysis of activated polymorphonuclear leukocytes (“PMNs”) stained with 30 humanized anti-CD11b constructs prepared according to this disclosure. The percentage of CD11b+ cells in live PMNs was calculated using a control-based gating method and is presented as the mean ± SEM from four independent experiments, the control being cells stained with human IgG4 isotype. White bars represent CD11b+ cells in untreated leukocytes (WBCs), while black bars represent CD11b+ cells in WBCs treated with PMA.

[0043] Figure 4 For summarizing and generating Figure 3 The graphs showing the results of the flow cytometry study are for the control and experimental groups.

[0044] Figure 5 and Figure 6 A chart summarizing the findings of studies analyzing the same 30 humanized anti-CD11b constructs to determine their role in complement-mediated phagocytosis.

[0045] Figure 7 To summarize the above text Figures 3 to 6 The table describes the functional test results of the same 30 humanized anti-CD11b constructs. As illustrated in the table, five of the 30 constructs were selected as lead candidates for further testing: VH1 and VL6; VH2 and VL2; VH2 and VL3; VH2 and VL6; and VH4 and VL6.

[0046] Figure 8 and Figure 9 The use of combination is shown Figure 7 The described lead candidate, along with additional controls, was analyzed by flow cytometry in cells expressing wild-type or live Mac-1. Figure 8 Histograms summarizing flow cytometry analyses for each test or control group are shown, and Figure 9 The mean fluorescence intensity (“MFI”) data for each test group or control group are shown.

[0047] Figure 10A graph showing the results of studies examining the dose-dependent effects of lead candidates on complement-mediated phagocytosis.

[0048] Figure 11 A graph showing the results of a study evaluating TNF-α secretion in cells treated with the lead candidate. Detailed Implementation

[0049] The detailed descriptions below are intended as a description of various configurations and are not intended to represent the only configurations in which the concepts described herein can be practiced. The detailed descriptions include specific details for the purpose of providing a thorough understanding of the various concepts. However, it will be apparent to those skilled in the art that these concepts can be practiced without these specific details.

[0050] This disclosure provides a modified (e.g., humanized) antibody that specifically recognizes at least one epitope of a polypeptide encoded by the human gene ITGAM located at 16p11.2, which encodes the CD11b chain of CD11b / CD18 integrin (also known as Mac-1, αMβ2, CR3, or SLEB6; UniProt accession number P11215, as described above). The CD11b integrin polypeptide pairs with the CD18 integrin polypeptide to form a functional Mac-1 integrin. Mac-1 is primarily expressed in myeloid cells (such as macrophages, neutrophils, and dendritic cells) and binds to a variety of ligands, including ligands of the ICAM family, complement protein iC3b, and fibrinogen. Mac-1 mediates phagocytosis of iC3b-coated granules (such as apoptotic cells). It also assists neutrophils in their movement on endothelial cells prior to extravasation by promoting leukocyte transport to sites of inflammation through binding to ICAM ligands on vascular endothelial cells. Mac-1 is reportedly also possesses many other ligands and is therefore considered highly heterogeneous.

[0051] Interestingly, Mac-1 has been shown to suppress several immune processes. Mac-1 restricts dendritic cell maturation and function, as well as dendritic cell-induced T cell activation. In macrophages, Mac-1 restricts TLR signaling via Src / Syk signaling, leading to the degradation of downstream TLR signaling components myeloid differentiation primary response 88 (MyD88) and TRIF. Mac-1 has also been involved in suppressing macrophage responses by inducing signaling inhibitors such as SOCS3 (cytokine signaling inhibitor 3) and protein A20, as well as interleukin (IL)-10.

[0052] Given its role in various immune processes, Mac-1 (and its CD11b chain branch) represents a potential therapeutic target. Therefore, this disclosure provides humanized antibodies capable of specifically binding to the CD11b chain of Mac-1 (i.e., the polypeptide expressed by ITGAM). Humanized antibodies are advantageous because when non-human antibodies, such as mouse antibodies, are administered to humans, they are recognized as foreign substances and induce the production of antibodies against proteins of the non-human animal (e.g., anti-mouse antibodies, such as human anti-mouse antibody, “HAMA”). Such antibodies enhance the clearance of animal-derived antibodies from the body and reduce the therapeutic effect provided by animal-derived antibodies.

[0053] The humanized antibodies described herein can be generated by grafting one or more CDRs of a non-human antibody (e.g., any or all of the CDRs represented by SEQ ID NO. 3 to SEQ ID NO. 8) onto a human frame to increase the human sequence content. See Safdari et al., “Antibody humanization methods – a review and update.” Biotechnology and Genetic Engineering Reviews 29.2 (2013):175-186. After humanization, the immunogenicity of the selected lead can be estimated by in vitro immunogenicity assays. See Jaber and Baker, “Assessment of the immunogenicity of different interferon beta-1a formulations using ex vivo T-cell assays.” J. Pharmaceutical and Biomedical Analysis 43.4 (2007):1256-1261.

[0054] The binding agents described herein include humanized antibodies or antigen-binding fragments thereof capable of specifically binding to the CD11b chain expressed by the human ITGAM gene. As described above, such antibodies and antigen-binding fragments can be administered to human subjects as therapeutic agents for various conditions and elicit a reduced immune response in a humanized form.

[0055] In some aspects, the antibody or its antigen-binding fragment may comprise one or more specific CDR regions. For example, the antibody or its antigen-binding fragment may comprise a heavy chain variable domain comprising a CDR1 region having a polypeptide sequence represented by SEQ ID NO:3, a CDR2 region having a polypeptide sequence represented by SEQ ID NO:4, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO:5. In some aspects, the antibody or its antigen-binding fragment may also (or alternatively) comprise a light chain variable domain comprising a CDR1 region having a polypeptide sequence represented by SEQ ID NO:6, a CDR2 region having a polypeptide sequence represented by SEQ ID NO:7, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO:8. In a further aspect, the antibody or its antigen-binding fragment may comprise a CDR region that differs from any CDR region sequence described herein due to one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions compared to any of the aforementioned CDR region sequences. Any or all substitutions can be conservative substitutions, where the substituted amino acid belongs to the same class as the original amino acid (e.g., aliphatic, hydroxyl or sulfur / selenium-containing, cyclic, aromatic, basic or acidic and corresponding amides).

[0056] In a further aspect, the humanized binder within the scope of this disclosure may comprise any heavy chain variable region (VH) and / or any light chain variable region (VL) sequence of the heavy chain variable region (VH) described herein, or variations thereof. For example, the VH may be selected from any of SEQ ID NO:21 to SEQ ID NO:24; and / or the VL may be selected from any of SEQ ID NO:25 to SEQ ID NO:28. In some cases, the humanized binder may comprise a VH and / or a VL having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO:21 to SEQ ID NO:28. In some cases, the humanized binder may comprise VH and / or VL having a polypeptide sequence having at least, at most, or exactly one, two, three, four, or five amino acid substitutions compared to any one of SEQ ID NO:25 to SEQ ID NO:28.

[0057] In some aspects, the humanized binding agent may comprise a pair of variable domains containing any VH or variant thereof described herein paired with any VL described herein (e.g., having the sequence identity level or multiple substitutions as described above with respect to a single variable domain). For example, the humanized binding agent may comprise: a) a VH comprising the polypeptide sequence of SEQ ID NO:22 and a VL comprising the polypeptide sequence of SEQ ID NO:28; b) a VH comprising the polypeptide sequence of SEQ ID NO:23 and a VL comprising the polypeptide sequence of SEQ ID NO:26; c) a VH comprising the polypeptide sequence of SEQ ID NO:23 and a VL comprising the polypeptide sequence of SEQ ID NO:27; d) a VH comprising the polypeptide sequence of SEQ ID NO:23 and a VL comprising the polypeptide sequence of SEQ ID NO:28; or e) a VH comprising the polypeptide sequence of SEQ ID NO:24 and a VL comprising the polypeptide sequence of SEQ ID NO:28. These specific pairings are constructed and tested in further detail below.

[0058] Example

[0059] Various aspects of this disclosure will be illustrated with reference to the following non-limiting examples.

[0060] Example 1 Example of the generation of humanized anti-CD11b binding agents.

[0061] Humanization of the CBRM1 / 5 antibody (see Lin et al., (2015)) was performed using Abzena’s composite human antibody technology (“CHAb”). See Perry et al., “New approaches to prediction of immune responses to therapeutic proteins during preclinical development.” Drugs in R&D 9 (2008):385-396. A total of five heavy chain (“VH1” to “VH5”) and six light chain (“VL1” to “VL6”) sequences were designed and selected for further analysis (Figure 1). Two light chain designs contained substitutions for glutamic acid (E) within CDR2, at Y50 (VL5), and at M55 (VL6) to reduce local iTope-AI hotspots. The iTope-AI score is a computer-simulated immunogenicity score generated from a technique used to predict peptide binding to a set of MHC class II alleles (covering HLA-DR, DP, and DQ).

[0062] Figure 1 and Figure 2The amino acid sequence alignment of CBRM1 / 5 and the humanized antibody is shown. The CDR definition and protein sequence number conform to Kabat. The variable region of the heavy chain in the humanized variant is shown. Figure 1 In, and the variable region of the light chain in the humanized variant is shown in Figure 2 In the diagram, “VH0” and “VL0” represent the heavy chain and light chain variable regions of CBRM1 / 5, respectively. Variations in the parental (chimeric) sequence (VH0 or VL0) are highlighted in light gray. Dark gray boxes in the CDR2 of the VL5 and VL6 light chains represent the Y50E and M55E mutations, respectively, to reduce local iTope-AI hotspots.

[0063]

[0064] Table 1 A summary of the homology of each VL domain used in the candidate and / or control constructs with human lineages and the iTope-AI score.

[0065]

[0066] Table 2 A summary of the homology of each VL domain used in the candidate and / or control constructs with human lineages and the iTope-AI score.

[0067] Tables 1 and 2 above show the homology with human lines and the iTope-AI score, which represents a computer-simulated immunogenicity score generated from CHAb technology to predict the binding of the peptide to a set of selected designed MHC class II alleles (covering HLA-DR, DP, and DQ). Chimeras (VH0 / VL0), controls (VH0 / VL1 and VH1 / VL0), and 30 humanized variants generated from combinations of 5 heavy chains (VH1–VH5) and 6 light chains (VL1–VL6) were designed, and their corresponding antibodies were generated in ExpiCHO cells. VH0 / VL0 is a chimeric antibody containing the antigen-binding fragment (Fab) of CBRM1 / 5 and the crystallizable fragment (Fc) of hIgG4 (S241P, L248E), and was used as a parental control.

[0068] Baseline CBRM1 / 5 antibodies can distinguish between the inactive (resting) and activated forms of Mac-1. To verify that humanized variants retain this property, WBCs were isolated from four healthy human donors and activated with or without phorbol 12-myristate-13-acetate (PMA). Cells were then stained with 30 μg / mL of each of 30 humanized variants and a control antibody (human IgG4 isotype) and analyzed by flow cytometry. The percentage of CD11b+ cells in live PMNs was calculated using a control-based gating method and is expressed as mean ± SEM from four independent experiments, with the control being cells stained with the human IgG4 isotype. VL5-containing variants lost Mac-1 binding in polymorphonuclear leukocytes (PMNs) regardless of which VH variant they were paired with. Figures 3 to 4 Furthermore, all variants containing VH3 showed a significantly reduced binding to Mac-1. Figures 3 to 4 However, VH1 / VL6 and all variants with VH2 retain the properties of CBRM1 / 5, which can distinguish between the inactive (resting) and active forms of Mac-1, while variants with VH4 or VH5 may lose the specificity of the parental antibody CBRM1 / 5. Figures 3 to 4 ).

[0069] Subsequent experiments tested the function of 30 humanized variants by evaluating the inhibitory effect of antibodies on complement-mediated phagocytosis. For phagocytosis, THP-1 cells differentiated for 2 days with 200 ng / mL PMA were incubated with sheep erythrocytes (RBCs) conditioned with 10% C5 depleted human serum. Thirty minutes before phagocytosis incubation, 40 μg / mL of the 30 humanized variants, along with chimeric controls (VH0 / VL0, VH0 / VL1, VH1 / VL0), negative controls (mouse IgG1 isotype, human IgG4 isotype), and positive controls (CBRM1 / 5) were administered. Statistical analysis was performed using one-way ANOVA compared to VH0 / VL0. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001

[0070] All humanized variants, except those containing VL5, were found to inhibit the phagocytosis of sheep RBCs conditioned with C5-depleted human serum in differentiated THP-1 cells. Figures 5 to 6 Interestingly, humanized variants containing VH3 exhibited reduced binding capacity in PMN. Figures 3 to 4 However, like other variants, it inhibits complement-mediated phagocytosis. Figures 5 to 6 ).

[0071] Based on estimates of its ability to bind and activate PMN ( Figures 3 to 4) and its inhibitory effect on phagocytosis ( Figures 5 to 6 Functional results of 30 humanized variants of the antibody were analyzed, and five lead humanized variants were selected: VH1 / VL6, VH2 / VL2, VH2 / VL3, VH2 / VL6, and VH4 / VL6, which best preserved the characteristics of the original antibody (VH0 / VL0). Results included iTope-AI score, phagocytic activity, and binding selectivity to active Mac-1, such as... Figure 7 As shown in the provided table. Phagocytosis data were used to determine whether each variant had an inhibitory effect on phagocytosis. Flow cytometry data were obtained through four independent experiments using untreated and PMA-treated WBCs isolated from four healthy human donors (D92, D93, D04, and D05). Gray shading was graded according to criteria in the bottom row of the table. The values ​​in the criteria were obtained as the median percentage of CD11b+ cells in the untreated group stained with VH0 / VL0 and the PMA-treated group in each donor. The final selection, indicated by the black box in the last column of the table, was based on the iTope-AI scores of VH and VL for each variant and its binding selectivity to active Mac-1 as analyzed by flow cytometry.

[0072] Next, five lead candidate constructs (VH1 / VL6, VH2 / VL2, VH2 / VL3, VH2 / VL6, and VH4 / VL6) were tested to confirm functionality. The I332G mutation in the αMI domain of CD11b is known to induce high affinity (activity) of Mac-1. See Xiong et al., “An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain.” *Journal of Biochemistry*, 2000; 275:38762-7. Therefore, plasmid vectors encoding CD11b WT or I332G mutants were transfected together with plasmid vectors encoding CD18 into 283FT cells to express inactive and active conformations of Mac-1. Similar to the VH0 / VL0 variant (which is a chimeric antibody with Fab and hIgG4 of CBRM1 / 5 and Fc(S241P, L248E)) Figures 3 to 4 All five humanized variants bound to the active conformation of Mac-1 more efficiently than WT Mac-1, while variants with VH1 / VL5 showed reduced binding to both conformations of Mac-1. Figures 8 to 9 ),and Figures 3 to 4 The results shown are consistent.

[0073] In addition, an experiment was conducted to evaluate the dose-dependent effects of five humanized variants on phagocytosis of sheep RBCs conditioned with C5-depleted human serum in differentiated THP-1 cells. As expected, all five humanized variants exhibited dose-dependent inhibition of complement-mediated phagocytosis, although there were differences in the half-maximal inhibitory concentration (IC50) among the variants. 50 There are some differences. Figure 10 For phagocytosis, THP-1 cells differentiated for 2 days with 200 ng / mL PMA were incubated with sheep RBCs conditioned with 10% C5 depleted human serum for 2 hours. Thirty minutes before phagocytic incubation, cells were treated with antibodies at 2-fold serial dilutions (from 40 μg / mL to 0.313 μg / mL) of five humanized variants. The phagocytic index was calculated as follows: (Total number of phagocytosed RBCs / Total number of macrophages) * 100.

[0074] Next, experiments were performed to test TNF-α levels in the culture supernatant of macrophages derived from human monocytes, as phagocytic receptors aggregated on the macrophage membrane are known to lead to macrophage activation via activation of the tyrosine-based activation motif (“ITAM”) on the receptor. Human monocyte-derived macrophages were prepared by differentiating monocytes isolated from the blood of two healthy donors for 5 days with 50 ng / mL macrophage colony-stimulating factor (“M-CSF”). Cells were treated for 20 hours with 40 μg / mL of a humanized variant or an allotype control, or 200 ng / mL LPS. TNF-α levels in the culture supernatant were detected using a human TNF-α ELISA kit according to the manufacturer's instructions. Figure 11 As shown, unlike lipopolysaccharide (“LPS”), none of the five humanized variants induced more TNF-α secretion compared to the isotype control antibody, indicating that monoclonal antibodies targeting active Mac-1 do not induce unintended macrophage activation.

[0075] The aforementioned experiments demonstrate that the exemplary humanized anti-CD11b binders according to this disclosure are both functional and unlikely to induce unintended macrophage activation.

[0076] ***

[0077] Finally, it should be understood that although various aspects of this specification are highlighted by reference to specific embodiments, those skilled in the art should understand that these disclosed embodiments are only for illustrating the principles of the subject matter disclosed herein. Therefore, it should be understood that, unless expressly stated otherwise, the disclosed subject matter is by no means limited to the specific compounds, compositions, articles, apparatuses, methods, schemes, and / or reagents described herein. Furthermore, those skilled in the art should understand that certain changes, modifications, substitutions, alterations, additions, deletions, and sub-combinations thereof can be made based on the teachings herein without departing from the spirit of this specification.

[0078] When referring to an embodiment or an aspect thereof, terms such as "may" or "may" also include meanings such as "may not" or "cannot." Therefore, if this specification discloses an embodiment or an aspect thereof that can or may be incorporated into the subject matter of the invention, it also explicitly implies a negative limiting or exclusionary clause, meaning that the embodiment or an aspect thereof cannot or may not be incorporated into the subject matter of the invention. Similarly, the use of the term "optionally" when referring to an embodiment or an aspect thereof means that the embodiment or an aspect thereof may or may not be incorporated into the subject matter of the invention. Whether such negative limiting or exclusionary clauses apply will depend on whether such negative limiting or exclusionary clauses are detailed in the claimed subject matter.

[0079] While the numerical ranges and values ​​illustrating the broad scope of the invention are approximate, the numerical ranges and values ​​described in specific examples are reported as precisely as possible. However, any numerical range and value inherently contains some error, necessarily caused by the standard deviation found in their respective test measurements. References to numerical ranges of values ​​herein are intended only as a simplified way of referring individually to each individual value falling within said range. Unless otherwise specified herein, each individual value of a numerical range is incorporated into this specification as if each individual value were individually referenced herein.

[0080] Unless otherwise stated herein or explicitly contradicted by the context, the terms “a / an,” “the,” and similar references used in the context of describing the currently claimed invention (particularly in the context of the following claims) should be interpreted to cover both the singular and plural. Furthermore, ordinal indicators for identified elements (such as “first,” “second,” “third,” etc.) are used to distinguish between elements and do not indicate or imply a required or limited number of such elements, nor do they indicate a particular location or order of such elements unless specifically stated otherwise. Unless otherwise indicated herein or explicitly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and not to limit its scope. No language in this specification should be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0081] When used in claims, whether as submitted or added according to amendments, the open transitional term "comprising" (and its equivalent open transitional phrases, such as including, containing, and having) covers, alone or in combination with unlisted subject matter, all expressly listed elements, limitations, steps, and / or features; named elements, limitations, and / or features are necessary, but other unlisted elements, limitations, and / or features may be added and still form a construction within the scope of the claims. Specific embodiments disclosed herein may be used in claims in place of or as a modification of "comprising" to further limit the scope. When used in claims, whether as submitted or added according to amendments, the closed transitional phrase "comprising" excludes any elements, limitations, steps, or features not expressly recited in the claims. The closed transitional phrase "comprising" limits the scope of the claims to the expressly listed elements, limitations, steps, and / or features, as well as any other elements, limitations, steps, and / or features that do not substantially affect the essential and novel characteristics of the claimed subject matter. Therefore, the meaning of the open transition phrase "comprising" is defined as encompassing all expressly listed elements, limitations, steps, and / or features, as well as any optional, unspecified additional elements, limitations, steps, and / or features. The meaning of the closed transition phrase "consisting of" is defined as including only those elements, limitations, steps, and / or features specifically recited in the claims, while the meaning of the closed transition phrase "substantially constitutes" is defined as including only those elements, limitations, steps, and / or features specifically recited in the claims, as well as those elements, limitations, steps, and / or features that do not substantially affect the essential and novel features of the claimed subject matter. Therefore, the open transition phrase "comprising" (and its equivalents) includes, within its meaning, the claimed subject matter specified by the closed transition phrase "consisting of" or "substantially constitutes" as a limitation. Since such embodiments described herein or claimed with the phrase "comprising" are explicitly or inherently unambiguously described, implemented, and supported herein, the phrases "substantially constitutes" and "consisting of" are...

[0082] All patents, patent publications, and other publications referenced and identified in this specification are individually and expressly incorporated herein by reference in their entirety for describing and disclosing compositions and methods, such as those described in such publications, that can be used in conjunction with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventor has a right to use prior art or for any other reason prior to such disclosure. All statements regarding the dates or contents of these documents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0083] Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. Therefore, the invention is not limited to what is shown and described.

[0084] sequence list

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Claims

1. A binding agent comprising a humanized antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide expressed by the human gene ITGAM.

2. The binder according to claim 1, wherein the polypeptide expressed by the human gene ITGAM comprises the polypeptide or fragment thereof represented by SEQ ID NO:1, SEQ ID NO:

2.

3. The binding agent according to claim 1 or 2, wherein the binding agent comprises a full-length antibody.

4. The binder according to any one of claims 1 to 3, wherein the binder comprises a human IgG antibody.

5. The binder according to any one of claims 1 to 4, wherein the binder comprises having F c An antibody containing a domain, wherein the antibody binds to FcγR at a ratio similar to that of wild-type FcγR. c Antibodies against structural domains are reduced.

6. The binding agent according to any one of claims 1 to 4, wherein the binding agent comprises antibody F c Antibodies with structural domains, said antibodies having wild-type F c Antibodies with structural domains have increased effector functions compared to those with other domains.

7. The binder according to any one of claims 1 to 6, wherein the binder comprises a humanized antibody or an antigen-binding fragment thereof that binds to a detectable marker.

8. The binder according to any one of claims 1 to 7, wherein the binder comprises a humanized antibody or an antigen-binding fragment thereof bound to a conjugate comprising a cytotoxic agent.

9. The binding agent according to any one of claims 1 to 8, wherein the antibody or fragment thereof comprises a) A heavy-chain variable structural domain, the heavy-chain variable structural domain comprising Having a CDR1 region containing the polypeptide sequence represented by SEQ ID NO:3 Having a CDR2 region with the polypeptide sequence represented by SEQ ID NO:4 and / or Having a CDR3 region containing the polypeptide sequence represented by SEQ ID NO:5; and / or b) Light chain variable structural domains, wherein the light chain variable structural domains include: Having a CDR1 region containing the polypeptide sequence represented by SEQ ID NO:6 Having a CDR2 region with the polypeptide sequence represented by SEQ ID NO:7 and / or It has a CDR3 region containing the polypeptide sequence represented by SEQ ID NO:

8.

10. The binder according to claim 9, wherein a) The CDR1, CDR2, and / or CDR3 regions of the heavy chain variable domain are represented by polypeptide sequences having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:3 to SEQ ID NO:5; and / or b) The CDR1, CDR2 and / or CDR3 regions of the light chain variable structural domain are represented by polypeptide sequences having at least, at most or exactly 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO:6 to SEQ ID NO:

8.

11. The binder of claim 10, wherein one or more of the substitutions are conservative substitutions.

12. The binder according to any one of claims 1, 2 or 7 to 11, wherein the binder comprises an antigen-binding fragment, wherein the fragment comprises a Fab fragment, a Fab′ fragment, an F(ab′)2 fragment, a single-chain antibody ("scFv"), a dimerized V region fragment ("biantibody"), or a disulfide-stabilized V region fragment ("dsFv").

13. The binder according to any one of claims 1 to 12, wherein the binder comprises: a) Heavy chain variable regions ("VH") selected from SEQ ID NO:21 to SEQ ID NO:24; and / or b) Selected light chain variable regions ("VL") from SEQ ID NO:25 to SEQ ID NO:

28.

14. The binder according to any one of claims 1 to 12, wherein the binder comprises: a) One or more heavy chain variable regions ("VH"), said one or more heavy chain variable regions having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NO:21 to SEQ ID NO:24; and / or b) One or more light chain variable regions ("VL"), said one or more light chain variable regions having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NO:25 to SEQ ID NO:

28.

15. The binder according to any one of claims 1 to 12, wherein the binder comprises: a) One or more heavy chain variable regions ("VH"), said one or more heavy chain variable regions having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO:21 to SEQ ID NO:24; and / or b) One or more light chain variable regions ("VL"), said one or more light chain variable regions having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO:25 to SEQ ID NO:

28.

16. The binder according to any one of claims 1 to 12, wherein the binder comprises: a) The heavy chain variable region ("VH") of the polypeptide sequence of SEQ ID NO:22 and the light chain variable region ("VL") of the polypeptide sequence of SEQ ID NO:28; b) VH containing the polypeptide sequence of SEQ ID NO:23, and VL containing the polypeptide sequence of SEQ ID NO:26; c) VH containing the polypeptide sequence of SEQ ID NO:23, and VL containing the polypeptide sequence of SEQ ID NO:27; d) VH containing the polypeptide sequence of SEQ ID NO:23, and VL containing the polypeptide sequence of SEQ ID NO:28; or e) VH containing the polypeptide sequence of SEQ ID NO:24, and VL containing the polypeptide sequence of SEQ ID NO:

28.

17. The binder according to any one of claims 1 to 12, wherein the binder comprises: a) A heavy chain variable region ("VH") comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:22, and a light chain variable region ("VL") comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:28; b) A VH comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:23, and a VL comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:26; c) A VH comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23, and a VL comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:27; d) A VH comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23, and a VL comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:28; or e) A VH comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:24, and a VL comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

28.

18. The binder according to any one of claims 1 to 12, wherein the binder comprises: a) A heavy chain variable region ("VH") comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:22, and a light chain variable region ("VL") comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:28; b) VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:26; c) VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:27; d) A VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:23, and a VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:28; or e) A VH comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:24, and a VL comprising a polypeptide sequence having at least, at most, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO:

28.

19. A method for treating a pathology associated with phagocytosis in a human subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a binder according to any one of claims 1 to 18.

20. The method of claim 19, wherein treating the pathology comprises reducing, eliminating, or modulating one or more symptoms of the pathology.

21. The method according to claim 19 or 20, wherein the pathology is autoimmune hemolytic anemia ("AIHA").

22. The method according to claim 19 or 18, wherein the pathology is immune thrombocytopenic purpura (ITP).

23. The method according to claim 19 or 20, wherein the pathology is sickle cell disease.

24. The method according to claim 19 or 20, wherein the pathology is a neurodegenerative disease.

25. The method of claim 24, wherein the neurodegenerative disease is Alzheimer's disease, dementia, or spinal muscular atrophy.

26. The method according to claim 19 or 20, wherein the pathology is a demyelinating disease.

27. The method of claim 26, wherein the demyelinating disease is chronic inflammatory demyelinating polyneuropathy ("CIDP") or multifocal motor neuropathy ("MMN").

28. A composition comprising a binder according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

29. A nucleic acid molecule encoding a binding agent according to any one of claims 1 to 15.

30. The nucleic acid molecule of claim 29, wherein the nucleic acid comprises genomic DNA, a recombinant vector, or mRNA.

31. A host cell comprising a nucleic acid molecule according to claim 29 or 30.

32. The host cell according to claim 31, wherein the host cell comprises Escherichia coli, Sf9, COS, HEK293 or CHO cells.

33. A pharmaceutical composition comprising: The nucleic acid molecule according to claim 29 or 30, and At least one pharmaceutically acceptable excipient.

34. The pharmaceutical composition of claim 33, wherein the excipient is a carrier.

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