Methods of depleting disease-causing agents via antibody targeted phagocytosis

Abstract

To provide a method for depleting a pathogenic substance by antibody targeted phagocytosis.SOLUTION: The present disclosure relates to a method of depleting or reducing the number of disease-causing agents including host cells, or host cells products, microbes or their products in a human subject upon administration of a molecule that causes targeted phagocytosis and comprises a binding domain that binds a specific phagocytotic receptor, such as Dectin-1, and a binding domain that binds a specific disease-causing agent. In a specific embodiment, a method of the disclosure depletes or reduces the number of disease-causing agents in tissues, blood, or bone marrow by targeted phagocytosis.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of U.S. Provisional Patent Application No. 62 / 830,139, filed on April 5, 2019, the disclosure of which is hereby incorporated by reference in its entirety into this specification.

[0002] The present disclosure relates to methods for depleting or reducing disease - causing substances in humans by targeted phagocytosis.

Background Art

[0003] Professional phagocytes are a subset of white blood cells that generally refer to monocytes, macrophages, dendritic cells, neutrophils, eosinophils, and osteoclasts, and specifically recognize and phagocytose abnormal or disease - causing host or foreign substances (Rabinovitch, 1995, Trends in Cell Biol; Arandejelovic, et al, 2015, Nat Immunol; Rosales, et al, 2017 BioMed Research International). Phagocytosis is the main mechanism used to remove pathogens and cellular debris. Phagocytosis is defined as the cellular uptake of particulate matter (>0.5 mm) within a plasma membrane envelope and is closely related to, and partially overlaps with, the endocytosis of soluble ligands via the fluid - phase macropinocytosis pathway and receptor - mediated pathways (Rosales, et al, 2017 BioMed Research International; Gordon, 2016, Immunity; Tse, et al, 2003, J Biol Chem). Next, the ingested material is digested in the phagosome and They are. Bacteria, dead tissue cells, and small inorganic particles are all examples of objects that can be phagocytosed. Several terms have been applied to the uptake of apoptotic cells, also known as efferocytosis, and the mechanisms associated with necrotic cells (necrosis and pyroptosis) resulting from infections and inflammation (Henson and Bratton, 2009). The ingested mate rial is destroyed in the process of phagocytosis via the endolysosomal pathway. Dendritic cells and macrophages engulf pathogens by phagocytosis, break them down, and present antigens to cells of the adaptive immune system.

[0004] Receptors on the plasma membrane of phagocytes that mediate phagocytosis can be classified into non-opsonin type and opsonin type. Examples of non-opsonin receptors include lectin-type receptors, dectin receptors, or scavenger receptors (Freeman and Grinstein, Immunological Reviews, 2014). Some phagocyte pathways require a second signal (causing activation of NF-κB) from pattern recognition receptors (PRRs) activated by attachment to pathogen-associated molecular patterns ( PAMPs) (Patin, et al, 2018, Semin Cell Dev Biol; Brandt, et al, 2013, PLoS One). By professional phagocytes Examples of variably expressed non-opsonin receptors include lectin-like recognition molecules such as CD169, CD33, and related receptors for sialic acid-added residues. In addition, phagocytes express a group of dectin-1 (a receptor for fungal beta-glucan with well-defined signaling capacity), related C-type lectins (such as MICL, dectin-2, Mincle, and DNGR-1), and scavenger receptors (Asano, et al, 2018, J Biochem, Lock, et al, 2004, Immunobiol). SR-A, MARCO, and CD36 differ in domain structure and have different but overlapping recognition for apoptotic microbial ligands (Freeman and Grinstein, Immunological Reviews, 2014). These mixed receptors bind polyanionic ligands and are not well-defined enough in terms of their intracellular signaling capacity, suggesting that multiple ligand and receptor interactions are likely requirements for uptake. In particular, Toll-like receptors (TLRs) are sensors and not phagocyte entry receptors, but often cooperate with other non-opsonin receptors to promote uptake and signaling (Gordon 2016).

[0005] Plasma membrane receptors can be classified as opsonin-dependent, mainly FcRs (activating or inhibitory) for conserved domains of IgG antibodies, and complement receptors such as CR3 for iC3b deposited by the classical (IgM or IgG) or alternative lectin pathways of complement activation. CR3 may also mediate recognition in the absence of opsonin, perhaps by depositing macrophage-derived complement. Plasma or cell-derived opsonins include fibronectin, mannose-binding lectin, and milk fat globulin (MFG-E8). A list of the most common phagocyte receptors is shown in Table A (Rosales 2017).

[0006] To date, four β-glucan receptors have been identified as candidates for mediating antifungal phagocytosis, namely complement receptor 3 (CR3; CD11b / CD18), lactosylceramide, selected scavenger receptors, and dectin-1 (βGR). Dectin-1 consists of a single C-type, lectin-like carbohydrate recognition domain, a short stalk, and a cytoplasmic tail with an immunoreceptor activation tyrosine motif (ITAM). The receptor recognizes particles, such as zymosan, Saccharomyces cerevisiae, and heat-killed Candida albicans, in a β-glucan-dependent manner (Taylor 2002). Dectin-1 has been clearly shown to be sufficient to activate phagocytosis. Dectin-1 is expressed on bone marrow dendritic cells, monocytes, macrophages, and B cells.

[0007] It would be beneficial to develop targeted removal and degradation of accumulated disease-causing substances without boosting overall phagocytosis. The present disclosure provides a solution to the problem and describes other advantages.

[0008] All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety as if each individual reference were specifically and separately indicated as being incorporated by reference. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] The present disclosure relates to a method for removing and degrading some of these substances, including host cells or host cell products, microorganisms or their products, in a human subject by administration of a molecule comprising a first binding domain that specifically binds to a disease-causing agent, a second binding domain that binds to dectin-1, a phagocytosis receptor expressed on macrophages, and induces phagocytosis, and an immunoglobulin Fc domain. In a specific embodiment, the method of the present disclosure depletes or reduces the number of disease-causing agents in tissues, blood, and / or bone marrow by targeted phagocytosis.

[0010] In some embodiments, provided herein is a method of reducing the number of disease-causing substances in a subject by targeted phagocytosis, the method comprising administering to the subject a binding protein comprising a first binding domain that specifically binds to a substance and a second binding domain that binds to a phagocytosis receptor expressed on macrophages, monocytes, and / or granulocytes and induces the phagocytosis activity of macrophages, monocytes, and / or granulocytes. In some embodiments, the phagocytosis receptor is dectin-1, such as human dectin-1. In some embodiments, provided herein is a method of reducing the number of disease-causing substances in a subject, the method comprising administering to the subject a binding protein comprising a first binding domain that specifically binds to a substance and a second binding domain that binds to dectin-1. In some embodiments, the binding protein further comprises an immunoglobulin Fc domain. In some embodiments, the binding protein is an antibody (e.g., a multispecific or bispecific antibody). In some embodiments, the subject is human. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a disease-causing substance. In some embodiments, the subject is human. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a disease-causing substance, and the bispecific antibody has the format shown and / or described with reference to FIG. 1A.

[0011] In some embodiments, administration of the binding protein results in a decrease in the number of substances. In some embodiments, administration of the binding protein results in the number of substances decreasing below the limit of detection. In some embodiments, administration of the binding protein results in a decrease in the number of substances for at least about one week after dosing of the binding protein. In some embodiments, administration of the binding protein results in a decrease in the number of substances within 12 hours, 24 hours, 36 hours, or 48 hours after administration. In some embodiments, the decrease in the disease-causing substance is reversible, for example, after administration of the binding protein is discontinued. In some embodiments, administration of the binding protein results in a decrease in the severity and / or incidence of one or more symptoms in a subject.

[0012] In some embodiments, the method results in the removal and / or a decrease in the level of one or more disease-related proteins or protein aggregates. In some embodiments, the method results in inhibition of the accumulation of abnormal proteins. In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of a disease, such as a neurodegenerative disease, fibrosis, or amyloidosis. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target protein or protein aggregate.

[0013] In some embodiments, the method results in the removal of cancer, tumor, or lymphoma cells and / or a decrease in their number. In some embodiments, the method results in alleviating one or more symptoms of cancer and / or preventing the progression of cancer. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by cancer cells (e.g., a tumor antigen expressed on the surface of cancer cells).

[0014] In some embodiments, the method results in the removal and / or reduction in level of one or more microorganisms (e.g., bacterial cells, fungal cells, protozoal cells, or viruses). In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of a disease or infection caused by a microorganism (e.g., bacterial cells, fungal cells, protozoal cells, or viruses). In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a bacterial cell (e.g., an antigen expressed on the surface of a bacterial cell). In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a fungal cell (e.g., an antigen expressed on the surface of a fungal cell). In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a protozoal cell (e.g., an antigen expressed on the surface of a protozoal cell). In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a virus (e.g., an antigen expressed on the surface of a virus).

[0015] In some embodiments, the method results in the removal and / or reduction in level of senescent cells and / or their product(s). In some embodiments, the method results in alleviating or preventing the progression of aging, for example, in one or more age-related symptoms or conditions. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by a senescent cell (e.g., an antigen expressed on the surface of a senescent cell).

[0016] In some embodiments, the method results in the removal and / or a decrease in the level of LDL and other substances that induce cardiovascular diseases, such as atherosclerosis or familial hypercholesterolemia. In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of cardiovascular diseases, such as atherosclerosis or familial hypercholesterolemia. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to lipoprotein particles (e.g., LDL).

[0017] In some embodiments, the method results in the removal and / or a decrease in the level of mast cells. In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of mast cell-related diseases, such as allergy, fibrosis, COPD, asthma, or other immunoproliferative mast cell-related diseases. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by mast cells (e.g., an antigen expressed on the surface of mast cells).

[0018] In some embodiments, the method results in the removal and / or a decrease in the level of eosinophils. In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of eosinophil-related diseases, such as allergy, fibrosis, COPD, asthma, or other immunoproliferative eosinophil-related diseases. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by eosinophils (e.g., an antigen expressed on the surface of eosinophils).

[0019] In some embodiments, the method results in the depletion and / or reduction in level of ILC2 cells. In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of an ILC2-related disease, such as allergy, fibrosis, COPD, asthma, or other immunoproliferative ILC2-related diseases. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds dectin-1 and a second binding domain that binds a target antigen expressed by ILC2 cells (e.g., an antigen expressed on the surface of ILC2 cells).

[0020] In some embodiments, the method results in the depletion and / or reduction in level of inflammatory immune cells in one or more tissues selected from the group consisting of, for example, muscle, GI tract, lung, heart, joint, and brain. In some embodiments, the method results in alleviating or preventing the progression of one or more symptoms of myositis, IBD, RA, allergy, fibrosis, COPD, asthma, or other immunoproliferative inflammatory immune cell-related diseases. In some embodiments, the binding protein is a bispecific antibody comprising a first binding domain that binds dectin-1 and a second binding domain that binds a target antigen expressed by inflammatory immune cells (e.g., an antigen expressed on the surface of inflammatory immune cells).

[0021] In some embodiments, the binding protein is an antibody; two antibodies or IgG covalently linked; IgG-scFv; an intrabody; a peptibody; a nanobody; a single domain antibody; an SMTP; a multispecific antibody (e.g., a bispecific antibody, a diabody, a triabody, a tetrabody, a tandem diabody-scFV, a tandem triabody-scFV, an ADAPTIR); a Fab, Fab’, F(ab’)2, or Fv fragment; a Fab’-SH or F(ab’)2 diabody; a linear antibody; a scFv antibody; a VH antibody; or a multispecific antibody formed from antibody fragments. In some embodiments, one or more (plural available) binding domains of the binding protein are non-human, chimeric, humanized, or human. In some embodiments, one or more (plural available) binding domains of the binding protein are humanized or human. In some embodiments, both binding domains of the binding protein are non-human, chimeric, humanized, or human. In some embodiments, both binding domains of the binding protein are humanized or human.

[0022] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following "Modes for Carrying Out the Invention". In certain embodiments, for example, the following items are provided. (Item 1) A method for removing or reducing the number of disease-causing substances in a human subject by targeted phagocytosis, comprising administering to the subject a binding protein comprising a first binding domain that specifically binds to the substance, a second binding domain that binds to dectin-1, a phagocytosis receptor expressed on macrophages, and induces the phagocytosis activity of the macrophages, and an immunoglobulin Fc domain. (Item 2) The method according to item 1, wherein the administration of the antibody reduces the number of the substance below the detection limit and maintains the level below detection for at least about 1 week after the antibody is administered. (Item 3) The method according to item 1, wherein the reduction of the disease-causing substance occurs within the first 24 hours or 48 hours after administration. (Item 4) The method according to item 1, wherein the reduction of the disease-causing substance is reversible. (Item 5) The method according to item 1, wherein the reduction of the disease-causing substance results in a reduction of symptoms. (Item 6) The method according to any one of items 1 to 5, which is used to remove disease-related proteins and protein aggregates, inhibit the accumulation of abnormal proteins, and thus alleviate or prevent the progression of diseases including neurodegeneration, fibrosis, or amyloidosis. (Item 7) The method according to any one of items 1 to 5, which is used to remove or reduce the level of cancer, tumor, or lymphoma cells, and thus inhibit or prevent the progression of the disease. (Item 8) The method according to any one of items 1 to 5, which is used to remove or reduce the level of microorganisms (e.g., bacteria, fungi, viruses), parasitic protozoa, and thus inhibit or prevent the progression of the disease. (Item 9) The method according to any one of items 1 to 5, which is used to remove senescent cells and their products or reduce their levels, thereby inhibiting or preventing the progression of aging. (Item 10) The method according to any one of items 1 to 5, which is used to remove microorganisms (e.g., bacteria, fungi, viruses), parasitic protozoa or reduce their levels, thereby inhibiting or preventing the progression of the said disease. (Item 11) The method according to any one of items 1 to 5, which is used to remove LDL and other substances that induce cardiovascular diseases including atherosclerosis or familial hypercholesterolemia or reduce their levels, thereby inhibiting or preventing the progression of the said disease. (Item 12) The method according to any one of items 1 to 5, which is used to remove mast cells or reduce their levels, thereby inhibiting or preventing the progression of allergies, fibrosis, other mast cell-related diseases including COPD, asthma, and immunoproliferative diseases. (Item 13) The method according to any one of items 1 to 5, which is used to remove eosinophils or reduce their levels, thereby inhibiting or preventing the progression of allergies, fibrosis, COPD, asthma, and other eosinophil-related diseases including immunoproliferative diseases. (Item 14) The method according to any one of items 1 to 5, which is used to remove ILC2 cells or reduce their levels, thereby inhibiting or preventing the progression of allergies, fibrosis, COPD, asthma, and other ILC2 cell-related diseases including immunoproliferative diseases. (Item 15) The method according to any one of items 1 to 5, which is used to remove inflammatory immune cells or reduce their levels in muscles, the GI tract, lungs, heart, joints, brain, and other organs, thereby inhibiting or preventing the progression of myositis, IBD, RA, allergies, fibrosis, COPD, asthma, and other immune cell-related diseases including immunoproliferative diseases. (Item 16) The method according to any one of the preceding items, wherein the binding protein is selected from specific antibodies; two IgG (IgG2) covalently linked; IgG-scFv; intrabodies, peptibodies, nanobodies, single domain antibodies, SMTP, and multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetra-bodies, tandem diabody-scFV, tandem tri-scFv, ADAPTIR); multispecific antibodies formed from Fab, Fab’, F(ab’)2, and Fv fragments, Fab’-SH, F(ab’)2, diabodies, linear antibodies, scFv antibodies, VH, and antibody fragments. (Item 17) The method according to item 16, wherein the binding domain of the binding protein is non-human, chimeric, humanized, or human, preferably humanized or human. (Item 18) The method according to any one of the preceding items, wherein the binding protein is a bispecific antibody comprising a first binding domain that binds to dectin-1 and a second binding domain that binds to a target antigen expressed by the disease-causing substance.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0044] Several aspects are described below with reference to applications that serve as examples for illustration. It should be understood that numerous specific details, relationships, and methods are described so as to obtain a complete understanding of the features described herein. However, one skilled in the art will readily recognize that the features described herein can be practiced without one or more of the specific details, or using other methods. Since some acts can occur in a different order and / or concurrently with other acts or events, the features described herein are not limited by the order of acts or events illustrated. Furthermore, not all of the acts or events illustrated are necessarily required to carry out the methodology according to the features described herein.

[0045] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, the terms "including", "includes", "having", " The terms "has", "with", or their variants are intended to be inclusive in a manner similar to the term "comprising" as long as they are used in either "a form for carrying out the invention" and / or "the claims". The term "comprising", as used herein, is synonymous with "including" or "containing", and is inclusive or non-limiting. Any reference to "or" in this specification is intended to include "and / or" unless otherwise stated. As used herein, the term "about" with respect to a number refers to a value that is the number plus or minus 10% of that number. The term "about" with respect to a range refers to a range from a value that is 10% less than the lowest value of the range to a value that is 10% more than the highest value of the range. There are various abnormal host cells that have accumulated and not been eliminated, such as tumor, lymphoma, dead, necrotic, apoptotic, dying, infected, damaged cells associated with diseases. In addition, various cell products, such as aggregated proteins (β-amyloid plaques or tau aggregates), lipoprotein particles, may cause diseases as their accumulation increases. Disease-causing cells may have glycoproteins, surface proteins, or glycolipids typical of abnormal cells associated with diseases, disorders, or other undesirable conditions. In addition to host substances, various foreign pathogens, such as infectious microorganisms (e.g., viruses, fungi, and bacteria), as well as microbial products and debris (e.g., viral particle envelopes, endotoxins), may not be sufficiently removed in a patient. The abnormalities listed above may cause diseases such as cancer, Alzheimer's disease, fibrosis, Parkinson's disease, Huntington's disease, HIV, hepatitis A, B, or C, sepsis, etc. Many of these disorders or diseases are characterized by the accumulation of disease-causing substances in different organs in a human subject.

[0046] Any reference to "or" in this specification is intended to include "and / or" unless otherwise stated. As used herein, the term "about" with respect to a number refers to a value that is the number plus or minus 10% of that number. The term "about" with respect to a range refers to a range from a value that is 10% less than the lowest value of the range to a value that is 10% more than the highest value of the range.

[0047] There are various abnormal host cells that have accumulated and not been eliminated, such as tumor, lymphoma, dead, necrotic, apoptotic, dying, infected, damaged cells associated with diseases. In addition, various cell products, such as aggregated proteins (β-amyloid plaques or tau aggregates), lipoprotein particles, may cause diseases as their accumulation increases. Disease-causing cells may have glycoproteins, surface proteins, or glycolipids typical of abnormal cells associated with diseases, disorders, or other undesirable conditions. In addition to host substances, various foreign pathogens, such as infectious microorganisms (e.g., viruses, fungi, and bacteria), as well as microbial products and debris (e.g., viral particle envelopes, endotoxins), may not be sufficiently removed in a patient. The abnormalities listed above may cause diseases such as cancer, Alzheimer's disease, fibrosis, Parkinson's disease, Huntington's disease, HIV, hepatitis A, B, or C, sepsis, etc. Many of these disorders or diseases are characterized by the accumulation of disease-causing substances in different organs in a human subject.

[0048] Methods are provided herein for altering and improving the phagocytic activity, selectivity, or phenotype of host phagocytic cells by using biological agents.

[0049] The present disclosure describes the use of a disease-causing agent on one arm and a molecule that specifically binds to the phagocytosis receptor dectin-1 receptor on the other arm (see, e.g., FIG. 1B). To achieve targeted phagocytosis, it is necessary to generate monoclonal antibodies that have agonistic activity when dectin-1 binds. The present disclosure proposes that agonist antibodies activate the receptor and induce phagocytosis. Bispecific antibodies that bind to the phagocytic cell receptor dectin-1 and a disease-causing agent, such as β-amyloid aggregate plaques, can induce phagocytosis of the substance and its degradation (FIG. 1A). In addition to or instead of conventional bispecific antibodies, two covalently linked IgGs (IgG2) in which one IgG binds to a phagocytosis receptor and the other binds to a disease-causing agent can be used (FIG. 1A). Another option is to use an IgG-scFv format in which the IgG binds to a phagocytosis receptor and the scFv portion binds to a disease-causing agent (FIG. 1A).

[0050] To enable targeted removal of disease-causing agents by phagocytosis, the antigen-binding domains of the present disclosure can be selected from IgG, intrabodies, peptibodies, nanobodies, single-domain antibodies, SMTP, and multispecific antibodies (e.g., bispecific antibodies, diabodies, tribodies, tetrabodies, tandem diabody-scFVs, tandem tribody-scFvs, ADAPTIR).

[0051] Multispecific antibodies have binding specificities for at least two different epitopes, usually derived from different antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies).

[0052] Methods for making bispecific antibodies are known in the art. One well-established approach for making bispecific antibodies is the "Knobs-into-holes" or "protuberance-into-cavity" approach. See, for example, U.S. Patent No. 5,731,168. Each of two immunoglobulin polypeptides (e.g., heavy chain polypeptides) includes an interface, and the interface of one immunoglobulin polypeptide interacts with the corresponding interface on the other immunoglobulin polypeptide, thereby associating the two immunoglobulin polypeptides. In some embodiments, the interfaces can be engineered such that a "knob" or "protuberance" located at the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" located at the interface of the other immunoglobulin polypeptide. In some embodiments, knobs can be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, holes can be constructed by replacing large amino acid side chains with smaller side chains. The knobs or holes can be present at the native interface or introduced synthetically. Polynucleotides encoding modified immunoglobulin polypeptides having one or more corresponding knob or hole-forming mutations can be expressed and purified using standard recombinant techniques and cell lines known in the art. See, for example, U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, 7,695,936, 8,216,805, U.S. Patent Publication No. 2013 / 0089553, and Spiess et al., Nature Biotechnology 31: 753-758, 2013. The modified immunoglobulin polypeptides are prokaryotic It can be produced using a host cell, such as E. coli, or a eukaryotic host cell, such as a CHO cell. Immunoglobulin polypeptides having corresponding knobs and holes can be expressed in the host cells in a co-culture and co-purified as heteromultimers, or expressed in a single culture, purified separately, and assembled in vitro.

[0053] According to a different approach, an antibody variable domain (antibody-antigen binding site) having a desired binding specificity is fused to an immunoglobulin constant domain sequence.

[0054] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkages.

[0055] The binding proteins of the present disclosure (e.g., monoclonal antibodies or antigen-binding portions thereof) can be non-human, chimeric, humanized, or human. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, Fab’-SH, F(ab’)2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments.

[0056] "Fab" (antigen-binding fragment) is a part of an antibody that binds to an antigen and includes the variable region and CHI of the heavy chain linked to the light chain by an interchain disulfide bond. The antibody can be an antibody of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0057] The anti-disease agent antibody can covalently attach to a phagocytosis receptor ligand, such as β-1,6-linked glucan (e.g., curdlan and dextran), and induce phagocytosis of the substance (Figure 1A).

[0058] The binding of a molecule that mediates the targeted removal of a disease-causing substance by phagocytosis may or may not have avidity, i.e., it may or may not induce dimerization of a phagocytosis receptor, such as dectin-1, or a target antigen present on a substance.

[0059] In addition to the beneficial removal of disease-causing substances by phagocytosis, the molecule can induce the production of inflammatory mediators and change the disease microenvironment in tumors, cancers, lymphomas, etc.

[0060] The immunoglobulin Fc portion of a molecule that causes targeted phagocytosis can engage Fc receptors and play an important role in the process by inducing additional phagocytosis. In some embodiments, the molecule has a modified Fc domain with reduced ADCC activity compared to wild-type human IgG1.

[0061] Without wishing to be bound by theory, antibody candidates with higher agonist activity to induce phagocytosis may be the most attractive for drug development. Antibody candidates that induce low internalization may exhibit the most prominent phagocytosis due to higher receptor occupancy on the cell surface and higher levels of receptor-antibody complexes.

[0062] To generate monoclonal antibodies (mAbs) against dectin-1, a recombinant target is utilized for immunization of mice. The generated mAbs are analyzed for selective binding to dectin-1 by ELISA and flow cytometry. The selected mAbs are tested in vitro for activation (phagocytosis) and internalization ability induced by dectin-1. mAb candidates are further tested for binding to cynomolgus monkey and mouse dectin-1. Positive candidates are used in vitro and in vivo for phagocytosis. The activity of the selected mAbs is compared to commercially available mAbs. For example, anti-dectin-1 mAbs are tested with the following anti-dectin-1 mAbs: 259931 (R&D Systems; catalog number: MAB1859), 15E2 (Invitrogen catalog number: 50-9856-42; BioLegend catalog number: 355402), BD6 (Bio-Rad catalog number: MCA4662), GE2 (Abcam catalog number: Ab82888); REA515 (Miltenyi Biotec catalog number: 130-107-725).

[0063] To generate monoclonal antibodies (mAbs) against disease-causing agents, such as β-amyloid aggregates, the agent is utilized for immunization of mice. The generated mAbs are analyzed by ELISA or flow cytometry for selective binding to the appropriate target, if applicable. mAb candidates with the highest affinity are further tested for binding to cynomolgus monkey target, if applicable. The phagocytosis activity of anti-dectin-1 mAb candidates is compared to commercially available anti-dectin-1 mAbs. Positive candidates are used to generate bispecific antibodies composed of an arm that binds to dectin-1 and a disease-causing agent, such as β-amyloid aggregates.

[0064] Antibodies can be produced using recombinant methods. For example, nucleic acids encoding antibodies can be isolated and inserted into replicable vectors for further cloning or expression. DNA encoding antibodies can be readily isolated and sequenced using conventional procedures (e.g., by oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are known in the art; as vector components, one or more of the following are generally mentioned but not limited to: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. In the present specification, suitable host cells for cloning or expression of DNA in vectors are cells of prokaryotes, yeast, or higher eukaryotes. When using recombinant techniques, antibodies can be produced intracellularly in the periplasmic space or secreted directly into the medium. If the antibody is produced intracellularly, either the host cell or particulate debris of lysed fragments is removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, it is common for the supernatant from such an expression system to be first concentrated using a commercially available protein concentration filter.

[0065] The final candidates are used for phagocytosis in vitro and in vivo. The activity of the selected candidates is compared to ligand-induced phagocytosis as reported (Herre 2004).

[0066] To demonstrate the phagocytosis activity of the final candidates in vitro for the depletion of disease-causing substances, an in vitro model that reproduces the activity in humans is used. Peripheral blood lymphocytes (PBLs) isolated from normal blood donors are incubated with the final candidates to test for depletion. The levels of the substances are measured by ELISA or flow cytometry. The phagocytosis activity of the antibodies is tested using purified primary monocytes as previously described (Ackerman 2011). To demonstrate the phagocytosis activity of the candidates on macrophages, macrophages are generated from primary monocytes. In addition, the Ab activity against single-cell tissue homogenates, as well as primary tissue cells composed of macrophages and DCs from bone marrow or synovial fluid, is tested.

[0067] For the depletion or reduction in the level of disease-causing substances, such as LDL or E. coli, to show the in vivo activity of the selected antibody candidates, mice or cynomolgus monkeys are used. To identify the pre-dose levels of LDL by ELISA, a cohort of cynomolgus monkeys is bled 1 day prior to single-dose antibody treatment. After treatment with the antibody, the monkeys are bled at the following time points: 1 hour, 1, 7, 14, and 30 days. The levels of disease-causing substances, such as LDL, in blood and other biological samples, such as synovial fluid, bone marrow, and spleen, are determined by ELISA.

[0068] The final mAb candidates are human or humanized and characterized for their binding to the human and cynomolgus monkey phagocytosis receptor dectin-1, and to disease-causing substances such as LDL, phagocytosis ability, and in vivo activity. In addition, the final candidates need to be soluble at concentrations higher than 10 mg / mL, have low levels of soluble aggregates (<5%), maintain binding to the target (>90% potency) as measured by ELISA, and show no degradation products as measured by SDS PAGE when incubated at 2 - 8°C for 3 months.

[0069] Toxicological analysis of the final humanized candidates is performed in cynomolgus monkeys at high doses exceeding five times the dose expected to be used in human subjects.

[0070] While not wishing to be bound by theory, molecules that effect targeted phagocytosis may, for example, show distinct benefits for patients with Alzheimer's disease, Parkinson's disease, cancer, infectious diseases (viral, bacterial, fungal, protozoal infections), inflammatory or immune diseases (e.g., autoimmune diseases, inflammatory bowel disease, multiple sclerosis), degenerative diseases (e.g., joints and cartilage) rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, etc. In addition, targeted phagocytosis antibody treatment may have better activity in depleting cells in tissues than ADCC that depends on NK cells. The treatment may have selective activity for the removal of specific disease causative agents rather than a therapy that targets bone marrow cells and generally improves phagocytosis.

[0071] Accordingly, the present disclosure provides a method for reducing or depleting the number of disease causative agents in a human subject by inducing targeted phagocytosis, which is targeted by binding to a phagocytosis receptor and a substance, and administering a molecule having an immunoglobulin Fc region.

[0072] The following description is presented to enable a person skilled in the art to make and use various embodiments. The description of specific devices, techniques, and applications is provided only by way of example. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but should be accorded the scope consistent with the claims.

Examples

[0073] (Example 1: Analysis of Dectin-1 Expression) This example describes the results of experiments characterizing the expression of dectin-1 by various cell types.

[0074] Materials and Methods Healthy donor samples Fresh buffy coats from healthy donors were obtained from the Stanford Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated by ficoll-paque (GE Healthcare, Chicago, IL) separation and cryopreserved in Bambanker cell freezing medium (Bulldog-Bio, Portsmouth, NH). Briefly, buffy coats were diluted in phosphate-buffered saline (PBS) at a 1:1 ratio, and then the diluted buffy coats were layered over ficoll and centrifuged at 760 g. The PBMC layer was isolated and washed in PBS prior to downstream analysis. Peripheral blood leukocytes (PBLs) were isolated through erythrocyte lysis. Tissue samples were provided by the Cooperative Human Tissue Network, funded by the National Cancer Institute. Tissue dissociation was performed according to the manufacturer's instructions for the Miltenyi Biotec tumor dissociation kit (Miltenyi Biotec Inc., Auburn, CA). Cryopreserved cynomolgus PBMCs were obtained from Human Cells.

[0075] Primary cells and cell cultures Human monocytes were isolated from the PBMCs of healthy donors according to the manufacturer's instructions of the Pan-Monocyte Isolation Kit (Miltenyi Biotec Inc., Auburn, CA). For macrophage differentiation, monocytes from PBMCs were allowed to adhere on cell culture plates for 3 hours. The floating cells were washed away, and the adhered monocytes were cultured in 20 ng / ml of MCSF (Peprotech, Rocky Hill, NJ) for 7 days to be fully differentiated into macrophages. HEK-Blue Null1 cells (Invivogen, San Diego, CA) were maintained in DMEM / 10% FBS supplemented with normocin and zeocin. HEK-Blue hDectin-1a cells and HEK-Blue hDectin-1b cells (Invivogen, San Diego, CA) were maintained in DMEM / 10% FBS supplemented with normocin and puromycin.

[0076] Freestyle293-F cells were transiently transfected according to the manufacturer's (Thermo Fisher, Waltham, MA) suggestions. Briefly, the viable cell density and viability percentage were determined. Using Freestyle293 Expression medium, the cells were diluted to a final density of 1×10 * viable cells / mL. Freestyle Max Reagent was diluted, mixed using OptiPro SFM medium, and incubated at room temperature for 5 minutes. The diluted Freestyle Max Reagent was added to the plasmid DNA diluted with OptiPro SFM medium and mixed. The Freestyle Max Reagent / plasmid DNA complex was incubated at room temperature for 10 - 20 minutes. During addition, while gently swirling the culture flask, the complex was slowly transferred to the cells, and then the cells were incubated in an incubator at 37°C on an orbital shaker with a relative humidity of 80% or more and 8% CO2.

[0077] Flow cytometry analysis Approximately 1×10 5 ~5×10 5Individual cells were plated in a non - tissue - cultured 96 - well V - bottom plate and incubated for 10 minutes at room temperature in human FcgR blocking antibody (Biolegend, San Diego, CA). Then, the cells were stained with eFluor506 viability dye (ThermoFisher, Waltham, MA) at a 1:1000 dilution for 30 minutes on ice, followed by a washing step in FACS buffer (PBS containing 2% fetal bovine serum). An antibody cocktail was added to the cells and then incubated for 30 minutes on ice, followed by another washing step in FACS buffer. Ultracomp beads (ThermoFisher, Waltham, MA) were used for antibody compensation. The antibodies used in this assay are presented in Table 1. All data acquisition and fluorescence compensation were performed using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo flow cytometry data analysis software. The strategy used to determine dectin - 1 expression in monocytes, lymphocytes, and granulocytes was by gating separately on forward and side scatter. Single cells were gated using forward scatter area and forward scatter height, and then live cells were gated using eFluor506 and forward scatter area. Monocytes, T cells, B cells, NK cells, and granulocytes were gated using CD14, CD3+ / CD4+ / 8+, CD3 - CD19+, CD3 - CD56+, and CD15+ markers, respectively. Cultured macrophages were identified by CD11b staining. In lung tissue, CD45 was used to gate hematopoietic cells. T cells, B cells, and NK cells were gated in CD45+ cells using CD3+, CD3 - CD19+, CD3 - CD56+ strategies, respectively. Macrophages were gated in CD45+ cells after excluding T, B, and NK cells using CD163 and CD11b.In the binding assay, the primary dectin-1 antibody was titrated at 100, 33.3, 11.1, 3.7, 1.23, and 0.41 nM, and the isotype control was titrated at 166, 55.3, 18.4, and 6.150 nM, followed by using a fluorescently labeled anti-mouse Fc-specific secondary antibody.

[0078] Quantification of Receptors The number of dectin-1 receptors was quantified by staining healthy donor PBMCs with an APC-conjugated targeting antibody and gated based on the appropriate immune cell types described above. Quantum APC molecules of equivalent soluble fluorochrome (MESF) calibration standard beads (Bangs Laboratories, Inc., Fishers, IN) were obtained and analyzed simultaneously according to the manufacturer's protocol to convert median fluorescence intensity measurements to MESF units. Background fluorescence was removed by subtracting the MESF values of FMO (fluorescence minus one) and isotype controls. Subsequently, the MESF values were divided by the ratio of the fluorophore to the protein (provided by the manufacturer) to convert to antibody binding capacity or receptor number.

[0079] Antibodies Table 1 provides the antibodies used in the experiments described in the examples.

Table 1-1

Table 1-2

Table 1-3

[0080] Results The expression of dectin-1, also known as CLEC7A, in various cell types was evaluated using dectin-1 specific antibodies and flow cytometry analysis. Single live monocyte and lymphocyte populations from donor samples or cultured cell samples were analyzed by flow cytometry using lineage and cell type specific antibodies conjugated to fluorophores to identify each immune cell population. Dectin-1 was detected using a dectin-1 specific antibody. Dectin-1 expression was determined by comparison to fluorescence minus one (FMO) and isotype control antibodies. In some experiments, the dectin-1 receptor number and the percentage of dectin-1 positive cells were calculated. All antibodies used for the detection of dectin-1 and flow cytometry are listed in Table 1.

[0081] To determine the expression of dectin-1 in immune cell populations, peripheral blood mononuclear cell (PBMC) samples from two healthy donors were collected and analyzed by flow cytometry. High levels of dectin-1 expression were found on monocytes (CD14+ cells) in healthy PBMC samples (Figure 2). Monocytes are professional phagocytes. The expression of dectin-1 in monocytes was positive in 21 out of 22 donors tested, the percentage of dectin-1 positive monocytes was greater than 90%, and the number of receptors was in the range of 32,000 to 59,000 per cell. Dectin-1 was not detected in CD4+ T cells (CD3+CD4+ cells), CD8 T cells (CD3+CD8+ cells), B cells (CD3-CD19+ cells), or NK cells (CD3-CD56+ cells). Thus, in healthy donor PBMC samples, dectin-1 is selectively expressed on monocytes and not on T cells, B cells, or NK cells.

[0082] Since dectin-1 is highly expressed on monocytes, the expression of dectin-1 in granulocytes was also examined. Granulocytes are another type of phagocytic immune cell. Peripheral blood leukocyte (PBL) samples from three healthy donors were collected and analyzed by flow cytometry. As shown in Figure 3, in the PBL samples of three healthy donors, dectin-1 was highly expressed on monocytes and moderately expressed in granulocytes. Dectin-1 was expressed at a lower level in granulocytes compared to monocytes, and the number of receptors per cell was 4,000 - 5,000.

[0083] Monocytes can differentiate into macrophages, which are tissue-specific phagocytic cells. To determine the expression of dectin-1 on macrophages, donor samples were cultured in MCSF (20 ng / ml) for 7 days to differentiate into macrophages. Next, single live cells were stained with CD11b to confirm differentiation into macrophages, and then analyzed by flow cytometry to determine dectin-1 expression. As shown in Figure 4, dectin-1 was expressed on cultured monocytes-derived macrophages. Confirming that dectin-1 expression is retained in cultured monocytes-derived macrophages serves as a proof of principle that targeted phagocytosis is possible in tissues.

[0084] Macrophages are tissue-specific phagocytes. To test the expression of dectin-1 in macrophages within tissues, lung tissue samples were collected from healthy donors, dissociated, and analyzed by flow cytometry. Hematopoietic cells were gated using CD45 to separate them from non-hematopoietic cells in the tissue. T cells, B cells, and NK cells were identified in CD45+ cells using CD3+, CD3-CD19+, and CD3-CD56+ gates, respectively. After excluding T, B, and NK cells in CD45+ cells, macrophages were gated using CD163 and CD11b. Dectin-1 expression was determined for all isolated cell populations. Figure 5 shows the results of this experiment. Dectin-1 was highly expressed in macrophages in lung tissue samples, with 19,000 receptors per cell. Since no dectin-1 expression was detected in T cells, B cells, or NK cells, it is shown that dectin-1 is selectively expressed in macrophages in healthy human lung tissue. Dectin-1 was not detected in non-hematopoietic cells. This result demonstrates that targeted phagocytosis mediated by dectin-1 in tissues is possible if both appropriate cell types and targets are present.

[0085] The Dectin-1 receptor can be expressed as two different isoforms, isoform A and isoform B. To examine whether a Dectin-1 antibody recognizes either the A or B isoform, HEK293 cells were engineered to overexpress human Dectin-1 isoform A or B (HEK-Blue hDectin-1a cells and HEK-Blue hDectin-1b cells, respectively), and analyzed by flow cytometry to evaluate Dectin-1 expression. The 15e2 Dectin-1 antibody clone was used to confirm Dectin-1 expression. Control HEK293 cells (HEK-Blue Null1 cells) and Freestyle293 cells transiently transfected with a construct expressing human Dectin-1A (293F hDectin-1a FL) were analyzed to test the specificity of Dectin-1 detection. As shown in Figure 6, the 15e2 Dectin-1 antibody clone recognized both the A and B isoforms of Dectin-1 in HEK293 cells overexpressing Dectin-1. Since Dectin-1 was not detected in untransformed control cells, the antibody is specific for Dectin-1. Engineered HEK293 cells are a useful tool for the functional evaluation of phagocytosis and signaling events involving Dectin-1 in a normally non-phagocytic cell line.

[0086] The specificity of multiple Dectin-1 antibody clones (259931, GE2, and BD6) was also evaluated in HEK293 cells overexpressing Dectin-1 and in monocytes from healthy donors. The results of these experiments are summarized in Table 2. Clone 259931 had the highest affinity for Dectin-1 in all cells tested. Clone 259931 also had a high affinity for both isoform A and B of Dectin-1, while the other antibodies either did not bind to the B isoform or had a reduced binding affinity for the B isoform. The different affinities observed with different Dectin-1 antibody clones may be due to binding to different epitopes, as demonstrated by their different affinities for the receptor isoforms.

Table 2

[0087] Finally, a binding assay was performed to examine the cross-reactivity of human dectin-1 antibody clones 15e2 and 259931 against cynomolgus dectin-1. Monocytes were obtained from the monkey PBMC samples by flow cytometry. The isolated cells were incubated with either the 15e2 and 259931 dectin-1 antibody clones, as well as their respective isotype control antibodies IgG2a and IgG2b, followed by a fluorescent anti-mouse secondary antibody. To generate the binding curves, the dectin-1 antibodies were used in serial dose titrations of 100, 33.3, 11.1, 3.7, 1.23, and 0.41 nM, while the isotype controls were used in serial dose titrations of 166, 55.3, 18.4, and 6.150 nM. As shown in Figure 7, both human dectin-1 antibody clones cross-reacted with cynomolgus dectin-1 expressed on monocytes. However, the clones showed different binding characteristics to cynomolgus dectin-1 for each clone, demonstrating that different antibodies bind to different epitopes. Cynomolgus monkeys are commonly used as preclinical models for toxicology studies, and since these dectin-1 antibodies bind to cynomolgus monocytes, they can therefore be readily used in toxicology studies.

[0088] As shown in this example, dectin-1 is highly expressed in monocytes and macrophages, specialized phagocytic cells, but not in other immune cells. Dectin-1 expression is also specific for macrophages within healthy human lung tissue. The dectin-1 antibodies characterized in this example specifically recognize dectin-1 in cells, recognize both isoforms of dectin-1, and can cross-react with cynomolgus dectin-1. The antibodies described in this example can be used for targeted phagocytosis mediated by dectin-1.

[0089] (Example 2: Effects of Dectin-1 Antibodies on Phagocytosis and Signaling) This example describes the results of an experiment testing the effects of dectin-1 antibodies on phagocytosis and signaling.

[0090] Materials and Methods The materials and methods used in this experiment are detailed below. Unless otherwise specified, donor samples and primary cells were prepared as described in Example 1. Unless otherwise specified, cell culture, flow cytometry, and receptor quantification were performed as described in Example 1. The antibodies used in this example are listed in Table 1.

[0091] SEAP Reporter Assay in HEK Cells Using Dectin-1 Antibodies Immobilized by Air Drying The dectin-1 monoclonal antibodies 15e2, 259931, GE2, BD6, and a control isotype were immobilized by coating the surface of untreated 96-well, U-bottom polypropylene microtiter plates. For coating, 10 μg of antibody diluted in 50 μl of sterile PBS was added to each well. The plates were left uncovered in a Class II laminar flow cabinet overnight to allow the solution to evaporate. The coated plates were washed twice with 200 μl of sterile PBS to remove salt crystals and unbound antibody. Next, HEK-Blue hDectin-1a cells were cultured on the plates for 22 hours, and the alkaline phosphatase level was evaluated in the supernatant at an OD of 630 nm using QUANTI-Blue Solution (Invivogen, San Diego, CA) according to the manufacturer's instructions.

[0092] Labeling of Polystyrene Beads with pHrodo and Conjugation with Antibodies pHrodo labeling was performed using polystyrene beads coated with goat anti-mouse IgG (Fc) (Spherotech, Lake Forest, IL). The beads were washed with phosphate-buffered saline pH 7.2 (PBS) (Corning, Corning, NY) using Spin-X centrifuge tube filters (Corning, Corning, NY). The pH was adjusted by the addition of bicarbonate buffer. pHrodo Red, succinimidyl ester (pHrodo Red, SE) (ThermoFisher, Waltham, MA) was added to the beads and incubated at room temperature for 60 minutes with shaking. Next, the beads were washed with PBS using a Spin-X centrifuge tube filter to remove excess pHrodo RED. After pHrodo labeling, the antibody was conjugated to the beads according to the manufacturer's recommendations. Briefly, based on the binding ability of the antibody to the beads, excess antibody was added to the beads in PBS and incubated at room temperature for 60 minutes with shaking. Next, the beads were washed with PBS using a Spin-X centrifuge tube filter to remove unbound antibody.

[0093] Antibody-dependent cell phagocytosis In the phagocytosis experiment, HEK cells or monocytes overexpressing dectin-1 were seeded in 96-well plates and allowed to adhere for 1 hour. pHrodo beads conjugated to dectin-1 antibody or isotype were added at the desired ratio. Differentiated macrophages were detached using Accutase (Thermo Fisher, Waltham, MA), re-seeded in 96-well plates at the desired density, allowed to adhere for 2 hours, and then the beads were added. CellTracker Calcein AM (Thermo Fisher, Waltham, MA) was added to identify viable cells.

[0094] Plates containing cells and pHrodo-conjugated beads were placed in an IncuCyte S3 live imaging system (Sartorius, Germany). Phagocytosis was monitored by taking images at the desired time points and analyzed using IncuCyte S3 software. The overlap of bright red fluorescence (phagocytosed beads) with calcein AM-positive cells was considered as a measure of phagocytosis.

[0095] In some experiments, pHrodo-labeled beads were mixed with the dectin-1 antibody in a 96-well plate for 1 hour. The beads were spun down and the supernatant was aspirated to remove unbound antibody. Next, the cells were mixed with the beads at the desired ratio, spun down briefly, and monitored for phagocytosis. Alternatively, cells incubated with the beads for 30 minutes or 1 hour were collected, and phagocytosis was evaluated by flow cytometry using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA).

[0096] For the preparation of bispecific antibodies, a single antibody was conjugated to biotin or streptavidin (Abcam, Cambridge, MA) and pHrodo labeling (if shown). Antibodies were mixed at a ratio of 2:1 (biotinylated antibody:streptavidin antibody) and allowed to bind for 30 minutes at room temperature. The bispecific antibody was added to the cells and phagocytosis was investigated by IncuCyte live imaging. In one experiment, the biotinylated antibody was mixed with streptavidin-FITC beads (Thermo Fisher, Waltham, MA) of 40 nm size. Results

[0097] Results The dectin-1 specific antibody described in Example 1 was assayed for its ability to activate the secretion of alkaline phosphatase and phagocytosis in various cell types. Unless otherwise specified, phagocytosis was performed using polystyrene anti-mouse Fc IgG beads (approx. 3.4 μm) labeled with a pH-sensitive fluorescent dye (pHrodo Red) and conjugated to the dectin-1 antibody or isotype control. As a result of stimulation of dectin-1 by the ligand, secreted alkaline phosphatase (SEAP) was produced in the cells. The dectin-1 specific antibody can act as a ligand for the receptor and stimulate the SEAP signaling pathway in the cells.

[0098] The ability of the dectin-1 antibody to stimulate dectin-1 was tested by a SEAP reporter assay using HEK-Blue hDectin-1a cells. HEK-Blue hDectin-1a cells were engineered to express the dectin-1A isoform and genes involved in the dectin-1 / NF-κB / SEAP signaling pathway, and thus to express secreted alkaline phosphatase (SEAP) in response to stimulation by the dectin-1 ligand. As a positive control, the cells were incubated with zymosan (10 μg / ml), the natural ligand of dectin. As shown in Figure 8, the 15e2 dectin-1 antibody clone promotes SEAP secretion, presumably by engaging dectin-1 on the surface of cells that exhibit agonist activity. The activity resulting from stimulation by the dectin-1 antibody is comparable to that of zymosan. As shown in Figure 8, the effect of the dectin-1 antibody is also dose-dependent. Thus, the dectin-1 specific antibody induces alkaline phosphatase secretion in HEK-Blue hDectin-1a. These cells provide a useful tool for functionally screening dectin-1 antibodies.

[0099] Stimulation with dectin-1 can trigger the activation of phagocytosis. To investigate dectin-1-specific phagocytosis, cultured HEK-Blue hDectin-1a cells were treated with pHrodo-labeled beads conjugated with dectin-1 antibody or isotype control antibody. The fluorescent signal generated by pHrodo increases in an acidic environment, such as that found in phagosomes. As shown in FIGS. 9A and 9B, dectin-1 antibody-coupled beads promote phagocytosis in HEK-Blue hDectin-1a cells. As shown in FIG. 9B, pHrodo-labeled beads conjugated with dectin-1 antibody clone 259931 (4.5-fold higher than the isotype control) promoted a higher level of phagocytosis against the isotype control than the 15e2 clone (2.1-fold higher than the isotype control). Treatment of HEK-Blue hDectin-1a was sufficient to induce targeted phagocytosis. Some cells also phagocytosed multiple beads, indicating highly efficient internalization. Since HEK cells do not express the Fcγ receptor, another receptor involved in phagocytosis, and are not normally phagocytic, these results demonstrate high specificity for dectin-1-dependent phagocytosis.

[0100] The specificity of dectin-1-mediated phagocytosis observed upon stimulation with a dectin-1 antibody was further tested by a competition assay. If the observed phagocytosis was due to dectin-1 receptor stimulation by dectin-1 antibody-conjugated beads, the addition of free dectin-1 antibody was expected to decrease the phagocytosis of the beads. In this experiment, pHrodo-labeled beads were mixed with increasing amounts of dectin-1 antibody or isotype control (IgG2a) in the range of 20 ng to 400 ng. Since 20 ng of antibody is required to occupy all the binding sites of 400,000 beads (according to the manufacturer's instructions), amounts greater than 20 ng result in an excess of unbound antibody. As shown in Figure 10, the level of phagocytosis induced by the dectin-1-specific antibody decreased in the presence of excess free dectin-1 antibody. The excess free antibody competed with the pHrodo-labeled dectin-1 antibody-conjugated beads and caused a decrease in the phagocytosis of the beads. This observation confirmed that antibody-induced phagocytosis is specific for dectin-1 stimulation.

[0101] Phagocytosis was similarly examined using pHrodo-labeled beads of three sizes (0.85 μm, 3.4 μm, and 8 μm) conjugated to dectin-1 antibody or isotype control. It was found that all three sizes of beads were taken up via dectin-1-mediated phagocytosis (Figures 11A and 11B). These data support the conclusion that dectin-1 can efficiently phagocytose particles of different sizes within the size range of disease-causing agents such as cells (about 10 - 20 μm), bacteria (about 0.2 - 2 μm), larger viruses (about 0.5 - 1 μm), and protein aggregates.

[0102] Since dectin-1 is expressed as two distinct isoforms, the ability of dectin-1 antibodies to stimulate phagocytosis by both isoform A and B of dectin-1 was tested. HEK-Blue hDectin-1a and HEK-Blue hDectin-1b cells were incubated with pHrodo-labeled beads conjugated to dectin-1 antibodies or isotype controls. The 15e2 or 259931 dectin-1 antibody clones conjugated to the beads were tested in this experiment. These two clones can bind to both the A and B isoforms of dectin-1 with different affinities (see Table 2). As shown in Figures 12A and 12B, the 15e2 and 259931 dectin-1 antibody clones promoted phagocytosis at comparable levels in HEK cells overexpressing isoform A of dectin-1. However, 259931 promoted a higher level of phagocytosis than the 15e2 clone in HEK cells overexpressing isoform B of dectin-1. As shown in Table 2, the 259931 clone had a higher affinity for isoform B than the 15e2 clone. This result indicates that the specific epitope engaged by the dectin-1 antibody has a differential effect on phagocytic ability depending on the dectin-1 isoform expressed. Dectin-1 antibodies promoted phagocytosis in both the dectin-1 isoform A overexpressing cell line and the dectin-1 isoform B overexpressing cell line, and thus can promote phagocytosis in primary cells expressing either form of dectin-1.

[0103] The 259931 dectin-1 antibody clone showed better performance in promoting phagocytosis in cells expressing either the A or B isoform of dectin-1. Therefore, the ability of this antibody clone to promote the phagocytosis of particles of different sizes was tested. These results are shown in Figures 13A - 13C. Both the 259931 and 15e2 dectin-1 antibody clones promoted the phagocytosis of medium-sized particles with comparable efficiency. However, the 259931 clone promoted the phagocytosis of very small or very large particles more efficiently than the 15e2 clone. This result indicates that the two dectin-1 antibodies have different abilities to take up smaller or larger particles, suggesting that the epitopes engaged are associated with superior functional phagocytic ability.

[0104] As described in Example 1, dectin-1 is highly expressed in human monocytes, a type of phagocyte. To determine whether phagocytosis in monocytes can be promoted by the engagement of antibodies with dectin-1, purified monocytes (CD14+) derived from human PBMCs were incubated with pHrodo-labeled beads conjugated with dectin-1 antibodies. As shown in Figures 14A - 14C, dectin-1 antibody-conjugated beads promoted phagocytosis by monocytes at a significantly higher level (1.6-fold higher) than phagocytosis by isotype control beads. Therefore, in addition to promoting phagocytosis in cells overexpressing dectin-1, dectin-1-specific antibodies promote phagocytosis in human monocytes.

[0105] The activation of phagocytosis in monocytes is specific to the stimulation of dectin-1 and is independent of the Fcγ receptor (FcγR). As shown in Figure 15, the addition of an antibody that blocks FcγR did not affect the induction of phagocytosis by dectin-1 antibody-conjugated beads. The induced phagocytosis in monocytes is thus induced by dectin-1 antibodies, is dectin-1-specific, and does not rely on FcγR-mediated phagocytosis.

[0106] Since phagocytosis mediated by dectin-1 requires the actin cytoskeleton, the effect of adding cytochalasin D (CytoD), an actin depolymerizing drug, was also tested. Monocytes were incubated with dectin-1 antibody-conjugated beads in the presence or absence of CytoD. As shown in Figure 16, phagocytosis mediated by dectin-1 was inhibited by treatment with CytoD, demonstrating the requirement for the actin cytoskeleton. Since active actin polymerization is required for phagocytosis and phagocytosis mediated by dectin-1 is sensitive to treatment with CytoD, targeted phagocytosis mediated by the dectin-1 antibody is specific to this type of cell transport and does not occur via non-specific or passive mechanisms.

[0107] Finally, the ability of the dectin-1 antibody to promote phagocytosis in human macrophages was analyzed. Purified monocytes were cultured in MCSF (20 ng / ml) for 7 days to differentiate into macrophages. Next, monocyte-derived macrophages were incubated with dectin-1 antibody-conjugated beads to test for dectin-1-mediated phagocytosis in these cells. As shown in Figure 17, the dectin-1 antibody promoted bead-induced phagocytosis in cultured human macrophages. Higher frequencies of phagocytosis and larger numbers of ingested beads were observed in cells incubated with dectin-1 antibody-conjugated beads compared to isotype control beads. These results demonstrate that dectin-1-mediated phagocytosis in tissues via macrophage-expressed dectin-1 is possible.

[0108] The results presented in this example highlight that robust targeted depletion is possible in different compartments, such as blood, bone marrow, and tissue.

[0109] Next, phagocytosis of the Dectin-1 antibody conjugated to the pHrodo-labeled anti-H3N2 virus antibody was examined in a recombinant cell line overexpressing Dectin-1, providing a proof-of-concept demonstrating virus phagocytosis mediated by the Dectin-1 bispecific antibody. Biotinylated Dectin-1 antibody (15e2-B) or biotinylated isotype (IgG2a-B) was conjugated with the pHrodo-labeled streptavidin-12CA5 antibody (12CA5-SA-pHr), an anti-H3N2 antibody that binds to the hemagglutinin protein of the H3N2 influenza virus. HEK-Blue hDectin-1a cells were labeled with the cell-permeable dye Calcein AM and seeded in 96-well plates (50,000 cells per well). 15e2-B or isotype control was mixed with 12CA5-SA-pHr to form the bispecific antibody for 30 minutes. The soluble bispecific antibody was added to the cells at a final concentration of 40 nM. Phagocytosis of the 15e2-B / 12CA5-SA-pHr bispecific antibody was monitored by evaluating pHrodo activation using IncuCyte live cell imaging. A diagram of the conjugation of the bispecific Dectin-1 / 12CA5 antibody to the cells is shown in Fig. 18A. This format can be used to connect the cells to the H3N2 virus.

[0110] At 18 hours, representative images showed pHrodo-positive cells (phagocytosed 12CA5 pHrodo-labeled antibody emits bright red fluorescence in the phagosome; Fig. 18B). Fig. 18C shows phagocytosis of the 15e2-B / 12CA5-SA-pHr bispecific antibody over 24 hours. Phagocytosis was quantified by IncuCyte analysis software and represented as the overlap of red object counts (pHrodo) with Calcein-positive cells.

[0111] These results demonstrate that bispecific antibodies targeting dectin-1 and a disease-causing agent (e.g., H3N2 influenza virus) can cause phagocytosis of the agent in HEK cells overexpressing dectin-1. These data indicate that bispecific antibodies targeting dectin-1 and a small biological entity, such as an influenza virus (≈100 nm), can be used to connect dectin-1-expressing cells to the disease-causing agent for phagocytic uptake and elimination by phagocytosis.

[0112] Next, phagocytosis of a dectin-1 antibody conjugated to a pHrodo-labeled anti-H3N2 virus antibody was examined in primary human monocytes, providing proof-of-concept that virus phagocytosis mediated by dectin-1 bispecific antibodies occurs in primary human phagocytes. FIGS. 19A and 19B show phagocytosis of dectin-1 bispecific antibodies by human monocytes. A biotinylated dectin-1 antibody (15e2-B) or biotinylated isotype (IgG2a-B) was conjugated to pHrodo-labeled streptavidin-12CA5 (12CA5-SA-pHr), an anti-H3N2 antibody that binds to the hemagglutinin protein of H3N2 influenza virus. Human monocytes were labeled with the cell-permeable dye calcein AM and seeded into 96-well plates (50,000 cells per well). 15e2-B or the isotype control antibody was mixed with 12CA5-SA-pHr to form bispecific antibodies for 30 minutes. The soluble bispecific antibodies were added to the cells at a final concentration of 40 nM. Phagocytosis of the 15e2-B / 12CA5-SA-pHr bispecific antibody was monitored by evaluating pHrodo activation using IncuCyte live cell imaging. FIG. 19A shows phagocytosis of the 15e2-B / 12CA5-SA-pHr bispecific antibody over 21 hours, quantified by IncuCyte analysis software and represented as the overlap of red object counts (pHrodo) relative to calcein-positive cells. For the isotype ** p<0.01; ****p < 0.0001. Two-way ANOVA with Holm-Sidak multiple comparison test. Figure 19B shows representative images of pHrodo-positive cells at 6 hours of the experiment (phagocytosed 12CA5 pHrodo-labeled antibody emits bright red fluorescence in phagosomes).

[0113] These results indicate that monocytes phagocytosed the dectin-1 / H3N2 influenza virus bispecific antibody. These data support the concept that dectin-1 bispecific binding proteins can be utilized to promote phagocytosis of disease-causing agents, such as influenza virus, by human monocytes. From this, the possibility of eliminating these disease-causing agents for the treatment of infectious diseases is emphasized by the injection of soluble dectin-1 bispecific antibodies.

[0114] Phagocytosis of 40 nm beads by primary human monocytes was also examined. Figures 20A and 20B show phagocytosis of streptavidin FITC-labeled polystyrene beads (40 nm) conjugated with biotinylated dectin-1 antibody (15e2-B) or biotinylated isotype (IgG2a-B) by human monocytes. Polystyrene FITC beads were saturated with biotinylated dectin-1 antibody or isotype control for 30 minutes. Next, the antibody / bead complex was incubated with cultured human monocytes at a ratio of 1:6 (cells:beads). FITC staining of monocytes was monitored by IncuCyte live cell imaging. Figure 20A shows phagocytosis of SA-FITC beads by monocytes over 21 hours, quantified by IncuCyte analysis software and represented as the count of green (FITC-positive) objects. Figure 20B shows representative images of FITC-positive cells at 15 hours of the experiment.

[0115] The anti-dectin-1 antibody was found to promote phagocytosis of very small polystyrene beads (40 nm). These data indicate that very small particles can be phagocytosed by targeting dectin-1, suggesting the possibility of promoting phagocytosis of very small disease-causing agents, such as viruses.

[0116] For the sake of clarity, the present disclosure is described in some detail by way of illustration and example, but the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety.

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Claims

[Claim 1] The invention described in the specification.

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