Humanized anti-CD11B antibody and method of use thereof

Abstract

This disclosure provides a humanized anti-CD11b antibody and a method for using the same.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 487,174, filed on February 27, 2024, which is hereby incorporated by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (044546.00003SequenceListing.xml, size: 32Kb, and creation date: February 27, 2024) is hereby incorporated by reference in its entirety.

[0003] This disclosure relates to the field of immunology. In particular, this disclosure relates to humanized anti - CD11b antibodies and methods of using them.

Background Art

[0004] The human gene ITGAM encodes the CD11b chain of the CD11b / CD18 integrin (also called Mac - 1, αMβ2, CR3, or SLEB6; UniProt accession number P11215). Mac - 1 is one of the members of the β2 integrin family, which consists of an α - chain (such as α L , α M , α X , or α DIt consists of a common β2 subunit (e.g., CD18) that is non-covalently bound to the β2 integrin. β2 integrins are expressed on the cell surface as transmembrane heterodimers with a large extracellular domain 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 αMI domain, which is the spherical head of Mac-1, while some ligands bind to other parts of Mac-1, such as the β-propeller domain. Like other integrins, Mac-1 remains in an inactive, curved, closed state when unstimulated. Upon stimulation, such as inflammatory cytokines, chemokines, or TLR agonists, the extracellular domain of Mac-1 extends, allowing the head region to access the ligand. Ligand recognition by Mac-1 modulates various cellular functions related to leukocyte mobilization and host defense. See Non-Patent Documents 1 and 2.

[0005] Mac-1 is the primary phagocytic receptor for opsonized particles such as bacteria coated with iC3b. Mac-1 removes microorganisms and immune complexes through phagocytosis, and is an important regulator of host defense and tissue homeostasis. See Non-Patent Literature 2. Mac-1 is also highly expressed in microglia, where synaptic phagocytosis of weak signaling mediated by Mac-1 is crucial for neurodevelopment. See Non-Patent Literature 3. Simultaneously, iC3b / C3dg-inducible binding of Mac-1 to macrophages and the resulting phagocytosis are involved in hematological and central nervous system pathologies where abnormal complement activation is observed. 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, resulting in the removal of erythrocytes by macrophages in the liver or spleen (i.e., extravascular hemolysis). Another rare blood disorder, paroxysmal nocturnal hemoglobinuria (PNH), is primarily caused by a deficiency in complement regulators, resulting in the formation of membrane injury complexes (MACs) on red blood cells and consequently intravascular hemolysis. However, extravascular hemolysis through macrophage phagocytosis is also suggested to be involved in disease progression. See Non-Patent Literature 4 and 5. Immune thrombocytopenia (ITP) is also an autoimmune blood disorder characterized by thrombocytopenia caused by platelets targeting autoantibodies. Platelets coated with autoantibodies are destroyed by macrophages in the spleen and liver via Fcγ receptors and complement receptors. 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 Non-Patent Literature 3.

[0006] In addition to phagocytosis, Mac-1 plays a crucial role in intercellular interactions and cytokine secretion, which are essential for the recruitment of immune cells to infected or damaged tissue and for the inflammatory response. This process makes a vital contribution to the immune defense system by removing pathogens and cellular debris. See Non-Patent Literature 1 and 2. However, Mac-1-mediated adhesion has been proposed as a mechanism for the adhesion of erythrocytes to endothelial cells in sickle cell disease. See Non-Patent Literature 6. Furthermore, fibrinogen binding to CR3 on leukocytes has been suggested to induce nephropathy in sickle cell disease by increasing the secretion of inflammatory cytokines and reactive oxygen species. See Non-Patent Literature 7. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Vandendriessche et al.,Front Cell Dev Biol.,Feb 11;9:624025(2021) [Non-Patent Document 2] Rica et al.,Front Immunol.,Apr 29;12:662164(2021) [Non-Patent Document 3] Jensen et al., “Complement receptor 3 forms a compact high-affinity complex with iC3b.” J. Immunol., Jun 15;206(12):3032-3042(2021) [Non-Patent Document 4] Zaninoni et al., “The immunomodulatory effect and clinical efficacy of daratumumab in a patient with cold agglutinin disease.”Front Immunol.,Jun 3;11:946(2020) [Non-Patent Document 5] Lin et al., “Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria.” Blood, J. American Society of Hematology 126.7:891-894 (2015) [Non-Patent Document 6] Lombardi et al., “Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule,” Haematologica 104.5(2019):919 [Non-Patent Document 7] Nasimuzzaman et al., “Elimination of the fibrinogen integrin αMβ2-binding motif improves renal pathology in mice with sickle cell anemia,” Blood Advances, 3.9:1519-1532 (2019) [Overview of the project] [Problems that the invention aims to solve]

[0008] The association between Mac-1 and immuno-inflammatory dysregulation in many diseases has led to interest in therapeutic agents that inhibit 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) and Rica et al. (2021). Although not 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 novel leukocyte 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 monoclonal antibodies.”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))、および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,120.2:545-556 (1993) demonstrates Mac-1 blocking effects in various in vitro environments. M1 / 70 and 5C6 inhibited phagocytosis of mouse myelin by mouse microglia in vitro. See Rotshenker (2003). CBRM 1 / 5 has been shown to bind only to the activation-dependent neoepitope of CR3 and block the interaction between Mac-1 and ligands such as ICAM-1 and fibrinogen. See Diamond et al. (1993). Thus, CBRM 1 / 5 recognizes activated neutrophils and monocytes, rather than quiescent neutrophils and monocytes, enabling specific blockade of Mac-1 in activated cells. In vitro, CBRM 1 / 5 inhibited phagocytosis of C3dg-coated PNH erythrocytes by monocytes. See Linet al. (2015). However, while these results are promising, there is a need in this field for humanized Mac-1 antibodies that could potentially be used as therapeutic agents, for example, without causing undesirable immune responses and corresponding side effects that would otherwise impair clinical utility. [Means for solving the problem]

[0009] Accordingly, this disclosure provides modified Mac-1 mAbs (targeting the CD11b polypeptide chain of Mac-1) that can be administered as human therapeutic agents. Modifications include humanization, affinity maturation, and Fc mutations to reduce immunogenicity, increase binding affinity, and modulate Fc receptor-mediated effector function. Thus, this disclosure addresses the need for humanized antibodies that can specifically bind to polypeptides encoded by the human gene ITGAM (e.g., the CD11b polypeptide that forms part of the Mac-1 heterodimer as described above).

[0010] In a first general embodiment, the 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.

[0011] In some embodiments, the polypeptide comprises a polypeptide represented by SEQ ID NO: 1, SEQ ID NO: 2, or a fragment thereof. The fragment may comprise any portion of SEQ ID NO: 1 or SEQ ID NO: 2 (for example, 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).

[0012] In some embodiments, the binder includes a full-length antibody.

[0013] In some embodiments, the binder includes a human IgG antibody.

[0014] In some embodiments, the binder includes an antibody having an Fc domain that binds less to FcγR than an antibody having a wild-type Fc domain.

[0015] In some embodiments, the binder includes an antibody having an antibody Fc domain with enhanced effector function compared to an antibody having a wild-type Fc domain.

[0016] In some embodiments, the binder comprises a humanized antibody or its antigen-binding fragment conjugated to a detectable label.

[0017] In some embodiments, the binder comprises a humanized antibody or its antigen-binding fragment conjugated to a complex containing a cytotoxic agent.

[0018] In some embodiments, the antibody or its fragment comprises 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 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.

[0019] In some embodiments, 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 NOs: 3-5, respectively, 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 NOs: 6-8, respectively.

[0020] In some embodiments, the binding agent comprises an antigen-binding fragment, and the fragment comprises a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody ("scFv"), a dimerized V-region fragment ("diabody"), or a disulfide-stabilized V-region fragment ("dsFv").

[0021] In some embodiments, the binding agent comprises a) a heavy chain variable region ("VH") selected from SEQ ID NOs: 21-24, and / or b) a light chain variable region ("VL") selected from SEQ ID NOs: 25-28.

[0022] In some embodiments, the binder comprises a) one or more heavy chain variable regions ("VH") having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 21-24, and / or b) one or more light chain variable regions ("VL") having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 25-28.

[0023] In some embodiments, 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 respect to any one of SEQ ID NOs. 21-24, 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 respect to any one of SEQ ID NOs. 25-28.

[0024] In some embodiments, the binder comprises a) a heavy chain variable region ("VH") containing the polypeptide sequence of SEQ ID NO: 22 and a light chain variable region ("VL") containing 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.

[0025] In some embodiments, the binder comprises: a) a heavy chain variable region ("VH") containing polypeptide sequences having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity compared to SEQ ID NO: 22; and a light chain variable region ("VL") containing polypeptide sequences having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity compared to SEQ ID NO: 28; and b) a heavy chain variable region ("VL") containing polypeptide sequences having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity compared to SEQ ID NO: 23 VH contains polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity compared to SEQ ID NO: 26, VL contains polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity compared to SEQ ID NO: 23, VH contains polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 90% sequence identity compared to SEQ ID NO: 23, VL contains polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 9, VH containing polypeptide sequences with 100% sequence identity, and VL containing polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity compared to SEQ ID NO: 27, d) VH containing polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity compared to SEQ ID NO: 23, and less than SEQ ID NO: 28 VL containing polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, or VH containing polypeptide sequences with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity compared to SEQ ID NO: 24, and at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 compared to SEQ ID NO: 28,Alternatively, it may contain a VL containing a polypeptide sequence with 100% sequence identity.

[0026] In some embodiments, the binder comprises: a) a heavy chain variable region ("VH") containing a polypeptide sequence having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 22, and a light chain variable region ("VL") containing a polypeptide sequence having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 28; b) VH containing a polypeptide sequence having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 23, and VL containing a polypeptide sequence having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 26; c) a polypeptide sequence having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 23. VH containing a polypeptide sequence, and VL containing 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) VH containing 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 containing 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) VH containing 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 VL containing 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.

[0027] In a second general embodiment, the present disclosure provides a method for treating a phagocytic-related pathological condition in a human subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a binder as described herein.

[0028] In some embodiments, treating a pathological condition includes reducing, eliminating, or regulating one or more symptoms of the condition.

[0029] In some embodiments, the condition is autoimmune hemolytic anemia ("AIHA"), immune thrombocytopenia ("ITP"), sickle cell disease, or a neurodegenerative disease (e.g., Alzheimer's disease, dementia, or spinal muscular atrophy).

[0030] In a third general embodiment, the Disclosure provides a composition comprising any of the binders described herein and a pharmaceutically acceptable carrier.

[0031] In a fourth general embodiment, the Disclosure provides a nucleic acid molecule encoding one of the binders described herein.

[0032] In some embodiments, the nucleic acid includes genomic DNA, a recombinant vector, or mRNA.

[0033] In a fifth general embodiment, the present disclosure provides a host cell comprising any of the nucleic acid molecules described herein.

[0034] In some embodiments, the host cells include Escherichia coli, Sf9, COS, HEK293, or CHO cells.

[0035] In a sixth general embodiment, the Disclosure provides a pharmaceutical composition comprising any of the nucleic acid molecules described herein and a pharmaceutically acceptable carrier.

[0036] To achieve the aforementioned and related objectives, one or more embodiments include features that are fully described below and specifically pointed out in the claims. The following description details certain exemplary features of one or more embodiments. However, these features represent only a small fraction of the various ways in which the principles of various embodiments may be employed, and this description is intended to encompass all such embodiments and their equivalents.

[0037] The drawings described herein illustrate and illustrate exemplary embodiments of the present disclosure and are not intended to limit the scope of the invention as defined by the claims. [Brief explanation of the drawing]

[0038] [Figure 1] This is a multiple sequence alignment ("MSA") showing the alignment of five exemplary heavy chain variable regions (designated "VH1" to "VH5") used in humanized anti-CD11b antibody constructs prepared in accordance with this disclosure. Each heavy chain sequence represents a portion of SEQ ID NOs: 9, 11, 13, 15, 17, or 19. [Figure 2] This is an MSA showing the alignment of six exemplary light chain variable regions (designated "VL1" to "VL6") used in a humanized anti-CD11b antibody construct prepared in accordance with this disclosure. Each heavy chain sequence represents a portion of SEQ ID NOs: 10, 12, 14, 16, 18, or 20. [Figure 3] This graph shows the results of flow cytometry analysis of activated polymorphonuclear leukocytes ("PMNs") stained with 30 different humanized anti-CD11b constructs prepared in accordance with this disclosure. The percentage of CD11b+ cells in viable PMNs was calculated by gating based on a control, which is cells stained with human IgG4 isotype, and is expressed as the mean ± SEM from four independent experiments. White bars represent CD11b+ cells in untreated leukocytes (WBCs), while black bars represent CD11b+ cells in PMA-treated leukocytes. [Figure 4] Figure 3 is a chart summarizing the control and experimental groups used to create the flow cytometry test results. [Figure 5] This chart summarizes the results of a study in which 30 different humanized anti-CD11b constructs were chemically analyzed to determine their effects on complement-mediated phagocytosis. [Figure 6]This chart summarizes the results of a study in which 30 different humanized anti-CD11b constructs were chemically analyzed to determine their effects on complement-mediated phagocytosis. [Figure 7] This table summarizes the results of the functional tests conducted on the same 30 humanized anti-CD11b constructs mentioned above, relating to Figures 3-6. As shown in this table, five of the 30 constructs—VH1 and VL6, VH2 and VL2, VH2 and VL3, VH2 and VL6, and VH4 and VL6—were selected as lead candidates for further testing. [Figure 8] The results of flow cytometry analysis of cells expressing wild-type or active Mac-1 using the lead candidates and additional controls described in relation to Figure 7 are shown. Histograms summarizing the flow cytometry analysis for each test group or control group are shown. [Figure 9] The results of flow cytometry analysis of cells expressing wild-type or active Mac-1 using the lead candidates and additional controls described in relation to Figure 7 are shown. Mean fluorescence intensity ("MFI") data for each test group or control group are shown. [Figure 10] This graph shows the results of a study investigating the dose-dependent effect of lead candidates on complement-mediated phagocytosis. [Figure 11] This chart shows the results of a study evaluating TNF-α secretion from cells treated with lead candidates. [Modes for carrying out the invention]

[0039] The detailed descriptions provided below are intended to illustrate various configurations and not to represent the only configurations in which the concepts described herein can be put into practice. These detailed descriptions include specific details to provide a thorough understanding of the various concepts. However, it will be apparent to those skilled in the art that these concepts can be put into practice without these specific details.

[0040] 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, encoding the CD11b chain of the CD11b integrin (also known as Mac-1, αMβ2, CR3, or SLEB6, UniProt accession number P11215 as previously described). 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 wide variety of ligands, including ICAM family ligands, the complement protein iC3b, and fibrinogen. Mac-1 mediates the phagocytosis of iC3b-coated particles, such as apoptotic cells. Furthermore, by binding to ICAM ligands on vascular endothelium, it contributes to the trafficking of leukocytes to inflammatory sites, thereby assisting the migration of neutrophils on the endothelium before extravasation occurs. Mac-1 has been reported to have numerous other ligands, and is therefore thought to possess extremely diverse binding affinities.

[0041] Interestingly, Mac-1 has been shown to inhibit several immunological 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, which results in the degradation of myeloid differentiation primary response 88 (MyD88) and TRIF, which are downstream signaling components of the TLR pathway. Mac-1 is also involved in attenuating the macrophage response through the induction of signaling inhibitors such as cytokine signaling suppressor 3 (SOCS3) and protein A20, as well as interleukin (IL)-10.

[0042] Given the role of Mac-1 in various immunological processes, Mac-1 (and its CD11b chain component) presents a potential therapeutic target. Therefore, this disclosure provides a humanized antibody 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, inducing the production of antibodies against non-human animal proteins (e.g., human anti-mouse antibodies, such as "HAMA"). Such antibodies enhance the elimination of animal-derived antibodies from the body and reduce the therapeutic effects of animal-derived antibodies.

[0043] The humanized antibodies described herein may be generated by transplanting one or more CDRs of non-human antibodies (e.g., any or all of the CDRs represented by SEQ ID NOs. 3-8) onto a human framework, thereby increasing 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 selected leads may be assessed by ex vivo 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.

[0044] The binders described herein include humanized antibodies or their antigen-binding fragments capable of specifically binding to the CD11b chain expressed by the human gene ITGAM. As described above, such antibodies and antigen-binding fragments can be administered to human subjects as therapeutic agents for various conditions, and in their humanized forms, they induce a reduced immune response.

[0045] In some embodiments, an antibody or its antigen-binding fragment may include one or more specific CDR regions. For example, an antibody or its antigen-binding fragment may include a heavy chain variable domain comprising a CDR1 region having the polypeptide sequence represented by SEQ ID NO: 3, a CDR2 region having the polypeptide sequence represented by SEQ ID NO: 4, and / or a CDR3 region having the polypeptide sequence represented by SEQ ID NO: 5. In some embodiments, an antibody or its antigen-binding fragment may also (or alternatively) include a light chain variable domain comprising a CDR1 region having the polypeptide sequence represented by SEQ ID NO: 6, a CDR2 region having the polypeptide sequence represented by SEQ ID NO: 7, and / or a CDR3 region having the polypeptide sequence represented by SEQ ID NO: 8. In further embodiments, an antibody or its antigen-binding fragment may include a CDR region that differs from any of the CDR region sequences described in this paragraph by 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 of the substitutions may be conservative substitutions in which 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).

[0046] In further embodiments, humanization conjugates within the scope of this disclosure may include any of the heavy chain variable region (VH) sequences and / or light chain variable region (VL) sequences described herein, or variants thereof. For example, the VH may be selected from any one of SEQ ID NOs: 21-24, and / or the VL may be selected from any one of SEQ ID NOs: 25-28. In some cases, the humanization conjugate may include a VH and / or VL that share at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to any one of SEQ ID NOs: 21-28. In some cases, the humanization conjugate may include a VH and / or VL having a polypeptide sequence with at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions with respect to any one of SEQ ID NOs: 25-28.

[0047] In some embodiments, the humanization conjugate may include a pair of variable domains containing any VH as described herein paired with any VL as described herein, or a variant thereof (e.g., having the sequence identity levels or substitution numbers described above in relation to the individual variable domains). For example, the humanization conjugate may include a) a VH containing the polypeptide sequence of SEQ ID NO: 22 and a VL containing the polypeptide sequence of SEQ ID NO: 28, b) a VH containing the polypeptide sequence of SEQ ID NO: 23 and a VL containing the polypeptide sequence of SEQ ID NO: 26, c) a VH containing the polypeptide sequence of SEQ ID NO: 23 and a VL containing the polypeptide sequence of SEQ ID NO: 27, d) a VH containing the polypeptide sequence of SEQ ID NO: 23 and a VL containing the polypeptide sequence of SEQ ID NO: 28, or e) a VH containing the polypeptide sequence of SEQ ID NO: 24 and a VL containing the polypeptide sequence of SEQ ID NO: 28. These particular pairs were constructed and tested as described in further detail below. [Examples]

[0048] Various aspects of this disclosure are illustrated by the following non-limiting examples.

[0049] Example 1: Production of an exemplary humanized anti-CD11b binder. Humanization of CBRM1 / 5 antibodies (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 sequences ("VH1" to "VH5") and six light chain sequences ("VL1" to "VL6") were designed and selected for further analysis (Figure 1). Two of the light chain designs contained glutamic acid (E) substitutions at Y50 (VL5) and M55 (VL6) within CDR2, reducing localized iTope-AI hotspots. The iTope-AI score is an in silico immunogenicity score generated from a technique for predicting peptide binding to a panel of MHC class II alleles (covering HLA-DR, DP, and DQ).

[0050] Figures 1 and 2 show the amino acid sequence alignments of CBRM1 / 5 and the humanized antibody. The definition of CDRs and protein sequence numbering are performed according to Kabat. The variable region of the heavy chain in the humanized variant is shown in Figure 1, and the variable region of the light chain in the humanized variant is shown in Figure 2. "VH0" and "VL0" represent the variable regions of the heavy and light chains of CBRM1 / 5, respectively. Changes from the parent (chimeric) sequence (VH0 or VL0) are highlighted in light gray. The dark gray-bordered regions of CDR2 in the VL5 and VL6 light chains represent the Y50E and M55E mutations, respectively, which reduce localized iTope-AI hotspots. [Table 1] [Table 2]

[0051] Tables 1 and 2 above show the iTope-AI scores, which represent homology to human germline and in silico immunogenicity scores generated from CHAb technology for predicting peptide binding to a panel of MHC class II alleles (covering HLA-DR, DP, and DQ) for each selected design. Thirty humanized variants were designed from chimeric (VH0 / VL0), controls (VH0 / VL1 and VH1 / VL0), and combinations of five heavy chains (VH1-VH5) and six light chains (VL1-VL6), and corresponding antibodies were produced in ExpiCHO cells. VH0 / VL0 was a chimeric antibody with an antigen-binding fragment (Fab) of CBRM1 / 5 and a crystallizable fragment (Fc) (S241P, L248E) of hIgG4, and was used as the parental control.

[0052] Baseline CBRM1 / 5 antibodies can distinguish between inactive (quiescent) and activated forms of Mac-1. To verify that humanized variants retain this property, leukocytes were isolated from four healthy human donors and activated in the absence or presence of phorbol 12-myristate-13-acetic acid (PMA). Subsequently, cells were stained with 30 μg / mL each of 30 humanized variants, along with a control antibody (human IgG4 isotype), and analyzed using flow cytometry. The percentage of CD11b+ cells in viable PMNs was calculated by gating based on the control, which consisted of cells stained with human IgG4 isotype, and expressed as the mean ± SEM from four independent experiments. Variants containing VL5, paired with any VH variant, lost their binding to Mac-1 in polymorphonuclear leukocytes (PMNs) (Figures 3-4). Furthermore, all variants containing VH3 showed significantly reduced binding to Mac-1 (Figures 3-4). However, all variants with VH1 / VL6 and VH2 retained the CBRM1 / 5 characteristic that allows them to distinguish between the inactive (quiescent) and activated forms of Mac-1, while variants with VH4 or VH5 tended to lose the specificity of the parent antibody CBRM1 / 5 (Figures 3-4).

[0053] Subsequent experiments were conducted to test the functionality of 30 humanized variants by evaluating the inhibitory effect of antibodies on complement-mediated phagocytosis. For phagocytosis, THP-1 cells differentiated in 200 ng / mL PMA for 2 days were incubated with sheep erythrocytes (RBCs) opsonized with 10% C5-deficient human serum. Along with chimeric controls (VH0 / VL0, VH0 / VL1, VH1 / VL0), negative controls (mouse IgG1 isotype, human IgG4 isotype), and positive controls (CBRM1 / 5), 30 humanized variants at 40 μg / mL were treated 30 minutes before phagocytic incubation. Statistical analysis was performed via one-way ANOVA compared to VH0 / VL0. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001

[0054] All humanized variants except those containing VL5 were found to inhibit the phagocytic activity of opsonized sheep erythrocytes with C5-deficient human serum in differentiated THP-1 cells (Figures 5-6). Interestingly, the humanized variant containing VH3 showed reduced binding affinity in PMN (Figures 3-4), but inhibited complement-mediated phagocytosis to the same extent as the other variants (Figures 5-6).

[0055] Based on the functional results and inhibitory effects on phagocytosis (Figures 5-6) of 30 humanized variants evaluated for their ability to bind to activated PMN (Figures 3-4), five lead humanized variants that best retained the characteristics of the original antibody (VH0 / VL0) were selected: VH1 / VL6, VH2 / VL2, VH2 / VL3, VH2 / VL6, and VH4 / VL6. The results include iTope-AI scores, phagocytic activity, and binding selectivity to active Mac-1, as shown in the table provided as Figure 7. Phagocytosis data were used to determine whether each variant exhibited inhibitory effects on phagocytosis. Flow cytometry data were obtained in four independent experiments using untreated and PMA-treated leukocytes isolated from four healthy human donors (D92, D93, D04, and D05). The gray shaded areas represent evaluations based on the criteria shown in the bottom row of the table. Each value in this criterion was obtained as the median percentage of CD11b+ cells between the untreated group and the PMA-treated group stained with VH0 / VL0 in each donor. The final selection, indicated by the black box in the last column of the table, was made based on the VH and VL iTope-AI scores of each variant and their binding selectivity to active Mac-1, as analyzed by flow cytometry.

[0056] Next, five lead candidate constructs (VH1 / VL6, VH2 / VL2, VH2 / VL3, VH2 / VL6, and VH4 / VL6) were tested to confirm their functionality. It is known that the I332G mutation in the αMI domain of CD11b induces a high-affinity (active) structure of Mac-1. See Xiong et al., “An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain.” J Biol Chem. 2000;275:38762-7. Therefore, plasmid vectors encoding CD11b WT or I332G mutants were transfected into 283FT cells along with a plasmid vector encoding CD18 to express inactive and active structures of Mac-1. Similar to the VH0 / VL0 variant (Figures 3-4), which is a chimeric antibody possessing Fab of CBRM1 / 5 and Fc(S241P,L248E) of hIgG4, all five humanized variants bound to the active structure of Mac-1 more efficiently than WT Mac-1, while the variants with VH1 / VL5 exhibited reduced binding to both structures of Mac-1 (Figures 8-9), consistent with the results shown in Figures 3-4.

[0057] Furthermore, we conducted experiments to evaluate the dose-dependent effects of five humanized variants on the phagocytic activity of sheep erythrocytes opsonized with C5-deficient human serum in differentiated THP-1 cells. As expected, all five humanized variants exhibited dose-dependent inhibitory effects on complement-mediated phagocytosis, but the maximal inhibitory concentration (IC) differed between the variants. 50Slight differences were observed in (Figure 10). For phagocytosis, THP-1 cells differentiated in 200 ng / mL PMA for 2 days were incubated with opsonized sheep erythrocytes in 10% C5-deficient human serum for 2 hours. Thirty minutes before phagocytic incubation, the cells were treated with antibodies of five different humanized variants, serially diluted 2-fold (from 40 μg / mL to 0.313 μg / mL). The phagocytic index was calculated using the following formula: total number of phagocytosed erythrocytes / total number of macrophages * 100.

[0058] Next, since it was known that clustered phagocytic receptors on the macrophage membrane can lead to macrophage activation via activation of the immunoreceptor tyrosine-based activation motif (ITAM) on the receptor, we conducted an experiment to investigate TNF-α levels in the culture supernatant from human monocyte-derived macrophages. Human monocyte-derived macrophages were prepared by differentiating monocytes isolated from the blood of two healthy donors with 50 ng / mL macrophage colony-stimulating factor ("M-CSF") for 5 days. The cells were treated with 40 μg / mL of a humanized variant or isotype control, or with 200 ng / mL of LPS for 20 hours. TNF-α levels in the culture supernatant were examined using a human TNF-α ELISA kit according to the manufacturer's instructions. As shown in Figure 11, in contrast to lipopolysaccharide (LPS), none of the five humanized variants induced more TNF-α secretion compared to the isotype control antibody, suggesting that monoclonal antibodies targeting active Mac-1 do not induce unintended macrophage activation.

[0059] The experiments described above demonstrate that the exemplary humanized anti-CD11b conjugates described herein are functional and unlikely to induce unintended macrophage activation. ***

[0060] Finally, while aspects of this specification are emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily comprehend that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific compounds, compositions, articles, apparatus, methods, protocols, and / or reagents described herein, unless otherwise explicitly stated. In addition, those skilled in the art will recognize that certain changes, modifications, substitutions, alterations, additions, deletions, and partial combinations thereof can be made in accordance with the teachings contained herein without departing from the spirit of this specification.

[0061] The use of the terms “may” or “can” with respect to an embodiment or aspect of an embodiment also includes the alternative meanings of “may not” or “cannot.” Therefore, where this specification discloses that an embodiment or aspect of an embodiment may or may be included as part of the inventive subject matter, any negative limitation or exclusion proviso also expressly means that the embodiment or aspect of an embodiment may or cannot be included as part of the inventive subject matter. Similarly, the use of the term “optionally” with respect to an embodiment or aspect of an embodiment means that such an embodiment or aspect of an embodiment may or may not be included as part of the inventive subject matter. Whether such a negative limitation or exclusion proviso applies will depend on whether the negative limitation or exclusion proviso is stated in the claimed subject matter.

[0062] Although the numerical ranges and figures describing the broad scope of the present invention are approximate, the numerical ranges and figures described in the specific examples are reported as accurately as possible. However, any numerical range or figure inherently contains certain errors that inevitably result from the standard deviation found in each test measurement. The description of numerical range values ​​in this specification is intended merely as a simplified way of individually referring to each distinct figure that falls within the range. Unless otherwise indicated herein, each individual numerical range value is incorporated herein as if it were described separately.

[0063] In the context describing this invention (in particular, in the context of the following claims), the terms “a,” “an,” and “the,” as well as similar reference expressions, should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless clearly contradicted by the context. Furthermore, ordinal designations such as “first,” “second,” and “third” for identified elements are used to distinguish elements and do not indicate, or imply, a required or limiting number of such elements, nor do they indicate a specific position or order of such elements unless specifically stated. All methods described herein may be carried out in any preferred order unless otherwise indicated herein or unless clearly contradicted by the context. Any and all embodiments or use of exemplary language (e.g., “such as”) provided herein are intended solely to better illustrate the invention and do not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0064] When used in the claims, whether at the time of filing or added by amendment, the open-ended transitional phrase “comprising” (and equivalent open-ended transitional phrases such as “including,” “containing,” and “having”) covers all explicitly stated elements, limitations, steps, and / or features, either alone or in combination with unlisted subject matter, and the enumerated elements, limitations, and / or features are essential, although other unlisted elements, limitations, and / or features may be added, still forming a configuration within the claims. The specific embodiments disclosed herein may be further limited in the claims by using the closed-ended transitional phrase “comprising of” or “comprising essentially of” instead of, or as an amendment thereto, “comprising.” When used in the claims, a closed-end transitional clause "comprising of," whether at the time of filing or added by amendment, excludes any element, limitation, process, or feature not expressly described in the claims. A closed-end transitional clause "comprising essentially of" limits the claims to any other element, limitation, process, and / or feature that does not materially affect the elements, limitations, steps, and / or features expressly described, as well as any other element, limitation, step, and / or feature that does not materially affect the basic and novel characteristics of the claimed subject matter. Thus, the meaning of an open-end transitional clause "comprising" is defined as encompassing all specifically mentioned elements, limitations, steps, and / or features, as well as any optional and additional unspecified ones.The meaning of a closed-end transitional phrase "comprising of" is defined as including only those elements, limitations, steps, and / or features specifically described in the claims, whereas the meaning of a closed-end transitional phrase "comprising essentially of" is defined as including only those elements, limitations, steps, and / or features specifically described in the claims, as well as those elements, limitations, steps, and / or features that do not substantially affect the basic and novel characteristics of the claimed subject matter. Thus, an open-end transitional phrase "comprising" (and its equivalents) includes, to a limited extent, the claimed subject matter identified by a closed-end transitional phrase "comprising of" or "comprising essentially of." Accordingly, embodiments described or claimed herein using the phrase “comprising” are explicitly or essentially uniquely described, usable, and supported herein with respect to the phrases “comprising essentially of” and “comprising of.”

[0065] All patents, patent publications, and other publications referenced and identified herein are incorporated herein by reference, individually and expressly, in whole, for the purpose of describing and disclosing, for example, compositions and methods described in such publications that may be used in connection with the present invention. These publications are provided only for disclosures prior to the filing date of this application. Nothing in this regard should be construed as acknowledging that the inventors do not have the right to precede the date of such disclosures, either by prior art or for any other reason. All statements regarding dates or representations relating to the contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or contents of these documents.

[0066] Finally, the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the invention as defined solely by the claims. Therefore, the invention is not limited to what is precisely illustrated and described.

[0067] Sequence List [ka] TIFF2026511370000005.tif253170TIFF2026511370000006.tif253170TIFF2026511370000007.tif254170TIFF2026511370000008.tif164170

Claims

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

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

3. A binder according to claim 1 or 2, wherein the binder contains a full-length antibody.

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

5. A binder according to any one of claims 1 to 4, wherein the binder is wild-type F ｃ F has reduced binding to FcγR compared to antibodies with a domain. ｃ A binder containing an antibody having a domain.

6. A binder according to any one of claims 1 to 4, wherein the binder is wild-type F ｃ Antibody F exhibits increased effector function compared to antibodies with domains. ｃ A binder containing an antibody having a domain.

7. A binder according to any one of claims 1 to 6, wherein the binder comprises a humanized antibody or its antigen-binding fragment conjugated to a detectable label.

8. A binder according to any one of claims 1 to 7, wherein the binder comprises a humanized antibody or its antigen-binding fragment bound to a complex containing a cytotoxic agent.

9. A binder according to any one of claims 1 to 8, wherein the antibody or its fragment is a) A CDR1 region having a polypeptide sequence represented by Sequence ID No. 3, A CDR2 region having a polypeptide sequence represented by sequence number 4, A heavy chain variable domain comprising a CDR3 region having a polypeptide sequence represented by Sequence ID No. 5, b) A CDR1 region having a polypeptide sequence represented by sequence number 6, A CDR2 region having a polypeptide sequence represented by sequence number 7, A light chain variable domain comprising a CDR3 region having a polypeptide sequence represented by sequence number 8, A binder containing a binder.

10. A binder according to claim 9, 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, respectively, compared to SEQ ID NOs: 3-5. 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, respectively, compared to SEQ ID NOs: 6-8. A binder.

11. A binder according to claim 10, wherein one or more of the substitutions are conservative substitutions.

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

13. A binder according to any one of claims 1 to 12, wherein the binder is a) A heavy chain variable region ("VH") selected from Sequence IDs 21-24, and / or b) A binder comprising a light chain variable region ("VL") selected from sequence numbers 25 to 28.

14. A binder according to any one of claims 1 to 12, wherein the binder is a) One or more heavy chain variable regions ("VH") having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs. 21-24, and / or b) One or more light chain variable regions ("VL") having at least, up to, or exactly 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of sequence numbers 25-28, A binder containing a binder.

15. A binder according to any one of claims 1 to 12, wherein the binder is 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 one of sequence numbers 21-24, and / or b) One or more light chain variable regions ("VLs") 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 sequence numbers 25-28, A binder containing a binder.

16. A binder according to any one of claims 1 to 12, wherein the binder is a) A heavy chain variable region ("VH") containing the polypeptide sequence of SEQ ID NO: 22, and a light chain variable region ("VL") containing 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, A binder containing a binder.

17. A binder according to any one of claims 1 to 12, wherein the binder is a) A heavy chain variable region ("VH") containing polypeptide sequences 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") containing polypeptide sequences 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) 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 respect to 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 respect to SEQ ID NO:

26. c) 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 respect to 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 respect to SEQ ID NO:

27. d) VH 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 respect to SEQ ID NO: 23, and VL 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 respect to SEQ ID NO: 28, or e) 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 respect to SEQ ID NO: 24, 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 respect to SEQ ID NO:

28. A binder containing a binder.

18. A binder according to any one of claims 1 to 12, wherein the binder is 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) VH, which comprises 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, which comprises 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) 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 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. A binder containing a binder.

19. A method for treating a pathological condition related to phagocytosis in a human subject, comprising administering to the subject a composition containing a therapeutically effective amount of the binder described in any one of claims 1 to 18.

20. A method according to claim 19, wherein treating the condition includes reducing, eliminating, or regulating one or more symptoms of the condition.

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

22. A method according to claim 19 or 18, wherein the pathological condition is immune thrombocytopenia (ITP).

23. A method according to claim 19 or 20, wherein the pathological condition is sickle cell disease.

24. A method according to claim 19 or 20, wherein the pathological condition is a neurodegenerative disease.

25. A method according to claim 24, wherein the neurodegenerative disease is Alzheimer's disease, dementia, or spinal muscular atrophy.

26. A method according to claim 19 or 20, wherein the pathological condition is a demyelinating disease.

27. The method according to 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 the binder according to any one of claims 1 to 15.

30. A nucleic acid molecule according to claim 29, wherein the nucleic acid comprises genomic DNA, a recombinant vector, or mRNA.

31. A host cell comprising the nucleic acid molecule described in claim 29 or 30.

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

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

34. A pharmaceutical composition according to claim 33, wherein the excipient is a carrier.

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