Mannose-3-glycan-mediated proteolysis

Abstract

The present disclosure provides a bifunctional binding protein for mannose 3 glycan-mediated degradation. Such glycan modification will improve current treatment and allow patients to have a better quality of life. Therefore, such bifunctional binding proteins are useful for treating and preventing various diseases.

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 164,991, filed on 23 March 2021, which is incorporated herein by reference in its entirety.

[0002] 2. Technical Fields The present invention generally relates to a bifunctional binding protein having mannose 3-glycosylation and its lysosomal targeting. [Background technology]

[0003] 3.Background technology The binding of carbohydrate-binding receptors to glycans enables a variety of biological pathways. The interaction between glycans and receptors is determined by the structure of the glycan. These important biological pathways are involved in regulating immune responses, mediating protein elimination, protein turnover, and controlling the transport of any native molecules, including soluble glycoproteins, glycolipids, and / or glycan moieties.

[0004] Endocytosis lectins are involved in receptor-mediated endocytosis by capturing glycosylated proteins via specific glycan structures and mediating their degradation. Endocytosis lectins are ubiquitous in the human body and can recognize a variety of glycan structures. See, for example, Cummings, Richard D.; McEver, Rodger P. (Eds.) (2017): Essentials of Glycobiology [Internet]. 3rd edition: Cold Spring Harbor Laboratory Press [8]. Additional background information on glycans and their receptors is provided in references [9]-

[12] .

[0005] Carbohydrate-binding receptors are highly diverse and, through glycoengineering, can be utilized to develop novel therapeutic agents with unprecedented efficacy against a wide range of diseases, including but not limited to inflammation, hematological disorders, autoimmune diseases, and cancer. This enables the development of novel therapeutic agents based on the concept of glycan-mediated proteolysis. Novel therapies may be obtained by utilizing human glycan-mediated proteolysis through site-directed glycosylation of any protein, such as chemokines, cytokines, polypeptides, or monoclonal antibodies. To date, it has not been described how efficiently Man3-containing glycans, such as Man3GlcNAc2 constructed on proteins, can bind to mannose or receptor-recognizing lectins and mediate lysosomal degradation. This invention presents novel findings regarding specific glycan-mediated proteolysis.

[0006] The compositions and methods provided herein address the unmet medical needs of patients suffering from various intractable diseases, such as cancer, autoimmune diseases, and inflammatory diseases, which are treated with current standard therapies. [Overview of the project]

[0007] 4. Outline of the Invention In one embodiment, a first portion specifically binds to a target protein, and the following structure [ka] A bifunctional binding protein comprising a second portion containing a glycan containing the above is provided herein. The squares represent N-acetylglucosamine residues, the black striped circles represent mannose residues, and X represents an amino acid residue of the bifunctional binding protein. In some embodiments, the N-glycan portion of the bifunctional binding protein consists of mannotriose-di-(N-acetyl-D-glucosamine) (Man3GlcNAc2), i.e., the mannose 3-glycan structure shown in this paragraph.

[0008] In certain embodiments, the bifunctional proteins provided herein include an N-glycan having a mannose-3 structure as a terminal glycan. Specifically, this may also include any branched structure of the N-glycan on the GlcNAc2 portion of the N-glycan. For example, GlcNac directly linked to X may be fucosylated. As used herein, the terms “mannose-3 structure,” “Man3 structure,” or “M3 structure” refer to an N-glycan having three terminal mannose residues.

[0009] In some embodiments, the target proteins to which the first portion specifically binds include HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, Frizzled receptor, Wnt, LRP5 / 6, CD38, CD73, TGF-β, bombesin®, CAIX, CD13, CD44v6, empurine, endoglin, EpCAM, EphA2, FAP-α, folic acid®, GRP78, IGF-1®, matryptase, mesothelin, and sMET / HGF. This includes R, MT1-MMP, MT6-MMP, PSCA, PSMA, Tn antigen, and uPAR, TSHRα, myelin oligodendrocyte glycoprotein (MOG), AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin and gangliosides GM1, GD3, GQ1B, SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47.

[0010] In some embodiments, the second part is a mannose 3 receptor, a differentiation cluster 206 (CD206) receptor, a DC-SIGN (differentiation cluster 209 or CD209) receptor, a type C lectin domain family 4-member G (LSECTin) receptor, or a macrophage-inducible Ca 2+ It specifically binds to the Man3GlcNAc2 receptor (Mincle). In some embodiments, the second portion of the bifunctional binding protein specifically binds to any endocytosis carbohydrate-binding receptor that recognizes the Man3GlcNAc2 structure. In some embodiments, the second portion of the bifunctional binding protein specifically binds to Langerin, macrophage mannose 2 receptor, Dectin-1, Dectin-2, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, DNGR1, CLEC12B, DEC-205, asialoglycoprotein receptor (ASPGR), and mannose 6-phosphate receptor.

[0011] In some embodiments, the second portion includes a glycan structure. In some embodiments, the glycan structure is mannose 3.

[0012] In some embodiments, the first portion of the bifunctional binding protein includes a heavy chain variable region or a light chain variable region. In some embodiments, the first portion of the bifunctional binding protein includes a Fab region of a monoclonal antibody.

[0013] In some embodiments, the bifunctional binding protein is an antibody. In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is recombinant. In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. In some embodiments, the antibody has a glycan-to-protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1.

[0014] In some embodiments, the bifunctional binding protein is glycosylated at a specific residue.

[0015] In some embodiments, the bifunctional binding protein is a self-antigen.

[0016] In some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the glycans of the bifunctional binding protein have a mannose-3 glycan structure. <000E077>

[0017] In some embodiments, the target protein is a cell surface molecule or a non-cell surface molecule. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the non-cell surface molecule is an extracellular protein. In some embodiments, the extracellular protein is an autoantibody, a hormone, a cytokine, a chemokine, a blood protein, or a central nervous system (CNS) protein.

[0018] In some embodiments, the target protein is bound by a first moiety.

[0019] In one aspect, provided herein is a method of delivering a target protein to the endosomes of liver macrophages, such as liver resident Kupffer cells (KC) and / or liver sinusoidal endothelial cells (LSEC) and / or monocyte-derived macrophages (MoMφ), comprising contacting the target protein with the bifunctional binding protein described herein under conditions that mediate endocytosis of the target protein.

[0020] In one aspect, provided herein is a method of degrading a target protein, comprising contacting the target protein with the bifunctional binding protein described herein under conditions that mediate lysosomal degradation of the target protein by a host cell.

[0021] In some embodiments, the target protein is upregulated in cancer or is involved in cancer progression.

[0022] In some embodiments, target proteins that are upregulated in cancer or are involved in cancer progression include HER2, EGFR, HER3, VEGFR CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2, Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, CD38, CD73, LILRB2 or TGF-β.

[0023] In some embodiments, the target protein is an autoantibody of an autoimmune disease. In some embodiments, the target protein is an autoantigen of an autoimmune disease.

[0024] In some embodiments, the autoantibodies of autoimmune diseases are antibodies that bind to TSHRα, MOG (myelin oligodendrocyte glycoprotein), AChR-α1, the non-collagen domain 1 (α3NC1) of the α3 chain of type IV collagen, ADAMTS13, desmoglein-1 / , GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, or gangliosides GM1, GD3 or GQ1B.

[0025] In some embodiments, the target protein is upregulated or expressed in neurodegenerative diseases. In some embodiments, target proteins that are upregulated or expressed in neurodegenerative diseases are α-synuclein, amyloid-β, components of the complement cascade, or in systemic amyloidosis, the accumulation of aggregated misfolded light chains or aggregated misfolded transthyretin.

[0026] In some embodiments, the target protein is upregulated or expressed in tumor-associated macrophages (TAMs). In some embodiments, the target protein is associated with the recruitment of TAMs in the tumor microenvironment. In other embodiments, the target protein is associated with the depletion of TAMs in the tumor microenvironment. In further embodiments, the target protein is associated with the reprogramming of TAMs in the tumor microenvironment.

[0027] In some embodiments, the target proteins associated with TAM include SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, or CXCR4. In other embodiments, the target proteins associated with TAM include CCL2, CXCL12, CSF-1, or CD47.

[0028] In some embodiments, the host cells are myeloid cells, immune cells, endothelial cells, parenchymal cells, or epithelial cells. In some embodiments, the immune cells are dendritic cells, macrophages, monocytes, microglia, granulocytes, or B lymphocytes.

[0029] In some embodiments, the host cell is any cell.

[0030] In some embodiments, the bifunctional binding protein enhances the degradation of the target protein compared to the degradation of the target protein in the presence of a bifunctional binding protein containing a different second portion.

[0031] In some embodiments, the degradation is mediated by endocytosis or phagocytosis.

[0032] In one embodiment, a pharmaceutical composition comprising a bifunctional binding protein and a pharmaceutically acceptable carrier as described herein is provided herein.

[0033] In one embodiment, a method for treating or preventing a disease in a patient is provided herein, comprising administering to the patient a bifunctional binding protein or a pharmaceutical composition described herein.

[0034] In some embodiments, the disease is an autoimmune disease, cancer or tumor, liver disease, inflammatory disorder, or blood coagulation disorder. In some embodiments, the autoimmune disease is selected from Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, acquired pemphigus vulgaris, immune thrombocytopenia, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), membranous nephropathy, systemic amyloidosis, and Guillain-Barré syndrome.

[0035] In some embodiments, the administration step includes intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection.

[0036] In one embodiment, a kit is provided herein that includes a bifunctional binding protein or a pharmaceutical composition described herein, and instructions for administering the bifunctional molecule or pharmaceutical composition to an individual requiring it.

[0037] In some embodiments, the bifunctional binding protein or pharmaceutical composition is present in one or more unit doses.

[0038] In some embodiments, the glycan further comprises a fucose residue in N-acetylglucosamine directly bound to X. In some embodiments, X is an asparagine residue in a bifunctional binding protein.

[0039] In one embodiment, a bifunctional binding protein is provided herein, which (i) specifically binds to a target protein and (ii) has the following structure [ka] Contains N-glycan,

[0040] The squares represent N-acetylglucosamine residues, the black striped circles represent mannose residues, and the X represents an amino acid residue of the bifunctional binding protein. The N-glycan is linked to the bifunctional binding protein at 1, 2, 3, 4, or 5 N-glycosylation sites.

[0041] In some embodiments, the glycan further comprises a fucose residue in N-acetylglucosamine directly bound to X. In some embodiments, X is an asparagine residue in a bifunctional binding protein.

[0042] In one embodiment, a population of bifunctional binding proteins is provided herein, wherein at least one of the N-glycosylation sites at a specific amino acid position of the bifunctional binding protein is glycosylated with a mannose 3N-glycan structure, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 99% of the N-glycosylation sites in the population.

[0043] In some embodiments, the N-glycosylation site comprises one or more asparagine residues, the asparagine residues being located within the standard consensus sequence NXS / T, the NXC motif (where X can be any amino acid other than proline), and a non-standard consensus motif.

[0044] In some embodiments, the N-glycosylation site is introduced into the bifunctional protein by recombinant engineering.

[0045] In some embodiments, recombinant engineering is carried out by adding, deleting, substituting amino acids, or adding glycotags. In some embodiments, the glycan consists of a mannose 3N-glycan structure.

[0046] In some embodiments, the target proteins are HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, Frizzled receptor, Wnt, LRP5 / 6, CD38, CD73, TGF-β, Bombecin®, CAIX, CD13, CD44, v6, CXCR4, ErbB-2, Her2, Empurin, Endoglin, EpCAM, EphA2, FAP-α, Folic Acid®, GRP78, IGF-1®, Matryptase, Mesoteric This includes sMET / HGFR, MT1-MMP, MT6-MMP, Muc-1, PSCA, PSMA, Tn antigen, and uPAR, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin and gangliosides GM1, GD3, GQ1B, MOG, SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47.

[0047] In some embodiments, the bifunctional protein binds to endocytosis carbohydrate-binding receptors, including mannose 3 receptor, differentiation cluster 206 (CD206) receptor, DC-SIGN (differentiation cluster 209 or CD209) receptor, C-type lectin domain family 4-member G (LSECTin) receptor, and macrophage-inducible Ca 2+ It specifically binds to the lectin-dependent receptor (Mincle).

[0048] In some embodiments, the bifunctional protein binds to an endocytosis carbohydrate-binding receptor via an N-glycan. In some embodiments, the N-glycan specifically binds to any endocytosis carbohydrate-binding receptor that recognizes the Man3GlcNAc2 structure.

[0049] In some embodiments, the bifunctional binding protein is an antibody. In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is recombinant. In some embodiments, the antibody includes a heavy chain variable region or a light chain variable region. In some embodiments, the antibody includes a Fab region. In some embodiments, the antibody includes an Fc domain.

[0050] In some embodiments, the antibody includes an N-glycosylation site in the Fc domain, to which an N-glycan is ligated. In some embodiments, the antibody includes an N-glycosylation site in the heavy chain variable region and / or the light chain variable region, to which an N-glycan is ligated.

[0051] In some embodiments, the antibody is glycosylated at a specific residue.

[0052] In some embodiments, the antibody has a glycan-to-protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1.

[0053] In some embodiments, the bifunctional binding protein contains an autoantigen and specifically binds to an autoantibody.

[0054] In some embodiments, the target protein is a cell surface molecule or a non-cell surface molecule. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the non-cell surface molecule is an extracellular protein.

[0055] In some embodiments, the extracellular protein is an autoantibody, hormone, cytokine, chemokine, blood protein, or central nervous system (CNS) protein.

[0056] In one embodiment, a method for delivering a target protein to a liver macrophage is provided herein, namely, contacting the target protein with a bifunctional binding protein described herein under conditions that mediate the endocytosis of the target protein.

[0057] In one embodiment, a method for degrading a target protein is provided herein, comprising contacting the target protein with a bifunctional binding protein described herein under conditions that mediate lysosomal degradation of the target protein by a host cell.

[0058] In some embodiments, the target proteins are upregulated in cancer or involved in cancer progression. In some embodiments, the target proteins that are upregulated in cancer or involved in cancer progression include HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2, Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, LILRB1, LILRB2, CD38, CD73, or TGF-β.

[0059] In some embodiments, the target protein is an autoantibody of an autoimmune disease. In some embodiments, the target protein is an autoantigen of an autoimmune disease. In some embodiments, the autoantibody of an autoimmune disease is an antibody that binds to MOG, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin, or ganglioside GM1, GD3, or GQ1B.

[0060] In some embodiments, the target protein is upregulated or expressed in neurodegenerative diseases. In some embodiments, the target protein upregulated or expressed in neurodegenerative diseases is α-synuclein, amyloid-beta, or a complement cascade component.

[0061] In some embodiments, the host cell is a myeloid cell, immune cell, endothelial cell, parenchymal cell, or epithelial cell. In some embodiments, the immune cell is a dendritic cell, macrophage, monocyte, microglia, granulocyte, or B lymphocyte. In some embodiments, the host cell is any cell.

[0062] In some embodiments, the bifunctional binding protein enhances the degradation of the target protein compared to the degradation of the target protein in the presence of a non-N-glycosylated bifunctional binding protein, or compared to the degradation of the target protein in the presence of a bifunctional binding protein containing an N-glycan different from the mannose 3N-glycan structure. In some embodiments, the degradation is mediated by endocytosis or phagocytosis.

[0063] In one embodiment, a pharmaceutical composition comprising a bifunctional binding protein and a pharmaceutically acceptable carrier as described herein is provided herein.

[0064] In one embodiment, a method for treating or preventing a disease in a patient is provided herein, comprising administering to the patient a bifunctional binding protein or a pharmaceutical composition described herein.

[0065] In some embodiments, the disease is an autoimmune disease, cancer or tumor, liver disease, inflammatory disorder, or blood coagulation disorder. In some embodiments, the autoimmune disease is selected from MOGAD (myelin oligodendrocyte glycoprotein antibody-associated disease), Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, acquired pemphigus vulgaris, immune thrombocytopenia, autoimmune encephalitis, and Guillain-Barré syndrome.

[0066] In some embodiments, cancer is defined as acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphoblastic leukemia; acute myelogenous leukemia; acute myeloid leukemia (adult / child); adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratomatous / rhabdoid tumor; basal cell carcinoma; extrahepatic cholangiocarcinoma; bladder cancer; osteosarcoma / malignant fibrous histiocytoma of bone; brain tumor (adult / child); cerebellar astrocytoma (adult / child); cerebral astrocytoma / malignant glioma; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumor; brain tumor, optic tract hypothalamic glioma; brainstem glioma Breast cancer; bronchial adenoma / carcinoid; bronchial tumor; Burkitt lymphoma; childhood cancer; carcinoid gastrointestinal tumor; carcinoid tumor; adult carcinoma, primary site unknown; carcinoma of unknown primary site; embryonal tumor of the central nervous system; primary lymphoma of the central nervous system; cervical cancer; childhood adrenocortical carcinoma; childhood cancer; childhood cerebral astrocytoma; chordoma, childhood; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorder; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; fibroplastic round cell tumor; emphysema; endometrium Cancer; ependymoblastoma; ependymoma; esophageal cancer; Ewing's sarcoma of the Ewing family tumors; extracranial germ cell tumors; extragonadal germ cell tumors; extrahepatic cholangiocarcinoma; gallbladder cancer; gastric (stomach) cancer; gastric carcinoid; gastrointestinal carcinoid tumors; gastrointestinal stromal tumors; germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic neoplasm; gestational trophoblastic neoplasm, primary site unknown; glioma; brainstem glioma; glioma, pediatric optic tract and hypothalamus; hair cell leukemia; head and neck cancer; cardiac cancer; hepatocellular (liver) cancer; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell carcinoma; Kaposi's sarcoma; renal cell carcinoma; Langerhans cell histiocytosis; laryngeal cancer; lip and oral cancer; liposarcoma; primary liver cancer; non-small cell lung cancer; small cell lung cancer; lymphoma of the central nervous system; macroglobulinemia, Waldenström; male breast cancer; malignant fibrous histiocytoma / osteosarcoma of bone; medulloblastoma; medullary epithelioma; melanoma; intraocular melanoma; Merkel cell carcinoma; Merkel cell cutaneous carcinoma; mesothelioma;Mesothelioma, adult malignant; metastatic squamous cell carcinoma of unknown primary origin; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasmacytic neoplasm; mycosis fungoides, myelodysplastic syndrome; myelodysplasia / myeloproliferative disorder; chronic myeloid leukemia; acute myeloid leukemia, adult; acute myeloid leukemia, pediatric; multiple myeloma (bone marrow cancer); chronic myeloproliferative disorder; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma, non-small cell lung cancer; non-Hodgkin lymphoma; oligodendroglioma; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial carcinoma (surface epithelial and stromal tumor); ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer Pancreatic cancer, islet cell cancer; papillomatosis; sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germ cell tumor; intermediate pineal parenchymal tumor; pineoblastoma and supratentorial primitive neuroectodermal tumor; pituitary tumor; pituitary adenoma; plasma cell tumor / multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); pelvic ureteral and transitional cell carcinoma; airway cancer involving the NUT gene on chromosome 15; retinoblastoma; rhabdomyosarcoma, pediatric; salivary gland cancer; sarcoma, Ewing family tumor; Sézary syndrome; skin cancer (melanoma); skin cancer (non-melanoma); small cell lung cancer; small intestine cancer Soft tissue sarcoma; soft tissue sarcoma; spinal cord tumor; squamous cell carcinoma; cervical squamous cell carcinoma of unknown primary origin, metastatic; stomach cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, cutaneous (mycosis fungoides and Sézary syndrome); testicular cancer; pharyngeal cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; pediatric thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; or Wilms' tumor (selected from these).

[0067] In some embodiments, the treatment involves reprogramming tumor-associated macrophages (TAMs) by administering a bifunctional binding protein under conditions that mediate endocytosis of the target protein.

[0068] In some embodiments, the target protein is upregulated or expressed in the TAM. In some embodiments, the target protein upregulated or expressed in the TAM includes SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47.

[0069] In some embodiments, the administration step includes intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection.

[0070] In one embodiment, a kit is provided herein that includes a bifunctional binding protein or a pharmaceutical composition described herein, and instructions for administering the bifunctional molecule or pharmaceutical composition to an individual requiring it.

[0071] In some embodiments, the bifunctional binding protein or pharmaceutical composition is present in one or more unit doses.

[0072] In one embodiment, the foregoing provides a method for treating an acute condition involving elevated levels of a target protein, the method comprising administering to a patient in need a bifunctional binding protein described in any one of the prior claims, the bifunctional binding protein (i) specifically binds to the target protein and (ii) has the following structure [ka] It contains an N-glycan having,

[0073] Squares represent N-acetylglucosamine residues, black striped circles represent mannose residues, X represents amino acid residues of the bifunctional binding protein, and N-glycans are linked to the bifunctional binding protein at numerous N-glycosylation sites, resulting in a half-life of the target protein of up to 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, or the half-life of the target protein of up to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, or up to 90% of the half-life of the target protein in the patient in the absence of any treatment.

[0074] In some embodiments, the glycan further comprises a fucose residue in N-acetylglucosamine directly bound to X. In some embodiments, X is an asparagine residue in a bifunctional binding protein.

[0075] In some embodiments, the bifunctional protein supports N-glycans at three or more N-glycosylation sites.

[0076] In one embodiment, the foregoing provides a method for treating an acute condition involving elevated levels of a target protein, the method comprising administering to a patient in need a bifunctional binding protein described in any one of the prior claims, the bifunctional binding protein (i) specifically binds to the target protein and (ii) has the following structure [ka] It contains an N-glycan having,

[0077] The squares represent N-acetylglucosamine residues, the black striped circles represent mannose residues, and the X represents an amino acid residue of the bifunctional binding protein. The N-glycan is linked to the bifunctional binding protein at multiple N-glycosylation sites, resulting in a half-life of the target protein of at least 1, 2, 3, or 4 days, or a half-life of the target protein of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the half-life of the unglycosylated bifunctional binding protein in the patient.

[0078] In some embodiments, the glycan further comprises a fucose residue in N-acetylglucosamine directly bound to X. In some embodiments, X is an asparagine residue in a bifunctional binding protein.

[0079] In some embodiments, the bifunctional protein supports N-glycans at two or fewer N-glycosylation sites. [Brief explanation of the drawing]

[0080] 5. Brief description of the drawing [Figure 1] The Man3 glycan presented on Fab is shown to be highly internally transported to macrophages. Macrophages were obtained and incubated with pHrodo-labeled antibody for 3 hours. The graph shows the mean mean fluorescence intensity (MFI) ± mean standard error (SEM) of pHrodo, normalized to Humira (H-A2F). Each point represents the value from an individual donor. At least 3 donors were used for each condition except H-PNGase (N=2).

[0081] [Figure 2]Man3 glycan on antibodies results in high internal distribution and lysosomal compartment targeting in human dendritic cells. Internal distribution is independent of the Fcγ receptor. Human monocyte-derived dendritic cells were obtained from five different PBMC donors. The graph shows the mean pHrodo MFI ± standard deviation (SD) from N=5 individual donors. AbNone indicates that no pHrodo-labeled antibody was added, while all other conditions indicate that a pHrodo-labeled antibody was added at 10 μg / ml. MbT: Mabthera used as the control antibody. AM: Man3-adalimumab. AS: Sialized adalimumab. HM: HUMIRA® (adalimumab). HS: Sialized HUMIRA® (adalimumab). +FcR blocking indicates that the Fcγ receptor blocking agent Human TruStain FcX was added before the addition of the pHrodo-labeled antibody.

[0082] [Figure 3] A and B show that Man3 glycan presented on Fab is highly internally translocated to dendritic cells. Dendritic cells were obtained from two PBMC donors. Dendritic cells were incubated with pHrodo-labeled antibody for 6 hours. A: Examples of gating of high pHrodo populations under conditions H-A2F and A8486-M3. High pHrodo-gated cells are black. B: The graph shows the percentage of high pHrodo cells (gated as shown in A) from each donor.

[0083] [Figure 4] A schematic diagram showing how mannose 3 is recognized by mannose-binding receptors such as CD206 and lectins.

[0084] [Figure 5]The phenotype of differentiated macrophages is shown. The macrophages were CD14+, CD163+, CD1a-, and CD206+. The flow cytometry plot shows marker expression from one representative culture. The graph on the right shows that the percentage of CD206+ macrophages remained constant between donors and experiments. Each point represents data from one peripheral blood mononuclear cell (PBMC) donor.

[0085] [Figure 6] Figures A and B show that antibodies presenting the M3 structure on Fab are specifically internally transported by CD206-expressing macrophages. Macrophages were incubated with pHrodo-labeled antibodies for 3 hours (A) or 24 hours (B). The graphs show pHrodo MFI adjusted for denomination of labeling (DOL). The histograms show the mean ± SD from all donors, with each point representing data from a single donor. Statistical analysis: paired two-sided t-test. *p<0.05; **p<0.01; ***p<0.001; ns: not significant (p>0.05).

[0086] [Figure 7] This study demonstrates that antibodies presenting the Man3 glycan structure rapidly and potently remove the target antigen from the rat blood circulation. Rats were intravenously administered HCA202 (0.5 mg / kg) and antibody (10 mg / kg). The graph shows the mean ± SD ng / ml serum concentration of HCA202 for 3 or 4 animals per group. Black circles indicate the H-A2F (adalimumab, Humira®) treatment group. White squares indicate the A-84-M3 treatment group. Black triangles indicate the A-8486-M3 treatment group. Black squares indicate the A-8486-A2G2S2 treatment group. White circles indicate the PBS treatment group (HCA202 only). White diamonds and dotted lines indicate the A-M3 treatment group. In the graph, if the HCA202 level fell below the lower limit of quantification (LLOQ) of the assay at the minimum necessary 10-fold dilution (MRD10) (dotted line LLOQ / MRD10 = 20 ng / ml), it was defined as 19 ng / ml.

[0087] [Figure 8] This study demonstrates that antibodies displaying the Man3 glycan structure partially distribute to the liver region at a faster rate compared to the control antibody. Mice were intravenously injected with 5 mg / kg of CF750-labeled antibody and imaged using fluorescence tomography. The graph shows the mean fluorescence in pmol ± SD for three animals / time point in the gated liver region of interest. White squares and dotted lines represent the A-M3 treatment group. Black circles represent the H-A2F treatment group. Black triangles represent the A-8486-M3 treatment group. [Modes for carrying out the invention]

[0088] 6. Modes for Carrying Out the Invention This specification describes bifunctional binding proteins (e.g., mannose-3 glycosylated bifunctional binding proteins) that have improved functionality compared to control antibodies. As illustrated herein, bifunctional binding proteins are engineered by introducing mannose-3 onto the glycosylation site on the bifunctional binding protein, resulting in an engineered glycosylation profile that mediates the degradation of the bifunctional binding protein and the endocytosis receptor of the target to which it binds. By customizing the mannose-3 glycan site, the engineered bifunctional binding proteins described herein can 1) have homogeneous glycosylation, 2) degrade large targets such as immune complexes, 3) have a defined ligand-to-antibody ratio, 4) have a defined glycosylation site, 6) activate a wider variety of potent degradation receptors, and / or 5) participate in protein degradation in a highly optimized manner.

[0089] As shown in Figure 4, although not theoretically bound, the bifunctional binding protein can bind to macrophage mannose-binding receptors, CD206, LSECTin-, lung surfactant protein SP-D, Mincle, DC-Sign, and any other lectin that recognizes mannose 3, as described herein. Endocytosis of ligands by type C lectins can result in the accumulation and degradation of receptors in phagolysosomes, or the recycling of receptors to the cell surface.

[0090] While not bound by theory, mannose-3 glycosylated antibodies are expected to reduce target proteins associated with human disease via natural degradation pathways, as described herein.

[0091] The term "approximately," when used with a number, refers to any number within ±1, ±5, or ±10% of the referenced number.

[0092] As used herein, the term “patient” refers to an animal (e.g., birds, reptiles, and mammals). In other embodiments, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is an animal other than a human. In some embodiments, the subject is livestock or a pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken). In certain embodiments, the subject is a human. The terms “subject” and “patient” may be used interchangeably herein.

[0093] The abbreviations "α[number]", "α[number], [number]", "β[number]", or "β[number], [number]" refer to glycosidic bonds or glycosidic linkages, which are covalent bonds that connect carbohydrate residues to another base. α-glycosidic bonds are formed when both carbon atoms have the same stereochemistry, while β-glycosidic bonds occur when the two carbon atoms have different stereochemistry.

[0094] As used herein, the term “bifunctional binding protein” means a protein that can bind to two different ligands via a first and a second portion, wherein the first portion is a different protein from the second portion. As used herein, the term “bifunctional binding protein” refers to a protein having three or more binding specificities or functions. An example of a bifunctional binding protein of this disclosure is a mannose-3 glycosylated soluble TSH receptor extracellular domain that binds to a mannose-3 glycosylated anti-TSH receptor antibody or an autoantibody.

[0095] As used herein, the term “inflammatory disorder” includes disorders, diseases, or conditions characterized by inflammation. Examples of inflammatory disorders include, among others, allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejection.

[0096] As used herein, the term “blood disorder” includes disorders, diseases, or conditions that affect the blood. Examples of blood disorders include, among others, bleeding disorders such as anemia and hemophilia, thrombosis, and blood cancers such as leukemia, lymphoma, and myeloma.

[0097] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency for use in animals, and more specifically in humans, or is listed in the United States Pharmacopeia or any other commonly recognized pharmacopoeia.

[0098] As used herein in the context of pharmaceutically acceptable carriers, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly in injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin.

[0099] In some embodiments, the bifunctional binding proteins provided herein may comprise one or more N-glycans having (i) binding specificity to one or more target proteins, and (ii) binding specificity to one or more endocytosis carbohydrate-binding proteins or receptors.

[0100] In some embodiments, the glycosylated bifunctional binding protein has one binding specificity for one target protein. In more specific embodiments, the glycosylated bifunctional binding protein has one binding specificity for one target protein and has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more valencies so that a single bifunctional binding protein can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target protein molecules.

[0101] In some embodiments, the bifunctional binding protein has one or more N-glycans capable of associating with the endocytosis receptor. In some embodiments, the bifunctional binding protein has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycans capable of associating with the endocytosis receptor. The N-glycans may be linked to the protein by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation consensus sequences (or glycan sites). These glycan sites may be naturally present in the protein or may be introduced by recombination through the addition, substitution, or deletion of amino acids. In certain embodiments, a glycotag is fused to the protein to create a bispecific binding protein. As used herein, a glycotag refers to a peptide containing a consensus N-glycosylation site sequence fused to the N-terminus, C-terminus, or both ends of a protein or polypeptide. In some embodiments, two or more glycans are the same and associate with the same endocytosis receptor(s). In some embodiments, two or more glycans are different and associate with different endocytosis receptors.

[0102] In some embodiments, bifunctional binding proteins comprising a first portion and a second portion that specifically bind to a target protein are provided herein.

[0103] In some embodiments, the first portion comprises a heavy chain variable region and a light chain variable region, or antigen fragments thereof. In some embodiments, the first portion comprises a Fab region of a monoclonal antibody. In some embodiments, the first portion specifically binds to any of the target proteins disclosed herein.

[0104] In some embodiments, the bifunctional binding protein is a TNFα monoclonal antibody. In other embodiments, the bifunctional binding protein is not a TNFα monoclonal antibody.

[0105] In some embodiments, the second portion includes a glycan structure having a mannose-3-glycan structure as disclosed herein. In some embodiments, the glycan structure is any of the glycans disclosed herein.

[0106] In some embodiments, any receptor that binds to mannose 3-glycan is included in the description of compositions and methods disclosed herein. In other embodiments, the second part binds to any endocytic carbohydrate-binding receptor that recognizes the Man3GlcNAc2 structure.

[0107] In some embodiments, the second portion specifically binds to the mannose 3 receptor. In some embodiments, the second portion specifically binds to the differentiation cluster 206 (CD206) receptor. In some embodiments, the second portion specifically binds to the DC-SIGN (differentiation cluster 209 or CD209) receptor. In some embodiments, the second portion specifically binds to the C-type lectin domain family 4-member G (LSECTin) receptor. In some embodiments, the second portion specifically binds to macrophage-inducible Ca 2+ It specifically binds to the lectin receptor (Mincle). In some embodiments, the second portion specifically binds to receptors selected from the group consisting of Langerin, macrophage mannose 2 receptor, Dectin-1, Dectin-2, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, DNGR1, CLEC12B, DEC-205, asialoglycoprotein receptor (ASPGR), and mannose 6-phosphate receptor (M6PR).

[0108] In some embodiments, the bifunctional binding protein is an antibody. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigenic fragment thereof. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is isolated from a human subject. In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. In other embodiments, the bifunctional binding protein is an autoantigen. In other embodiments, the bifunctional binding protein is an autoantibody.

[0109] In some embodiments, the antibody has a glycan-to-antibody ratio of 2:1, 4:1, 6:1, 8:1, or 10:1. In some embodiments, the antibody is glycosylated at a predetermined specific residue. In other embodiments, the antibody is glycosylated at a random residue.

[0110] In some embodiments, the following structure [ka] A bifunctional binding protein comprising a second portion having the above is provided herein. The squares represent N-acetylglucosamine residues, the black striped circles represent mannose residues, and X represents an amino acid residue of the bifunctional binding protein. In some embodiments, the X amino acid residue of the bifunctional binding protein disclosed herein is Asn of an N-linked glycosylation consensus sequence engineered into the bifunctional binding protein. Such an N-linked glycosylation consensus sequence may be present in the variable domain of the bifunctional binding protein in some embodiments.

[0111] In some embodiments, the bifunctional binding protein is [ka] The material comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of a glycan having the structure, where X represents an amino acid residue of the bifunctional binding protein. In some embodiments, the X amino acid residue of the bifunctional binding protein disclosed herein is the Asn of an N-linked glycosylated consensus sequence manipulated in the bifunctional binding protein.

[0112] In some embodiments, the bifunctional binding protein is an anti-TGF-β monoclonal antibody or an anti-Notch monoclonal antibody that has been glycosylated with mannotriose-di-(N-acetyl-D-glucosamine) (Man3GlcNAc2).

[0113] In some embodiments, the target protein is a cell surface molecule or a non-cell surface molecule. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the non-cell surface receptor is an extracellular protein. In some embodiments, the extracellular protein is an autoantibody, hormone, cytokine, chemokine, blood protein, or a protein expressed in the central nervous system (CNS).

[0114] In some embodiments, disease-related target proteins are upregulated in the disease compared to the non-disease state. In some embodiments, disease-related target proteins are expressed in the disease compared to the non-disease state. In some embodiments, disease-related target proteins are involved in disease progression. In some embodiments, the disease is cancer or a tumor. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is a neurodegenerative disease.

[0115] In some embodiments, the disease is Graves' disease. Graves' disease is the most common cause of hyperthyroidism. Its prevalence in the United States is 1.2% (1), and the lifetime risk for women is as high as 3%. The production of agonist anti-TSH receptor (TSHR) antibodies (TRAb) leads to overproduction of thyroxine hormone (more than 90% of patients are TRAb+) (2). Current treatments have not advanced for 50 years and are limited by a high risk of relapse and serious side effects such as hypothyroidism. In some embodiments, the target protein associated with Graves' disease is autoantibody-bound TSHRα. In other embodiments, the target protein associated with Graves' disease is TSHRα.

[0116] In some embodiments, the target protein includes a protein selected from the group consisting of HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2 Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, LILRB1, LILRB2, LILRB3, LILRB4, CD38, CD73, and TGF-β. In other embodiments, the target proteins include antibodies that bind to TSHRα, MOG, AChR-α1, the non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin, and gangliosides GM1, GD3, and GQ1B.

[0117] In some embodiments, methods for delivering target proteins to hepatocyte endosomes are provided herein. In some embodiments, the method for delivering target proteins to hepatocytes includes contacting the target protein with one of the bifunctional binding proteins disclosed herein under conditions that mediate endocytosis of any target protein disclosed herein. In some embodiments, the method for delivering target proteins to endosomes of hepatic macrophages such as hepatic commensal Kupffer cells (KCs) and / or hepatic sinusoidal endothelial cells (LSECs) and / or monocyte-derived macrophages (MoMφ) is carried out in vivo. In some embodiments, the mode of delivery of target proteins to hepatocyte endosomes in vivo includes intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection or intramuscular injection. In some embodiments, the method for delivering target proteins to hepatocyte endosomes is carried out ex vivo.

[0118] In some embodiments, methods for degrading target proteins are provided herein. In some embodiments, the method for degrading target proteins comprises contacting the target protein with one of the bifunctional binding proteins disclosed herein under conditions that mediate degradation of any of the target proteins disclosed herein by a host cell. In some embodiments, the degradation is lysosomal degradation. In some embodiments, the degradation is mediated by endocytosis or phagocytosis. In some embodiments, the degradation is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 18-fold, 20-fold, 25-fold, or 30-fold higher than the degradation mediated by a bifunctional binding protein containing any glycan excluding any second portion disclosed herein. In other embodiments, the bifunctional binding protein enhances the degradation of any of the disclosed target proteins compared to the degradation of the target protein in the presence of a bifunctional binding protein containing any glycan excluding any second portion disclosed herein. In some embodiments, the host cells are any host cells, including but not limited to bone marrow cells, immune cells, endothelial cells, parenchymal cells, or epithelial cells. In some embodiments, the immune cells may be dendritic cells, macrophages, monocytes, microglia, granulocytes, or B lymphocytes.

[0119] In some embodiments, mannose-3 degradation is optimal due to the involvement of endocytosis receptors. In some embodiments, the method of degrading target proteins via mannose-3 mediated degradation is selective. In some embodiments, mannose-3 degradation removes inflammatory cytokines from circulation, unwanted blood factors, autoantibodies, and cell surface receptors.

[0120] In some embodiments, the bifunctional binding protein mediating the degradation of the target protein is an anti-TGF-β monoclonal antibody containing Man3GlcNAc2, which captures TGF-β and degrades TGF-β in lysosomes via recognition of Man3GlcNAc2 by one of the endocytosis receptors disclosed herein. This approach can be applied to deplete cell surface receptors such as Notch for cancer treatment.

[0121] In some embodiments, the target protein is a protein that is upregulated in cancer. In some embodiments, the target protein is a protein involved in cancer progression. Examples of target proteins that are upregulated in cancer or involved in cancer progression and can be conjugated by the bifunctional binding proteins provided herein include HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2, Frizzled receptor, Wnt, and LRP5. / 6, CSF-1R, SIRPα, LILRB1, LILRB2, LILRB3, LILRB4, CD38, CD73, TGF-β, Bombecin R, CAIX, CD13, CD44v6, CXCR4, ErbB-2, Her2, Empurin, Endoglin, EpCAM, EphA2, FAP-α, Folic Acid R, GRP78, IGF-1R, Matryptase, Mesothelin, sMET / HGFR, MT1-MMP, MT6-MMP, Muc-1, PSCA, PSMA, Tn antigen, and uPAR are examples, but are not limited to these.

[0122] In some embodiments, the target protein is an autoantibody such as one associated with an autoimmune disease. Examples of autoantibodies that can be conjugated by the bifunctional binding proteins provided herein include, but are not limited to, autoantibodies against MOG, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin, and gangliosides GM1, GD3, and GQ1B.

[0123] In some embodiments, the target protein includes a protein that is upregulated or expressed in tumor-associated macrophages (TAMs). In some embodiments, the target protein is upregulated or expressed in pro-tumor TAMs. Examples of target proteins upregulated or expressed in TAMs include SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, or CXCR4(7). In other embodiments, the target protein includes CCL2, CXCL12, CSF-1, or CD47(7). As described in reference (7), these targets play a role in promoting pro-tumor TAMs, particularly by promoting TAM recruitment and programming.

[0124] In some embodiments, methods for reprogramming TAMs using any of the bifunctional binding proteins disclosed herein are provided. As described in reference (7), TAM reprogramming includes targeting and inhibiting macrophage receptors to reprogram pro-tumorogenic TAMs into anti-tumorogenic TAMs. In some embodiments, methods for depleting TAMs using any of the bifunctional binding proteins disclosed herein are provided. TAM depletion can occur by targeting receptors important for proliferation. Targeting these receptors can promote apoptosis of pro-tumorogenic TAMs. In further embodiments, methods for inhibiting the recruitment of TAMs into the tumor microenvironment using any of the bifunctional binding proteins disclosed herein are provided. In some embodiments, methods for reprogramming, depleting, or inhibiting the recruitment of TAMs include degrading targets upregulated or expressed by TAMs using any of the disclosed bifunctional binding proteins. In some embodiments, the target proteins upregulated or expressed in the TAM include SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, or CXCR4. In other embodiments, the target proteins associated with the TAM include CCL2, CXCL12, CSF-1, or CD47.

[0125] In some embodiments, pharmaceutical compositions comprising a bifunctional binding protein described herein and a pharmaceutically acceptable carrier are provided herein.

[0126] In some embodiments, methods for treating or preventing a disease in a patient are provided herein, comprising administering to the patient a bifunctional binding protein or a pharmaceutical composition described herein. In some embodiments, the disease is an autoimmune disease, cancer or tumor, liver disease, inflammatory disorder, or hematological disorder. In some embodiments, the autoimmune disease is selected from Graves' disease, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), membranous nephropathy, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, acquired pemphigus vulgaris, immune thrombocytopenia, and Guillain-Barré syndrome. In some embodiments, the cancer or tumor is selected from breast cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, hepatocellular carcinoma, and hematological T-cell and B-cell malignancies.

[0127] In some embodiments, methods for treating or preventing diseases provided herein include an administration step comprising intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular injection of a bifunctional binding protein or pharmaceutical composition described herein.

[0128] In some embodiments, methods for treating or preventing diseases provided herein require lower doses and / or lower administration frequencies to achieve the same effect compared to the same antibody having a different glycosylation profile, and / or can be administered over a long period of time (at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or at least 12 months, at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 years), and / or do not induce an immune response to the bifunctional binding protein in the patient.

[0129] In some embodiments, kits comprising the bifunctional binding proteins of the present disclosure are provided herein. In some embodiments, the kits further provide instructions for administering the bifunctional molecule or pharmaceutical composition to an individual requiring it.

[0130] In some embodiments, the pharmaceutical compositions described herein may be administered in a single dosage form, for example, a single dosage form of the bifunctional binding protein described herein.

[0131] In some embodiments, the preferred dose of the bifunctional binding protein described herein is the amount corresponding to the minimum dose effective in producing a therapeutic effect. For example, the effective dose of the anti-TSH receptor antibody may be the amount that inhibits the TSH activity of a subject suffering from Graves' disease.

[0132] In some embodiments, the amount of the difunctional binding protein described herein administered to a patient is less than the amount indicated on the label of the drug for the same difunctional binding protein that does not have the glycosylation profile of the difunctional binding protein described herein or has a different glycosylation profile.

[0133] In some embodiments, the accumulation of the difunctional binding protein described herein, administered to a patient over a period of time, is less than the accumulation indicated on the label of the same difunctional binding protein having a glycosylation profile that is either not present or different from that of the difunctional binding protein described herein. In some embodiments, the reduced accumulation may be administered at a reduced frequency and in a reduced dose. In some embodiments, the reduced accumulation may be administered at a reduced frequency and in one or more doses that are the same as or higher than the labeled dose. In some embodiments, the reduced accumulation may be administered at a reduced frequency and in one or more reduced doses that are the same as or higher than the labeled frequency. In some embodiments, the reduced accumulation may be administered over a period of time shorter than the period over which the pharmaceutical product achieves the same level of efficacy in treatment or prevention.

[0134] In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be about 1–150 mg, about 5–145 mg, about 10–140 mg, about 15–135 mg, about 20–130 mg, about 25–125 mg, about 30–120 mg, about 35–115 mg, about 40–110 mg, about 45–105 mg, about 50–100 mg, about 55–95 mg, about 60–90 mg, about 65–5 mg, about 70–80 mg, or about 75 mg. In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be about 5–80 mg. In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be about 25–50 mg. In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may range from about 15 mg to about 35 mg.

[0135] In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be 40 mg or less, for example, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 7 mg, 5 mg, and 2 mg. In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be 80 mg or less, for example, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, and 2 mg. In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be 160 mg or less, for example, 150 mg, 140 mg, 130 mg, 120 mg, 110 mg, 100 mg, 90 mg, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, and 2 mg. In some embodiments, the amount of the bifunctional binding protein described herein in a single dose administered to a patient may be 160 mg or more, for example, 170 mg, 180 mg, 200 mg, 250 mg, and 300 mg.

[0136] In some embodiments, the bifunctional conjugating protein of the Disclosure may be administered at a frequency of every week, i.e., every 14 days. In some embodiments, the bifunctional conjugating protein of the Disclosure may be administered at a frequency lower than every 14 days, for example, every bimonthly, every 21 days, monthly, every 8 weeks, every other month, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months. In some embodiments, the bifunctional conjugating protein of the Disclosure may be administered at the same or higher frequency as every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.

[0137] In some embodiments, the administration of the bifunctional binding protein of the Disclosure may include an induction dose higher than the following doses, for example, the following maintenance dose. In some embodiments, the administration of the bifunctional binding protein of the Disclosure may include a second dose lower than the induction dose and higher than the following maintenance dose. In some embodiments, the administration of the bifunctional binding protein of the Disclosure may include the same amount of bifunctional binding protein at all doses throughout the entire treatment period.

[0138] Methods for generating bifunctional binding proteins provided herein are well known in the art. Exemplary methods for generating bifunctional binding proteins provided herein are described in International Patent Application Publications WO2019 / 002512, WO2021 / 140143, WO2021 / 140144, and WO2022 / 053673, all of which are incorporated herein by reference and illustrated herein, and any one of them may be used to generate the bifunctional binding proteins provided herein. For example, it is readily apparent to those skilled in the art that the nucleic acid sequences of known proteins (e.g., monoclonal antibodies) and newly identified proteins (e.g., monoclonal antibodies) can be readily inferred using methods known in the art, and thus introducing nucleic acids encoding any bifunctional binding protein into host cells provided herein (e.g., via expression vectors, e.g., plasmids, e.g., site-directed integration by homologous recombination) would be well within the capabilities of those skilled in the art.

[0139] In some embodiments, Leishmania host cells containing the bifunctional binding proteins described herein are provided herein. Such host cells are, in some embodiments, Leishmania tarentolae. In some embodiments, the host cells are Leishmania aethiopica cells. In some embodiments, the host cells are part of the Leishmania aethiopica species complex. In some embodiments, the host cells are Leishmania aristidesi cells. In some embodiments, the host cells are Leishmania deanei cells. In some embodiments, the host cells are part of the Leishmania donovani species complex. In some embodiments, the host cells are Leishmania donovani cells. In some embodiments, the host cells are Leishmania chagasi cells. In some embodiments, the host cells are Leishmania infantum cells. In some embodiments, the host cells are Leishmania hertigi cells. In some embodiments, the host cells are part of the Leishmania major species complex. In some embodiments, the host cells are Leishmania major cells. In some embodiments, the host cell is a Leishmania martiniquensis cell. In some embodiments, the host cell is part of the Leishmania mexicana species complex. In some embodiments, the host cell is a Leishmania mexicana cell. In some embodiments, the host cell is a Leishmania pifanoi cell. In some embodiments, the host cell is part of the Leishmania tropica species complex. In some embodiments, the host cell is a Leishmania tropica cell.

[0140] In some embodiments, methods for producing a bifunctional binding protein are provided herein, comprising culturing Leishmania host cells as described herein and isolating the bifunctional binding protein.

[0141] In some embodiments, bifunctional binding proteins produced by the methods described herein are provided herein. Methods for producing Leishmania host cells and methods for producing bifunctional binding proteins using such host cells are well known in the art. Exemplary methods are described in International Patent Application Publications WO2019 / 002512, WO2021 / 140143, WO2021 / 140144 and WO2022 / 053673, which are incorporated herein by reference in their entirety and illustrated herein, and any one of them may be used to generate Leishmania host cells and produce the bifunctional binding proteins provided herein. For example, in some embodiments, the host cells described herein are cultured using any of the standard culture techniques known in the art, including but not limited to growth in nutrient-rich media such as Brain Heart Infusion, Triptycase Soy Broth, or Yeast Extract, all containing 5 μg / ml hemin. Additionally, incubation can be carried out at 26°C in the dark for 2-3 days as a static or shaking culture. In some embodiments, the host cell culture contains a suitable selective agent. For example, in some embodiments, the monoclonal antibodies described herein are purified from the host cell culture supernatant using any of the standard purification techniques known in the art, including but not limited to protein A affinity chromatography, ion exchange chromatography, and mixed-mode chromatography.

[0142] In some embodiments, the N-glycosylation site is an N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence may be one or more asparagine residues. In further embodiments, the N-linked glycosylation consensus sequence may be one or more asparagine residues that fall within the standard consensus sequence NXS / T, the NXC motif, and a non-standard consensus motif.

[0143] In some embodiments, the N-glycosylation site is manipulated into a bifunctional protein. In some embodiments, the N-glycosylation site can be introduced by inserting an N-linked glycosylation consensus sequence into the sequence of the bifunctional protein. In some embodiments, one or more sequences encoding an N-linked glycosylation consensus sequence can be inserted into the bifunctional protein sequence at one or more sites. In some embodiments, insertion of the N-linked glycosylation consensus sequence can be performed by molecular biology techniques, such as DNA cloning, DNA extraction, bacterial transformation, transfection, chromosome integration, cell screening, cell culture, DNA sequencing, DNA synthesis, and molecular hybridization. In some embodiments, the N-glycosylation site can be introduced by nuclease-based precision gene editing tools, such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and clustered, regularly spaced, short palindromic repeats / targeted Cas9 endonucleases (CRISPR / Cas9).

[0144] In some embodiments, the bifunctional protein is an antibody. In some embodiments, the N-linked glycosylation consensus sequence may be introduced at one or more sites on the Fab of the antibody. In some embodiments, the N-linked glycosylation consensus sequence may be introduced at one or more sites on the Fc of the antibody.

[0145] In some embodiments, the bifunctional protein is tailored to specific conditions by varying the number of glycan sites, and thus optimizing its potency and kinetics for each condition. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycan sites are manipulated to the Fab or Fc of the antibody. In certain embodiments, one manipulated glycan site presenting Man3 glycan on one Fab exhibits low potency and kinetics in macrophage internal transport, while two, three, four, or five manipulated glycan sites presenting Man3 glycan on one Fab exhibit fast potency or kinetics in macrophage internal transport. In certain embodiments, one manipulated glycan site presenting M3 glycan on one Fab exhibits low potency or kinetics in target proteolysis, while two, three, four, or five manipulated glycan sites presenting M3 glycan on one Fab exhibit fast potency or kinetics in target proteolysis. In some embodiments, the bifunctional protein contains glycan sites other than Man3 glycan.

[0146] Method for producing Man3-supported proteins Methods for generating recombinant proteins are known in the art. Methods for bioconjugating host cell proteins with N-glycans are also known in the art. Exemplary methods for generating the bifunctional binding proteins provided herein are described in International Patent Application Publications WO2019 / 002512, WO2021 / 140143, WO2021 / 140144, and WO2022 / 053673. The N-glycosylation biosynthetic pathways described in these publications may be used to synthesize the Man3-supported bifunctional proteins described herein. In certain embodiments, N-glycosylation occurs in a host cell, resulting in the production of the Man3-supported bifunctional protein in the host cell's secretory pathway. In certain embodiments, the host cell may be a Leishmania host cell. The Man3-glycosylated bifunctional protein is further purified from the cell culture using one of the standard purification techniques known in the art.

[0147] Other methods for generating the bifunctional proteins provided herein can also be used. For example, the bifunctional proteins provided herein can be generated using chemical bonding or chemoenzymatic modification.

[0148] Modifications that do not substantially affect the activity of the various embodiments of the present invention are also provided within the definition of the invention as provided herein. Accordingly, the following examples are intended to illustrate the invention, but not to limit it. [Examples]

[0149] 7. Examples 7.1 Example 1: Man3 glycosylated antibody provides efficient internal delivery and lysosomal compartment targeting. PBMCs from five healthy donors were collected from buffy coats using the standard Ficol gradient method. Monocytes were isolated from PBMCs using CD14 microbeads (Miltenyi ref. 130-050-201) according to the manufacturer's instructions. Monocytes were cultured for 6 days in RPMI culture medium (Gibco, ref. 21875-034) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate (Sigma, ref. S8636), MEM non-essential amino acid final concentration 1× (Sigma, ref. 11140-03), 50 ng / ml recombinant human GM-CSF (R&D Systems, ref. 215-GM), and 20 ng / ml recombinant human IL-4 (R&D Systems, ref. 204-IL). The differentiation of monocytes into immature dendritic cells on day 6 was evaluated by flow cytometry after staining for the markers CD14, CD1a, CD83, and HLA-DR. It was confirmed that immature dendritic cells exhibited the phenotypes CD14- / low and CD1a+HLA-DR+CD83- / low.

[0150] Antibodies were prepared as follows: Monoclonal anti-TNFα antibody, HUMIRA® (AbbVie), or adalimumab variants (AS, AM) derived from Leishmania tarentolae CGP (CustomGlycan Platform), or Mabthera, were rebuffered in 30 mM MES buffer pH 6.5 using a ZebaSpin column (ThermoFischer, USA). Galactosylation and alpha-2,6-sialylation (to generate HS and AS variants) were performed in a one-pot reaction with gentle rotation at 37°C using in vitro glycosylation (IVGE, Roche Diagnostics) according to the application note. Glycosylated mAbs were purified from the reaction mixture using Protein A Sepharose (MabSelectSuRe or HiTrap MabSelect PrismA column GE Healthcare) with FPLC (Bio-Rad NGC, Germany) according to the manufacturer's recommendations. Subsequently, a desalting procedure was performed using PD-10 (Sephadex 25, Sigma, Switzerland), followed by buffer exchange to PBS pH 6 (137 mM NaCl, 2.7 mM KCl, 8.6 mM NaH2PO4, 1.4 mM Na2HPO4, Sigma, Switzerland), and then sterile filtration was performed using a 0.2 μm PES filter (ThermoFisher, USA). The antibody quality and glycosylation are shown in Table 1. N-glycans were analyzed using the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, USA) according to the manufacturer's instructions, or by procainamide (PC) labeling of PNGaseF-releasing glycans. For further N-glycan analysis, monoclonal antibodies were cleaved into F(ab')2 and Fc / 2 with IdeZ (for IgG1) or separated into heavy and light chains (for IgG4), separated on SDS-PAGE, and the bands were excised. Enzymatic release of N-glycans from the monoclonal antibodies was performed using PNGase F. After release, the glycans were directly labeled with procainamide (PC). The PC-labeled N-glycans were analyzed by HILIC-UPLC-MS with fluorescence detection coupled to a mass spectrometer.Glycans were separated using an Acquity BEH Amide column. Data processing and analysis were performed using Unifi. Glucose units were assigned to the retention time of a procainamide-labeled dextran ladder. Glycan structures were assigned based on their m / z value and retention time. Glycan morphology and relative percentage were calculated based on peak area. SE-HPLC analysis was performed on a MabPac (ThermoFischer, USA) column at a concentration of 1 μg / μL, according to the manufacturer's instructions. Endotoxin levels were less than 0.2 EU / mg (Endosafe®). Antibodies were labeled with pHrodo dye (pHrodo iFL Red STP Ester [amine-reactive], ThermoFischer, ref. P36011) according to the manufacturer's instructions. Table 1 shows the main N-glycan structures presented by the antibodies used.

[0151] [Table 1]

[0152] Immature dendritic cells were incubated for 6 hours with pHrodo-labeled antibody at 10 μg / ml. Under some conditions, an Fc receptor blocking reagent (Human TruStain FcX, biolegend ref 640922) was added 30 minutes prior to the addition of the pHrodo-labeled antibody, according to the manufacturer's recommendations. Six hours after incubation with the pHrodo-labeled antibody, cells were acquired by flow cytometry and the pHrodo fluorescence intensity was recorded. Since pHrodo fluorescence is activated by acidic pH, it indicates that internally translocated molecules have reached the late endosome and lysosome compartments. Therefore, the pHrodo fluorescence intensity correlates with the amount of molecules that have internally translocated and routed into the lysosomal pathway.

[0153] Figure 1 shows that Man3 adalimumab (AM) underwent significantly greater internal migration than adalimumab variants presenting different N-glycans. HUMIRA® (HM) and sialylated HUMIRA® (HS), as well as sialylated (defucosylated) adalimumab (AS), showed similarly low levels of internal migration compared to Mabthera (MbT), the anti-CD20 antibody used as a reference. Mabthera displays the same major N-glycan structure (A2F) as HUMIRA®. This indicates that the Man3 glycan presented by the antibody leads to internal migration and targeting of the internally migrated antibody to lysosomal degradation. This internal migration was not dependent on defucosylation, as defucosylated but not sialylated adalimumab (AS) underwent less internal migration than HUMIRA® or Mabthera. This internal transfer of Man3 antibodies is independent of these receptors, as blocking of Fc receptors did not reduce the internal transfer of AM antibodies. These data suggest that specific receptors that bind to Man3 structures are expressed by dendritic cells and mediate routing to efficient endocytosis and lysosomal degradation pathways.

[0154] 7.2 Example 2: Man3 glycosylated Fab antibody results in efficient internal translocation and lysosomal targeting in human macrophages. The antibodies were labeled with pHrodo dye (pHrodo iFL Red STP Ester [amine-reactive], ThermoFisher, ref. P36011) according to the manufacturer's instructions. Since the fluorescence of pHrodo is activated at low pH, it is possible to visualize protein internal translocation and targeting to the lysosomal pathway.

[0155] The pHrodo labeling degree (DOL) of each antibody was determined as follows: Antibodies were diluted 1:2 with denaturing buffer and analyzed using Nanodrop at wavelengths of 280 nm and 560 nm (A280 and A560). Protein concentration and pHrodo DOL were calculated as follows:

[0156]

number

[0157]

number

[0158] MW is the molecular weight of the antibody used: 144,000 g / mol. λmax is the absorbance measured at 560 nm. ε dye has an extinction coefficient of 65,000 M. -1 cm -1 The dilution ratio is 2.

[0159] Table 2 shows the antibodies used in this experiment, along with their major N-glycan structures on the Fc or Fab moieties. Peripheral blood mononuclear cells (PBMCs) from human donors were collected from the buffy coat using the standard Ficol gradient method. Monocytes were isolated from PBMCs using the EasySep human monocyte isolation kit (StemCell ref. 19359). Monocytes were placed in a tissue culture dish (Falcon, ref. 353003) in macrophage differentiation medium at a rate of 5 × 10⁴. 5 Cells were seeded at a concentration of cells / ml. The macrophage differentiation medium was RPMI-1640 supplemented with L-glutamine (Sigma, R8758), 10% fetal bovine serum (FBS, PanBiotech, P305500), 1% Pen / Strep (Sigma, P4333), and 50 ng / ml hM-CSF (Peprotech, 300-25). Cells were incubated in a cell culture incubator at 37°C and 5% CO2. On day 3, half of the cell culture medium was replaced with fresh macrophage differentiation medium. After a total of 6-7 days of differentiation, macrophages were harvested. Cell culture plates were rinsed with PBS 1× (Sigma, D8537) and incubated in PBS 1× + 1 mM EDTA (Invitrogen, 15575-020) on ice for 15 minutes. Macrophages were harvested by cell scraping and pipetting.

[0160] [Table 2]

[0161] The phenotype of macrophages was confirmed by flow cytometry after staining for CD1a, CD14, CD206, and CD163. At least 90% of the cells were CD14+, CD206+, and CD163+, confirming that the macrophages were properly differentiated.

[0162] Macrophages were resuspended in internal distribution medium and placed in 96-well flat-bottom cell culture plates. 5 Cells were seeded in a total volume of 0.1 ml per well. The internal distribution medium was L-glutamine-containing RPMI-1640 supplemented with 2% FBS. Macrophages were pre-incubated at 37°C for 30 minutes with pooled human immunoglobulin (IVIg, Hizentra obtained from a pharmacy) at a final concentration of 2 mg / ml, and then pHrodo-labeled antibody was added at a final concentration of 1 μg / ml. Macrophages were incubated with pHrodo-antibody + IVIg at 37°C for 3 hours. Subsequently, the cell culture was washed, macrophages were harvested using trypsin treatment, and immediately obtained by flow cytometry.

[0163] Standard flow cytometry software was used to analyze the mean fluorescence intensity (MFI) of pHrodo in a gated single-cell population. MFI values ​​were adjusted for pHrodo DOL. The adjusted MFI values ​​were then normalized for each donor relative to Humira® (adalimumab) (H-A2F). Figure 2 shows macrophage internal migration data. Internal migration of Mabthera (rituximab) (M-A2F) and Humira® (H-A2F) was similar. The adalimumab variant (A-M3) presenting M3 glycan on the Fc moiety also internalized similarly to Humira® and Mabthera. Deglycosylation of Humira® and A-M3 (PNGase) resulted in approximately 60% reduction in internal migration. Deglycosylation disrupts the interaction of the Fc gamma receptor and the N-glycan receptor, thus indicating baseline internal migration levels. Humira® and Mabthera were internalized via the Fc gamma receptor on macrophages. In contrast, the A8486-M3 mutant, which presents M3 glycans on its Fab moiety, was internalized to macrophages at high levels in all donors. Internalization of A8486-M3 was 17 to 39 times higher than Humira® baseline, depending on the donor. The A8486-A2G2S2 mutant, which has 70% sialic acid-terminal N-glycans on its Fab moiety, was also internalized 6 times more efficiently than Humira® baseline.

[0164] These results indicate that M3 glycans presented on the Fab portion of antibodies lead to robust internal migration and targeting of the lysosomal pathway by human macrophages. Man3-mediated lysosomal targeting was more potent than sialic acid-mediated lysosomal targeting. These data also show that the position of the Man3 glycan enables efficient internal migration. Fab Man3 glycans lead to efficient internal migration by human macrophages.

[0165] 7.3 Example 3: Man3 glycosylated Fab antibodies result in efficient internalization and lysosomal targeting in human dendritic cells. Peripheral blood mononuclear cells (PBMCs) from two human donors were collected from buffy coats using standard Ficoll gradient methods. Monocytes were isolated from PBMCs using an EasySep human monocyte isolation kit (Stemcell ref. 19359C) according to the manufacturer's instructions.

[0166] The purified monocytes were resuspended in dendritic cell differentiation medium and seeded at 3×10 5 cells / cm 2 in a T75 cell culture flask. The dendritic cell differentiation medium contained RPMI-1640 supplemented with L-glutamine (Sigma, R8758), 10% fetal bovine serum (FBS, PanBiotech, P305500), 1% Pen / Strep (Sigma, P4333), 50 ng / ml (500 U / ml) rhGM-CSF (Peprotech, 300-03-20UG), and 50 ng / ml rhIL-4 (Peprotech, 200-04-20UG). Cells were incubated at 37 °C, 5% CO2 for 3 days. On day 3, half of the culture medium was replaced with fresh dendritic cell differentiation medium. On day 5, the medium was replaced with fresh dendritic cell differentiation medium supplemented with 50 ng / ml of TNFα (Peprotech, AF-300-01A) and 10 ng / ml of IL-1β (Peprotech, 200-01B-10UG) and IL-6 (Peprotech, 200-06-5UG). Cells were cultured for an additional 2 days (total differentiation time was 7 days). The cell cultures were washed and incubated on ice for 30 minutes in PBS 0.02% EDTA. Adherent cells were harvested by pipetting and washed with PBS.

[0167] Dendritic cell differentiation was confirmed by flow cytometry after staining for CD1a, CD14, CD206, and CD163 using the following antibodies: Alexa Fluor647 anti-human CD163 (Biolegend 333619, 1:200), PerCP / Cyanine5.5 anti-human CD1a (Biolegend 300129, 1:200), PE anti-human CD14 (Biolegend 301805, 1:400), and Alexa Fluor488 anti-human CD206 (Biolegend 321113, 1:200). Both donors showed at least 80% differentiated dendritic cells, as evaluated by CD1a+ and CD206+.

[0168] pHrodo-labeled antibodies were prepared as described in Example II. The antibodies, their glycan structures, and pHrodo DOL are shown in Table 2.

[0169] Dendritic cells were placed in a flat-bottomed 96-well plate in a 0.5 × 10⁶ arrangement. 6 Cells were seeded in wells. Dendritic cells were pre-incubated with 1 mg / ml IVIg (Hizentra, available from a pharmacy) at 37°C for 30 minutes, and then pHrodo-labeled antibody was added to a final concentration of 10 μg / ml. The dendritic cells were incubated with the antibody for 6 hours. The cells were then harvested and obtained by flow cytometry.

[0170] Flow cytometry data were analyzed using standard flow cytometry software. The percentage of cells that underwent efficient internal migration was defined by gating cells with high levels of pHrodo internal migration (high pHrodo population), as shown in Figure 3A. Figure 3B shows the percentage of high pHrodo cells for the different glycosylation mutants tested. The data indicate that Humira® (H-A2F) and Mabthera (M-A2F) were internally migrated by dendritic cells with low efficiency, while the A8486-M3 mutant was internally migrated highly. The A-M3 mutant showed internal migration higher than Humira® or Mabthera, but significantly lower than the A-8486-M3 mutant. Interestingly, the A8486-A2G2S2 mutant showed low internal migration.

[0171] Overall, these data from Examples I-III demonstrate that Man3 glycans yield more potent internal translocation and lysosome targeting in both macrophages and dendritic cells compared to classical glycan structures presented by standard IgG antibodies such as Humira® and Mabthera (A2F structure). These data also show that particularly efficient internal translocation can be achieved by selecting the location of Man3 glycans—Man3 glycans presented on Fab fragments yielded much more potent internal translocation than Man3 glycans on Fc fragments (on canonical N297). Finally, these data highlight that different glycan structures result in different target cell types and different efficiencies of internal translocation and degradation. Sialized glycans are internally translocated by macrophages but not in dendritic cells. Therefore, by adapting glycan structure and its location, it is possible to direct proteins to specific cell types and thus modulate the potency of internal translocation and degradation, as well as the functional outcomes of protein degradation.

[0172] 7.4 Example 4: Internal transfer and lysosomal targeting of Man3 glycosylated Fab antibodies in human macrophages are mediated by the mannose receptor and regulated by the presented Man3 loading. To evaluate whether the internal migration of antibodies presenting Man3 glycans on Fab is regulated by the amount of Man3 presented, experiments were conducted using glycan variants in which one, two, or three glycan sites were inserted into the Fab fragment. The antibodies were purified and labeled with pHrodo as described in Example I. Table 3 shows the characteristics of the antibodies studied. All antibodies showed an aggregation level of less than 5% by size exclusion HPLC, a purity of more than 94% by reduced polyacrylamide gel electrophoresis, and an endotoxin level of less than 1 EU / mg (LAL assay). The analytical methods for glycans and proteins are described in Example I.

[0173] [Table 3]

[0174] Human monocyte-derived M2 macrophages were generated as described in Example II. The phenotype of the macrophages was confirmed by flow cytometry after staining for CD1a, CD14, CD206, and CD163. Typically, the purity of the macrophages was over 80%, and the average percentage of CD206+ macrophages was over 80% (Figure 5).

[0175] Macrophages were resuspended in internal distribution medium and placed in 96-well flat-bottom cell culture plates. 5Cells were seeded in a total volume of 0.1 ml per well. The internal distribution medium was L-glutamine-containing RPMI-1640 supplemented with 2% FBS. Macrophages were pre-incubated at 37°C for 30 minutes at a final concentration of 2 mg / ml with pooled human immunoglobulin (IVIg, Hizentra obtained from a pharmacy), followed by the addition of pHrodo-labeled antibody at a final concentration of 1 μg / ml. Macrophages were incubated with pHrodo-antibody + IVIg at 37°C for 3 or 24 hours. Under some conditions, macrophages were incubated with mannan at 1 mg / ml for 30 minutes before the addition of pHrodo-labeled antibody. The cell cultures were then washed, macrophages were harvested using trypsin treatment, and immediately acquired by flow cytometry. Mean fluorescence intensity (MFI) of pHrodo for a gated single-cell population was analyzed using standard flow cytometry software. Adjusted MFI values ​​were generated by adjusting the MFI values ​​to pHrodo DOL. The adjusted MFIs of adalimumab variants (A-84 and A-84.86) were normalized to match the H-A2F reference (normalized MFI). The adjusted MFI of 5C9-84.86.162-M3 was normalized to the 5C9-IgG4-A2F unmodified reference antibody.

[0176] Figure 6 and Table 4 show the adjusted MFI data. After 3 hours, A-84.86-M3 showed higher internal migration than the H-A2F reference. Similarly, 5C9-84.86.162-M3 showed higher internal migration than the 5C9-A2F reference. The H-A2F antibody and 5C9-A2F antibody showed very similar internal migration baselines, indicating that IgG1 (adalimumab) or IgG4 isotype (5C9) does not significantly affect the baseline of macrophage internal migration under these conditions. These data indicate a tendency towards correlation between the M3 glycan load presented by the antibody and the potency of internal migration, with A-84-M3, with one modified glycan site per Fab, showing the lowest internal migration, and 5C9-84.86.162-M3, with three modified glycan sites per Fab, showing the highest internal migration. These data, along with the data from Example II, show that the efficacy of internal transfer of A-8486-M3 by human monocyte-derived M2 macrophages ranged from 2 to over 20 times compared to H-A2F, indicating variability in efficacy depending on the donor. At 3 hours, none of the non-M3 antibodies (A-84.86-A2G2, A-84.86-A2, and A-84.86-A2GalNAc2) showed greater uptake than H-A2F (Figure 6A), indicating that macrophages selectively internalize Man3-presenting antibodies. Importantly, the internal transfer of A-8486-M3 decreased with the addition of mannan, a mannose receptor competing ligand, at 1 mg / ml (Sigma, ref. M7504), but the uptake of H-A2F was not affected by mannan (Table 4). These data indicate that the internal transfer of M3 antibodies by M2 macrophages is mediated by the mannose receptor (CD206). At 24 hours, the level of internalization was elevated, consistent with the accumulation of the pHrodo signal due to the cumulative effect of the internalization cycle. Interestingly, all M3 antibodies, including A-84-M3, showed higher internalization than baseline H-A2F and 5C9-IgG4-A2F, indicating that M3 Fab antibodies with even one modified glycosylation site can be specifically recognized and internally transported to M2 macrophages (Figure 6B).The A-8486-A2G2 antibody also underwent internal transfer over 24 hours, albeit barely (but statistically significant) compared to H-A2F.

[0177] [Table 4] Table 4 shows the mean-normalized pHrodo MFI from two macrophage donors under specified conditions. Macrophages were incubated with pHrodo-labeled antibodies for 24 hours, with or without mannan.

[0178] 7.5 Example 5: Man3 glycosylated Fab antibody strongly depletes circulating antigens in vivo. To evaluate the circulating extracellular antigen depletion ability of antibodies produced by CGP and displaying the Man3-terminal glycan (M3 structure), experiments using rats were planned. Rats were injected with the antigen and either an antibody displaying the M3 glycan structure on Fab or an antigen-specific control antibody. To measure the degree of antigen depletion from peripheral blood compartments, the level of circulating antigen in the serum of treated animals was quantified over time. Table 5 shows the characteristics of the antibodies included in this study. All antibodies showed agglutination levels of less than 5% by size exclusion HPLC and purity of over 94% by reduced polyacrylamide gel electrophoresis. All glycosylated antibodies maintained high binding affinity to the antigen "HCA202".

[0179] [Table 5]

[0180] At the start of the experiment, 180-220g Wistar female rats (Janvier Labs, St Berthevin, France, ref. RjHan:WI) were intravenously bolus-injected with the anti-adalimumab Fab fragment HCA202 (Biorad, ref. HCA202) (antigen) at a dose of 0.5 mg / kg, 0.5 ml / rat. The HCA202 compound was subjected to an endotoxin removal step using Pierce® High Capacity Endotoxin Removal Spin Columns (Thermofisher, ref. 88274) prior to injection. After 15 minutes, the rats were injected with antibody (Tables 4 and 5) or PBS. Blood samples were collected by puncture from the jugular vein at 15-30 minutes, 1 hour, 6 hours, and 24 hours after antibody injection. Terminal blood samples were collected from the abdominal aorta after 48 hours. After allowing the blood samples to coagulate at room temperature for 30 minutes, serum was collected by centrifugation.

[0181] Total HCA202 levels (bound antibody + free HCA202) were measured by ELISA. Anti-Penta-His antibody (Qiagen, Ref. 34660) was coated onto a 96-well ELISA plate at 5 μg / ml in coating buffer (PBS pH 7.4, final composition: 8 mM Na-Phosphate, 8 mM K-Phosphate, 0.15 M NaCl, 10 mM KCl) overnight at 4°C. Because HCA202 has a histidine tag at the C-terminus of its heavy chain, this antibody allows for antibody binding and capture of free HCA202. The plate was washed three times with washing buffer (PBST = PBS containing 0.05% v / v Tween-20). Blocking buffer (2% (w / v) bovine serum albumin (BSA) in PBST) was added to each well, and the plate was incubated at room temperature for 1–3 hours. Seven checkpoint curves ranging from 500 ng / ml to 0.7 ng / ml were prepared on separate dilution plates using a 1:3 dilution. To this end, undiluted normal rat Wistar serum was spiked with 5 μg / ml HCA202 and 3-fold molar excess adalimumab (Humira). The spiked serum was incubated at room temperature for 10 minutes to form adalimumab / HCA202 immune complexes. The spiked serum was diluted 10-fold (MRD10) by adding diluent B (2% (w / v) bovine serum albumin (BSA) in PBST). 1:3 serial dilutions of the immune complex standard curves were performed using diluent B. Test samples were processed similarly. Test serum samples were diluted 10-fold in dilution plates (to obtain MRD10 samples) using diluent B. To ensure the signal fell within the linear range of the standard calibration curve, MRD10 samples were further diluted with diluent A (1 / 10 Wistar rat pooled serum diluted with 2% BSA + 0.05% PBST) as needed. After blocking, the ELISA plate was washed three times with washing buffer. Calibration material and diluted samples were added in double doses to the ELISA plate and incubated at room temperature for 1 hour. The ELISA plate was washed three times with washing buffer, and 1000 ng / ml of Humira solution was added to each sample. The ELISA plate was incubated at room temperature for 1 hour. The plate was washed three times with washing buffer.The detection antibody solution was prepared by diluting goat anti-human kappa LC-HRP (Thermofisher, ref. A18853) to 1:5000 with diluent B. The detection antibody solution was added to an ELISA plate and incubated in the dark at room temperature for 1 hour. The ELISA plate was then washed three times with wash buffer, and elucidated by adding TMB (3,3',5,5'-tetramethylbenzidine) substrate and subsequently quenching with H2SO4. The ELISA plates were read at 450 and 650 nM using a plate reader such as BioTek Synergy H1. Data analysis was performed using standard software such as Gen5 (Biotek).

[0182] Antibody levels in serum samples can be quantified by ELISA. This assay consists of a step of coating the sample with human TNFα to capture adalimumab and adalimumab variants present in the sample. Detection can be performed via an anti-human gamma HC-specific HRP-tagged detection antibody. Therefore, this assay quantifies only free antibodies (antibodies with at least one Fab arm that are not bound to HCA202). Briefly, recombinant human TNF-α (Peprotech, ref. AF-300-01A) is coated onto a 96-well ELISA plate overnight in PBS at pH 7.4 at 4°C, usually at 1 μg / ml. The blocking buffer, dilution buffers A and B, and wash buffer are the same as those used in the HC202 ELISA. The plate is washed three times and blocked with the blocking buffer as described in the HCA202 ELISA. Typically, seven calibration curves ranging from 333.3 ng / ml to 0.5 ng / ml at a 1:3 dilution are prepared by spiking 1 μg / ml adalimumab into pooled Wistar rat serum diluted 10-fold with Dilution Buffer B (minimum required 10-fold dilution, MRD10). Test serum samples are also diluted with Dilution Buffer B (minimum MRD10 sample). The diluted samples and standard calibration curve samples are transferred to an ELISA plate and incubated at room temperature for 1 hour after the blocking step. The plate is then washed three times and the detection antibody solution is added. The detection antibody solution can be prepared, for example, by diluting goat anti-human IgG (γ-chain specific)-HRP (Sigma, ref. A6029) antibody (usual dilution ratio 1:10,000) with Dilution Buffer B. The ELISA plate is protected from light with the detection antibody and incubated at room temperature for 1 hour, usually. The plate is then washed three times and eluted by adding the TMB substrate as described in the HCA202 ELISA.

[0183] Figure 7 shows the data obtained for HCA202 (antigen) levels. Table 6 shows the HCA202 depletion data. When no antibody was injected (PBS condition), HCA202 decayed slowly over 48 hours, as expected with the Fab fragment. Treatment with H-A2F (unmodified adalimumab, Humira®) resulted in increased HCA202 levels at 24 and 48 hours. Treatment with A-M3 and A-8486-A2G2S2 also resulted in increased HCA levels compared to PBS treatment (PBS resulted in 72% depletion from C-zero, while H-A2F, A-M3, and A-8486-A2G2S2 resulted in 52-63% depletion at 6 hours). C-zero is the theoretical concentration of (HCA) in serum that should be achieved immediately after injection, considering immediate and uniform whole blood distribution. This indicates that these antibodies do not have a depletion effect. In contrast, injection of A-84-M3 and A-8486-M3, which present exposed Man3 glycans, resulted in complete depletion of HCA202 within 1 hour (99% and 94% depletion, respectively) compared to non-depleted antibodies and PBS, and was undetectable after 6 hours (Table 6).

[0184] [Table 6]

[0185] These data highlight that antibodies displaying M3 glycans on Fab fragments exhibit remarkably high efficacy in removing circulating antigens from the bloodstream very quickly. In contrast, M3 glycans presented within Fc fragments do not cause active depletion.

[0186] 7.6 Example 6: Man3 glycosylated Fab antibody targets the liver in vivo. To study the in vivo distribution of antibodies that display mannose-terminal glycans (M3 structures), a mouse study was designed using fluorescently labeled antibodies that display M3 or a control glycan, along with in vivo and ex vivo tomographic imaging.

[0187] The antibody was labeled at a dose of 1 mg / mL in at least 1 mL of volume using a CF750 labeling kit (Biotium, ref. 92221) according to the manufacturer's instructions. After labeling, the degree of labeling (DOL) was measured. Since the DOL ranged from 2.7 to 5.2, the antibody was considered to be similarly labeled. Table 5 of Example V shows the characteristics of the antibody studied. 5 mg / kg of CF750-labeled antibody was injected (intravenous bolus) into SKH1 immunoresponsive hairless mice (Charles River Laboratories, ref. Crl:SKH1-hr) 5-6 weeks after the start of the experiment. The mice were imaged using FMT2500™ in vivo fluorescence imaging system (PerkinElmer), which collected both 2D surface fluorescence reflectance (FRI) and 3D fluorescence tomography (FMT) image datasets. Using a FMT (NIR excitation laser 745 nm / emission 770-800 nm), each animal was scanned twice in the supine position (thoracic region + abdominal region) at 0.25 hours, 1 hour, 3 hours, 6 hours, 24 hours, and 48 hours. The collected fluorescence data were reconstructed using FMT2500 system software (TrueQuant V2.0, PerkinElmer) to quantify the three-dimensional fluorescence signal of the entire animal body. Three-dimensional regions of interest (ROIs) including biological regions related to the thoracic, abdominal, and hepatic regions were visualized. The amount and dose percentage of labeled antibody in each ROI were determined at each time point. At 6 and 48 hours, the thyroid (including trachea), lungs, heart, liver, spleen, and kidneys were resected and subjected to FMT imaging.

[0188] Figure 8 shows FMT image data of the chest and liver ROIs for each antibody over time. Table 7 shows FMT image data acquired 6 hours after sampling of the organs. The unmodified control antibody H-A2F (adalimumab, Humira®) showed a distribution profile with low signal levels distributed in the liver region. The signal in the liver region did not increase over time (Figure 8). At 6 hours, only 6% of the injected dose of H-A2F was present in the liver (Table 7), but it was detected in all other organs. This distribution pattern is consistent with normal human IgG and antibodies that are widely distributed and still mainly present in the blood. Antibody A-M3 showed a similar distribution profile to H-A2F and had a broad organ distribution. The A-8486-M3 antibody showed a rapid and preferential distribution to the liver region (Figure 8). At 6 hours, 19% of the injected dose of A-8486-M3 was present in the liver (Table 7). The A-8486-M3 antibody was not present in the thyroid, lungs, heart, or kidneys, and was only detectable in the spleen. This distribution pattern is characteristic of antibodies that are not present in the blood. This is consistent with data showing that A-8486-M3 is internally translocated by the mannose receptor on M2 macrophages (Example IV) and exhibits very rapid and potent depletion of the circulating antigen (Example V). These data support the hypothesis that the antibody presenting the M3 structure on the Fab fragment is recognized by the mannose receptor (CD206), which triggers internal translocation and routing into the lysosomal degradation pathway.

[0189] [Table 7] The table shows the mean (N=3)% of the fluorophore dose injected into the harvested organs at 6 hours.

[0190] Throughout this application, various publications are referenced. The disclosures of these publications are incorporated herein by reference, in whole, to further illustrate the latest art to which the present invention belongs.

[0191] Although the present invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. References [1] O'Connor et al. “CCL2-CCR2 signaling in disease pathogenesis.” Endocrine, Metabolic & Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune, Endocrine & Metabolic Disorders) 15.2 (2015): 105-118. [2]Gschwandtner et al. “More than just attractive:how CCL2 influences myeloid cell behavior beyond chemotaxis.” Frontiers in immunology 10(2019):2759. [3]Liu et al. “Sox9 regulates self-renewal and tumorigenicity by promoting symmetrical cell division of cancer stem cells in hepatocellular carcinoma.” Hepatology 64.1(2016):117-129. [4]Jo et al.Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.J Biol Chem 275(2000):8806-8011. [5]Kim et al. “Targeting wnt signaling for gastrointestinal cancer therapy:Present and evolving views.” Cancers 12.12(2020):3638. [6]Ciardiello et al.“Clinical development of therapies targeting TGFβ:current knowledge and future perspectives.” Annals of Oncology(2020). [7]Zhang et al.“Tumor-associated macrophages:A promising target for a cancer immunotherapeutic strategy.” Pharmacological Research 161(2020):105111. [8]Cummings RD,Schnaar RL,Esko JD,Drickamer K,Taylor ME.Principles of Glycan Recognition.2017.In:Varki A,Cummings RD,Esko JD,Stanley P,Hart GW,Aebi M,Darvill AG,Kinoshita T,Packer NH,Prestegard JH,Schnaar RL,Seeberger PH,editors.Essentials of Glycobiology[インターネット].3rd ed.Cold Spring Harbor(NY):Cold Spring Harbor Laboratory Press;2015-2017 [9]Drickamer K,Taylor ME.Recent insights into structures and functions of C-type lectins in the immune system.Curr Opin Struct Biol.2015 Oct;34:26-34.doi:10.1016 / j.sbi.2015.06.003.Epub 2015 Jul 7.PMID:26163333;PMCID:PMC4681411.

[10] Taylor ME,Drickamer K.Mammalian sugar-binding receptors:known functions and unexplored roles.FEBS J.2019 May;286(10):1800-1814.doi:10.1111 / febs.14759.Epub 2019 Feb 6.PMID:30657247;PMCID:PMC6563452.

[10] Taylor ME,Drickamer K.Structural insights into what glycan arrays tell us about how glycan-binding proteins interact with their ligands.Glycobiology.2009 Nov;19(11):1155-62.doi:10.1093 / glycob / cwp076.Epub 2009 Jun 15.PMID:19528664;PMCID:PMC2757572.

[11] Lepenies B,Lee J,Sonkaria S.Targeting C-type lectin receptors with multivalent carbohydrate ligands.Adv Drug Deliv Rev.2013 Aug;65(9):1271-81.doi:10.1016 / j.addr.2013.05.007.Epub 2013 May 30.PMID:23727341.

[12] Allavena P,Chieppa M,Monti P,Piemonti L.From pattern recognition receptor to regulator of homeostasis:the double-faced macrophage mannose receptor.Crit Rev Immunol.2004;24(3):179-92.doi:10.1615 / critrevimmunol.v24.i3.20.PMID:15482253. This application provides the invention in the following embodiments. (Aspect 1) The first part specifically binds to the target protein, and the following structure (chemical 1) TIFF0007911007000016.tif50165 A bifunctional binding protein comprising a second portion containing a glycan, The bifunctional binding protein is characterized in that the squares represent N-acetylglucosamine residues, the black striped circles represent mannose residues, and X represents an amino acid residue of the bifunctional binding protein. (Aspect 2) The bifunctional binding protein according to embodiment 1, wherein the glycan further comprises a fucose residue in the N-acetylglucosamine amine directly bound to X. (Aspect 3) The bifunctional binding protein according to embodiment 1, wherein X is an asparagine residue in the bifunctional binding protein. (Aspect 4) The bifunctional binding protein according to Embodiment 1, wherein the glycan has the structure described in Embodiment 1. (Aspect 5) The aforementioned target proteins include HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, Frizzled receptor, Wnt, LRP5 / 6, CD38, CD73, TGF-β, Bombecin®, CAIX, CD13, CD44, v6, CXCR4, ErbB-2, Her2, Empurin, Endoglin, EpCAM, EphA2, FAP-α, Folic Acid®, GRP78, IGF-1®, Matryptase, Mesothelin, sMET / HGFR, MT1-MMP, MT6-MMP, Mu The bifunctional binding protein according to Embodiment 1, comprising c-1, PSCA, PSMA, Tn antigen, and uPAR, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin and ganglioside GM1, GD3, GQ1B, MOG, SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, CD47, or misfolded light chain and misfolded transthyretin. (Aspect 6) The second portion includes a mannose 3 receptor, a differentiation cluster 206 (CD206) receptor, a DC-SIGN (differentiation cluster 209 or CD209) receptor, a C-type lectin domain family 4-member G (LSECTin) receptor, and a macrophage-inducible Ca 2+ A bifunctional binding protein according to embodiment 1, which specifically binds to a lectin receptor dependent on lectin (Mincle). (Aspect 7) The bifunctional binding protein according to embodiment 1, wherein the second portion specifically binds to any endocytosis carbohydrate-binding receptor that recognizes the Man3GlcNAc2 structure. (Pattern 8) The bifunctional binding protein according to embodiment 1, wherein the second portion includes a glycan structure. (Aspect 9) The bifunctional binding protein according to embodiment 8, wherein the glycan structure is mannose 3. (Aspect 10) The bifunctional binding protein according to embodiment 1, wherein the first portion includes a heavy chain variable region or a light chain variable region. (Aspect 11) The bifunctional binding protein according to embodiment 1, wherein the first portion includes the Fab region of a monoclonal antibody. (Aspect 12) An antibody, a bifunctional binding protein as described in Embodiment 1. (Aspect 13) The bifunctional conjugating protein according to embodiment 12, wherein the antibody is a monoclonal antibody or a polyclonal antibody. (Aspect 14) The bifunctional binding protein according to embodiment 12, wherein the antibody is a recombinant. (Aspect 15) The bifunctional conjugating protein according to embodiment 12, wherein the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. (Aspect 16) The bifunctional binding protein according to embodiment 12, wherein the antibody has a glycan-to-protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1. (Aspect 17) The bifunctional binding protein according to embodiment 12, wherein the antibody is glycosylated at a predetermined specific residue. (Aspect 18) The bifunctional binding protein according to embodiment 1, wherein the bifunctional binding protein is a self-antigen. (Aspect 19) The bifunctional binding protein according to any one of embodiments 1 to 12, wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the glycan of the bifunctional binding protein has the structure of the glycan according to embodiment 1. (Aspect 20) The bifunctional binding protein according to embodiment 1, wherein the target protein is a cell surface molecule or a non-cell surface molecule. (Aspect 21) The bifunctional binding protein according to embodiment 20, wherein the cell surface molecule is a receptor. (Aspect 22) The bifunctional binding protein according to embodiment 20, wherein the non-cell surface molecule is an extracellular protein. (Aspect 23) The bifunctional binding protein according to embodiment 22, wherein the extracellular protein is an autoantibody, hormone, cytokine, chemokine, blood protein, or central nervous system (CNS) protein. (Aspect 24) A bifunctional binding protein according to any one of embodiments 20 to 23, wherein the target protein is bound by the first portion. (Aspect 25) A method for delivering a target protein to a liver macrophage, comprising contacting the target protein with a bifunctional binding protein according to any one of embodiments 1 to 22 under conditions that mediate the endocytosis of the target protein. (Aspect 26) A method for degrading a target protein, comprising contacting the target protein with a bifunctional binding protein according to any one of embodiments 1 to 24 under conditions that mediate lysosomal degradation of the target protein by a host cell. (Aspect 27) The method according to embodiment 26, wherein the target protein is upregulated in cancer or is involved in cancer progression. (Aspect 28) The method according to embodiment 27, wherein the target protein upregulated in cancer or involved in cancer progression includes HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2, Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, LILRB1, LILRB2, CD38, CD73, or TGF-β. (Aspect 29) The method according to embodiment 28, wherein the target protein is an autoantibody for an autoimmune disease. (Aspect 30) The method according to embodiment 28, wherein the target protein is an autoantigen of an autoimmune disease. (Aspect 31) The method according to embodiment 29, wherein the autoantibody for the autoimmune disease is an antibody that binds to MOG, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin, or ganglioside GM1, GD3, or GQ1B. (Aspect 32) The method according to embodiment 26, wherein the target protein is upregulated or expressed in neurodegenerative diseases. (Aspect 33) The method according to embodiment 32, wherein the target protein upregulated or expressed in neurodegenerative diseases is α-synuclein, amyloid-beta, or a complement cascade component. (Aspect 34) The method according to embodiment 26, wherein the host cell is a bone marrow cell, an immune cell, an endothelial cell, a parenchymal cell, or an epithelial cell. (Aspect 35) The method according to embodiment 34, wherein the immune cells are dendritic cells, macrophages, monocytes, microglia, granulocytes, or B lymphocytes. (Aspect 36) The method according to embodiment 26, wherein the host cell is any cell. (Aspect 37) The method according to aspect 26, wherein the bifunctional binding protein enhances the degradation of the target protein compared to the degradation of the target protein in the presence of a bifunctional binding protein containing a different second portion. (Aspect 38) The method according to embodiment 26, wherein the decomposition is mediated by endocytosis or phagocytosis. (Aspect 39) A pharmaceutical composition comprising a bifunctional binding protein according to any one of embodiments 1 to 24 and a pharmaceutically acceptable carrier. (Approach 40) A method for treating or preventing a disease in a patient, comprising administering to the patient a bifunctional binding protein according to any one of embodiments 1 to 24, or a pharmaceutical composition according to embodiment 39. (Aspect 41) The method according to embodiment 40, wherein the disease is an autoimmune disease, cancer or tumor, liver disease, inflammatory disorder, or blood coagulation disorder. (Aspect 42) The method according to embodiment 41, wherein the autoimmune disease is selected from MOGAD (myelin oligodendrocyte glycoprotein antibody-associated disease), Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, acquired pemphigus vulgaris, immune thrombocytopenia, autoimmune encephalitis, and Guillain-Barré syndrome. (Aspect 43) The method according to embodiment 41, wherein the cancer is selected from lung cancer, breast cancer, gastric cancer, colorectal cancer, bladder cancer, malignant melanoma, multiple myeloma, and Hodgkin lymphoma. (Aspect 44) The method according to aspect 43, wherein the treatment includes reprogramming tumor-associated macrophages (TAMs) by administering the bifunctional binding protein under conditions that mediate endocytosis of a target protein. (Aspect 45) The method according to embodiment 44, wherein the target protein is upregulated or expressed in the TAM. (Aspect 46) The method according to embodiment 45, wherein the target protein upregulated or expressed in the TAM includes SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47. (Aspect 47) The method according to embodiment 40, wherein the administration step includes intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection. (Aspect 48) A kit comprising a bifunctional binding protein according to any one of embodiments 1 to 24, or a pharmaceutical composition according to embodiment 39, and instructions for administering the bifunctional molecule or the pharmaceutical composition to an individual requiring it. (Aspect 49) The kit according to embodiment 48, wherein the bifunctional binding protein or the pharmaceutical composition is present in one or more unit doses. (Appearance 50) A bifunctional binding protein that (i) specifically binds to a target protein, and (ii) has the following structure (Case 2) TIFF0007911007000017.tif47165 Contains N-glycan, The square represents an N-acetylglucosamine residue, the black striped circle represents a mannose residue, and X represents an amino acid residue of the bifunctional binding protein, wherein the N-glycan is linked to the bifunctional binding protein at 1, 2, 3, 4, or 5 N-glycosylation sites. (Aspect 51) The bifunctional binding protein according to embodiment 50, wherein the glycan further comprises a fucose residue in the N-acetylglucosamine that is directly bound to X. (Appearance 52) The bifunctional binding protein according to embodiment 50, wherein X is an asparagine residue in the bifunctional binding protein. (Aspect 53) A group of bifunctional binding proteins according to embodiment 50, wherein at least one of the N-glycosylation sites at a specific amino acid position of the bifunctional binding protein is glycosylated with an N-glycan specified in embodiment 50, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 99% of the N-glycosylation sites of the group. (Aspect 54) The bifunctional binding protein according to embodiment 50 or 53, wherein the N-glycosylation site comprises one or more asparagine residues, and the asparagine residues are located within the NXS / T, NXC motif, and non-standard consensus motif of the standard consensus sequence. (Aspect 55) The bifunctional binding protein according to embodiment 50 or 53, wherein the N-glycosylation site is introduced into the bifunctional protein by recombinant engineering. (Aspect 56) The bifunctional binding protein according to embodiment 55, wherein the recombinant engineering is carried out by adding an amino acid, deleting an amino acid, substituting an amino acid, or adding a glycotag. (Aspect 57) The bifunctional binding protein according to embodiment 50 or 53, wherein the glycan has the structure described in embodiment 50. (Pattern 58) The aforementioned target proteins include HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, Frizzled receptor, Wnt, LRP5 / 6, CD38, CD73, TGF-β, Bombecin®, CAIX, CD13, CD44, v6, CXCR4, ErbB-2, Her2, Empurin, Endoglin, EpCAM, EphA2, FAP-α, Folic Acid®, GRP78, IGF-1®, Matryptase, Mesothelin, sMET / HGFR, MT1-M A bifunctional binding protein according to embodiment 50 or 53, comprising MP, MT6-MMP, Muc-1, PSCA, PSMA, Tn antigen, and uPAR, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin and ganglioside GM1, GD3, GQ1B, MOG, SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47. (Aspect 59) The aforementioned bifunctional protein binds to endocytosis carbohydrate-binding receptors, including mannose 3 receptor, differentiation cluster 206 (CD206) receptor, DC-SIGN (differentiation cluster 209 or CD209) receptor, C-type lectin domain family 4-member G (LSECTin) receptor, and macrophage-inducible Ca 2+ A bifunctional binding protein according to embodiment 50 or 53, which specifically binds to a lectin-dependent receptor (Mincle). (Appendix 60) The bifunctional binding protein according to embodiment 59, wherein the bifunctional protein binds to the endocytosis carbohydrate-binding receptor via the N-glycan. (Aspect 61) The bifunctional binding protein according to embodiment 50 or 53, wherein the N-glycan specifically binds to any endocytic carbohydrate-binding receptor that recognizes the Man3GlcNAc2 structure. (Aspect 62) The bifunctional binding protein according to embodiment 50 or 53, wherein the bifunctional binding protein is an antibody. (Aspect 63) The bifunctional conjugating protein according to embodiment 62, wherein the antibody is a monoclonal antibody or a polyclonal antibody. (Personal aspect 64) The bifunctional binding protein according to embodiment 62, wherein the antibody is a recombinant. (Patent 65) The bifunctional binding protein according to embodiment 50 or 53, wherein the antibody includes a heavy chain variable region or a light chain variable region. (Aspect 66) The bifunctional binding protein according to embodiment 50 or 53, wherein the antibody includes a Fab region. (Patent 67) The bifunctional binding protein according to embodiment 50 or 53, wherein the antibody comprises an Fc domain. (Pattern 68) The bifunctional binding protein according to embodiment 62, wherein the antibody includes an N-glycosylation site in the Fc domain, and the N-glycan is linked to the N-glycosylation site of the Fc domain. (Patent 69) The bifunctional binding protein according to embodiment 62, wherein the antibody includes an N-glycosylation site in the heavy chain variable region and / or the light chain variable region, and the N-glycan is linked to the N-glycosylation site in the heavy chain variable region and / or the light chain variable region. (Aspect 70) The bifunctional binding protein according to embodiment 62, wherein the antibody is glycosylated at a predetermined specific residue. (Aspect 71) The bifunctional binding protein according to embodiment 62, wherein the antibody has a glycan-to-protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1. (Aspect 72) The bifunctional binding protein according to embodiment 62, wherein the antibody is glycosylated at a predetermined specific residue. (Aspect 73) The bifunctional binding protein according to embodiment 50 or 53, wherein the bifunctional binding protein contains an autoantigen and specifically binds to an autoantibody. (Aspect 74) The bifunctional binding protein according to embodiment 50 or 53, wherein the target protein is a cell surface molecule or a non-cell surface molecule. (Aspect 75) The bifunctional binding protein according to embodiment 74, wherein the cell surface molecule is a receptor. (Aspect 76) The bifunctional binding protein according to embodiment 74, wherein the non-cell surface molecule is an extracellular protein. (Aspect 77) The bifunctional binding protein according to embodiment 76, wherein the extracellular protein is an autoantibody, hormone, cytokine, chemokine, blood protein, or central nervous system (CNS) protein. (Pattern 78) A method for delivering a target protein to a liver macrophage, comprising contacting the target protein with a bifunctional binding protein according to any one of embodiments 50 to 77 under conditions that mediate the endocytosis of the target protein. (Aspect 79) A method for degrading a target protein, comprising contacting the target protein with a bifunctional binding protein according to any one of embodiments 50 to 77 under conditions that mediate lysosomal degradation of the target protein by a host cell. (Appendix 80) The method according to embodiment 79, wherein the target protein is upregulated in cancer or is involved in the progression of cancer. (Aspect 81) The method according to embodiment 80, wherein the target protein upregulated in cancer or involved in cancer progression includes HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2, Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, LILRB1, LILRB2, CD38, CD73, or TGF-β. (Aspect 82) The method according to embodiment 81, wherein the target protein is an autoantibody for an autoimmune disease. (Aspect 83) The method according to embodiment 81, wherein the target protein is an autoantigen of an autoimmune disease. (Pattern 84) The method according to embodiment 82, wherein the autoantibody for the autoimmune disease is an antibody that binds to MOG, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin, or ganglioside GM1, GD3, or GQ1B. (Pattern 85) The method according to embodiment 79, wherein the target protein is upregulated or expressed in neurodegenerative diseases. (Pattern 86) The method according to embodiment 85, wherein the target protein upregulated or expressed in neurodegenerative diseases is α-synuclein, amyloid-beta, or a complement cascade component. (Aspect 87) The method according to embodiment 79, wherein the host cell is a bone marrow cell, an immune cell, an endothelial cell, a parenchymal cell, or an epithelial cell. (Pattern 88) The method according to embodiment 87, wherein the immune cells are dendritic cells, macrophages, monocytes, microglia, granulocytes, or B lymphocytes. (Pattern 89) The method according to embodiment 79, wherein the host cell is any cell. (Aspect 90) The method according to embodiment 79, wherein the bifunctional binding protein enhances the degradation of the target protein compared to the degradation of the target protein in the presence of an N-glycosylated bifunctional binding protein, or compared to the degradation of the target protein in the presence of a bifunctional binding protein containing an N-glycan different from the N-glycan specified in embodiment 50. (Aspect 91) The method according to embodiment 79, wherein the decomposition is mediated by endocytosis or phagocytosis. (Patent 92) A pharmaceutical composition comprising a bifunctional binding protein according to any one of embodiments 50 to 77 and a pharmaceutically acceptable carrier. (Aspect 93) A method for treating or preventing a disease in a patient, comprising administering to the patient a bifunctional binding protein according to any one of embodiments 50 to 77, or a pharmaceutical composition according to embodiment 92. (Aspect 94) The method according to embodiment 93, wherein the disease is an autoimmune disease, cancer or tumor, liver disease, inflammatory disorder, or blood coagulation disorder. (Aspect 95) The method according to embodiment 94, wherein the autoimmune disease is selected from MOGAD (myelin oligodendrocyte glycoprotein antibody-associated disease), Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, acquired pemphigus vulgaris, immune thrombocytopenia, autoimmune encephalitis, and Guillain-Barré syndrome. (Personal aspect 96) The aforementioned cancers include acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphoblastic leukemia; acute myelogenous leukemia; acute myeloid leukemia (adult / child); adrenal cortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratomatous / rhabdoid tumor; basal cell carcinoma; extrahepatic cholangiocarcinoma; bladder cancer; osteosarcoma / malignant fibrous histiocytoma of bone; brain tumor (adult / child); cerebellar astrocytoma (adult / child); cerebral astrocytoma / malignant glioma; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumor; brain tumor, optic tract hypothalamic glioma; brainstem glioma; breast Cancer; bronchial adenoma / carcinoid; bronchial tumor; Burkitt lymphoma; childhood cancer; carcinoid gastrointestinal tumor; carcinoid tumor; adult carcinoma, primary site unknown; carcinoma of unknown primary site; embryonal tumor of the central nervous system; primary lymphoma of the central nervous system; cervical cancer; childhood adrenocortical carcinoma; childhood cancer; childhood cerebral astrocytoma; chordoma, childhood; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorder; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; fibroplastic round cell tumor; emphysema; endometrial cancer; ependyma Blastoma; ependymoma; esophageal cancer; Ewing's sarcoma (part of the Ewing family of tumors); extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic cholangiocarcinoma; gallbladder cancer; gastric (stomach) cancer; gastric carcinoid; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor; germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic neoplasm; gestational trophoblastic neoplasm, primary site unknown; glioma; brainstem glioma; glioma, pediatric optic tract and hypothalamus; hair cell leukemia; head and neck cancer; cardiac cancer; hepatocellular carcinoma (liver); Hodgkin's lymphoma Tumor; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma; Intraocular melanoma; Islet cell carcinoma; Kaposi's sarcoma; Renal cell carcinoma; Langerhans cell histiocytosis; Laryngeal cancer; Lip and oral cancer; Liposarcoma; Primary liver cancer; Non-small cell lung cancer; Small cell lung cancer; Lymphoma, primary of the central nervous system; Macroglobulinemia, Waldenström; Male breast cancer; Malignant fibrous histiocytoma / osteosarcoma of bone; Medulloblastoma; Medullary epithelioma; Melanoma; Intraocular melanoma (ocular); Merkel cell carcinoma; Merkel cell cutaneous carcinoma; Mesothelioma; Adult malignant mesothelioma;Metastatic squamous cell carcinoma of unknown primary origin; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasmacytic cell neoplasm; mycosis fungoides, myelodysplastic syndrome; myelodysplasia / myeloproliferative disorder; chronic myeloid leukemia; acute adult myeloid leukemia; acute pediatric myeloid leukemia; multiple myeloma (bone marrow cancer); chronic myeloproliferative disorder; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma, non-small cell lung cancer; non-Hodgkin lymphoma; oligodendroglioma; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial carcinoma (surface epithelial and stromal tumor); ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer; pancreatic cancer, pancreas Islet cell tumors; papillomatosis; sinus and nasal cavity cancers; parathyroid cancers; penile cancers; pharyngeal cancers; pheochromocytomas; pineal astrocytomas; pineal germ tumors; intermediate pineal parenchymal tumors; pineoblastomas and supratentorial primitive neuroectodermal tumors; pituitary tumors; pituitary adenomas; plasma cell tumors / multiple myeloma; pleuropulmonary blastomas; primary central nervous system lymphomas; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); pelvic and ureteral transitional cell carcinomas; airway cancers involving the NUT gene on chromosome 15; retinoblastoma; rhabdomyosarcoma (pediatric); salivary gland cancers; sarcomas, Ewing family tumors; Sézary syndrome; skin cancers (melanoma); skin cancers (non-melanoma); small cell lung cancer; small intestine cancer The method according to embodiment 94, selected from soft tissue sarcoma; soft tissue sarcoma; spinal cord tumor; squamous cell carcinoma; cervical squamous cell carcinoma of unknown primary origin, metastatic; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, cutaneous (mycosis fungoides and Sézary syndrome); testicular cancer; pharyngeal cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; pediatric thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; or Wilms' tumor. (Patent 97) The method according to aspect 96, wherein the treatment includes reprogramming tumor-associated macrophages (TAMs) by administering the bifunctional binding protein under conditions that mediate endocytosis of a target protein. (Pattern 98) The method according to embodiment 97, wherein the target protein is upregulated or expressed in the TAM. (Pattern 99) The method according to embodiment 98, wherein the target protein upregulated or expressed in the TAM includes SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47. (Aspect 100) The method according to embodiment 93, wherein the administration step includes intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection. (Aspect 101) A kit comprising a bifunctional binding protein according to any one of embodiments 50 to 77, or a pharmaceutical composition according to embodiment 92, and instructions for administering the bifunctional molecule or the pharmaceutical composition to an individual requiring it. (Aspect 102) The kit according to embodiment 101, wherein the bifunctional binding protein or the pharmaceutical composition is present in one or more unit doses. (Aspect 103) A method for treating acute symptoms accompanied by elevated levels of a target protein, comprising administering a bifunctional binding protein described in any one of the preceding embodiments to a patient in need of treatment, wherein the bifunctional binding protein (i) specifically binds to the target protein and (ii) has the following structure (3) TIFF0007911007000018.tif46165 It contains an N-glycan having, The method wherein the squares represent N-acetylglucosamine residues, the black striped circles represent mannose residues, X represents an amino acid residue of the bifunctional binding protein, the N-glycan is linked to the bifunctional binding protein by numerous N-glycosylation sites, thereby causing the half-life of the target protein to be up to 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours in the patient after administration of the bifunctional binding protein to the patient, or the half-life of the target protein to be up to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, or up to 90% of the half-life of the target protein in the patient in the absence of any treatment. (Aspect 104) The method according to embodiment 103, wherein the glycan further comprises a fucose residue in the N-acetylglucosamine directly bound to X. (Aspect 105) The method according to embodiment 103, wherein X is an asparagine residue in the bifunctional binding protein. (Aspect 106) The method according to embodiment 103, wherein the bifunctional protein supports the N-glycan at three or more N-glycosylation sites. (Aspect 107) A method for treating chronic symptoms accompanied by elevated levels of a target protein, comprising administering a bifunctional binding protein described in any one of the preceding embodiments to a patient in need of treatment, wherein the bifunctional binding protein (i) specifically binds to the target protein and (ii) has the following structure (C4) TIFF0007911007000019.tif47165 It contains an N-glycan having, The method wherein a square represents an N-acetylglucosamine residue, a black striped circle represents a mannose residue, X represents an amino acid residue of the bifunctional binding protein, and the N-glycan is linked to the bifunctional binding protein at numerous N-glycosylation sites, thereby causing the half-life of the target protein to be at least 1, 2, 3, or 4 days in the patient, or the half-life of the target protein to be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the half-life of the unglycosylated bifunctional binding protein in the patient. (Aspect 108) The method according to embodiment 107, wherein the glycan further comprises a fucose residue in the N-acetylglucosamine directly bound to X. (Aspect 109) The method according to embodiment 107, wherein X is an asparagine residue in the bifunctional binding protein. (Aspect 110) The method according to embodiment 107, wherein the bifunctional protein supports the N-glycan at two or fewer N-glycosylation sites.

Claims

1. A bifunctional binding protein which is an antibody or an antigen-binding fragment (Fab) thereof, (i) A first portion that can specifically bind to the target protein, (ii) comprising a second portion capable of binding to an endocytosis carbohydrate-binding receptor, The second part is 【Chemistry 1】 The formula contains a glycan comprising a structure selected from the group consisting of (wherein the formula, squares represent N-acetylglucosamine residues, black striped circles represent mannose residues, and X represents the amino acid residue of the first part) and its fucosylated variants, The first portion includes the Fab region of the antibody, and The glycan is linked to at least one N-glycosylation site within the Fab region, and The bifunctional binding protein wherein at least one N-glycosylation site within the Fab region is introduced by recombinant engineering.

2. (a) The fucosylated mutant contains a fucose residue linked to N-acetylglucosamine directly bound to X, or (b) The bifunctional binding protein according to claim 1, wherein X is an asparagine residue in the first portion.

3. The second portion is a mannose 3 receptor, a differentiation cluster 206 (CD206) receptor, a DC-SIGN (differentiation cluster 209 or CD209) receptor, a C-type lectin domain family 4-member G (LSECTin) receptor, or a macrophage-inducible Ca ２＋ A bifunctional binding protein according to claim 1 or 2, which specifically binds to a lectin receptor dependent on Mincle.

4. The bifunctional binding protein according to claim 1 or 2, wherein the second portion specifically binds to an endocytosis carbohydrate-binding receptor that recognizes the Man3GlcNAc2 structure.

5. The bifunctional binding protein according to any one of claims 1 to 4, wherein the bifunctional binding protein is a monoclonal antibody, optionally, (a) The antibody is recombinant; (b) The antibody is a humanized antibody, a chimeric antibody, or a fully human antibody; (c) The antibody has a glycan-to-protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1; or (d) The antibody is glycosylated at a specific residue. The aforementioned bifunctional binding protein.

6. The bifunctional binding protein according to claim 1, wherein the target protein is a cell surface molecule, and optionally, the cell surface molecule is a receptor.

7. The bifunctional binding protein according to claim 1, wherein the target protein is a non-cell surface molecule, optionally the non-cell surface molecule is an extracellular protein, and optionally the extracellular protein is an autoantibody, hormone, cytokine, chemokine, blood protein, or central nervous system (CNS) protein.

8. The aforementioned target proteins include HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, and Frizzled. Receptors, Wnt, LRP5 / 6, CD38, CD73, TGF-β, Bombecin®, CAIX, CD13, CD44, v6, ErbB-2, Empurin, Endoglin, EpCAM, EphA2, FAP-α, Folate®, GRP78, IGF-1®, Matryptase, Mesothelin, sMET / HGFR, MT1-MMP, MT6 - A bifunctional binding protein according to claim 1, comprising MMP, PSCA, PSMA, Tn antigen, uPAR, TSHRα, AChR-α1, non-collagen domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIIb / IIIa, GPIa / IIa, NMDA receptor, glutamate decarboxylase (GAD), amphiphycin and ganglioside GM1, GD3, GQ1B, MOG, SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, CCL2, CXCL12, CSF-1, CD47, or a misfolded light chain and misfolded transthyretin.

9. A pharmaceutical composition comprising a bifunctional binding protein according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A bifunctional binding protein according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9, for use in a method of treating or preventing an autoimmune disease, cancer or tumor, liver disease, inflammatory disorder, or blood coagulation disorder in a patient, wherein the method comprises administering the bifunctional binding protein or the pharmaceutical composition to the patient.

11. (a) The autoimmune disease is selected from MOGAD (myelin oligodendrocyte glycoprotein antibody-associated disease), Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, acquired pemphigus vulgaris, immune thrombocytopenia, autoimmune encephalitis, and Guillain-Barré syndrome; or (b) The cancer is selected from lung cancer, breast cancer, gastric cancer, colorectal cancer, bladder cancer, malignant melanoma, multiple myeloma, and Hodgkin lymphoma. A bifunctional binding protein or pharmaceutical composition for use according to claim 10.

12. A bifunctional conjugating protein or pharmaceutical composition for use according to claim 11, wherein the treatment comprises reprogramming tumor-associated macrophages (TAMs) by administering the bifunctional conjugating protein under conditions that mediate endocytosis of a target protein, wherein optionally, the target protein is upregulated or expressed in the TAM, and further optionally, the target protein upregulated or expressed in the TAM comprises SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47.

13. A bifunctional binding protein or pharmaceutical composition for use according to any one of claims 10 to 12, wherein the administration step comprises intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection.

14. A kit comprising a bifunctional binding protein according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9, and instructions for administering the bifunctional binding protein or the pharmaceutical composition to a patient in need thereof.

15. The kit according to claim 14, wherein the bifunctional binding protein or the pharmaceutical composition is present in one or more unit doses.

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