Methods of administration and treatment using TACI-Fc fusion immunomodulatory proteins

Abstract

The present invention relates to methods of administration and treatment using TACI-Fc fusion immunomodulatory proteins. In particular, provided herein are methods of treatment and uses relating to immunomodulatory TACI-Fc fusion proteins that exhibit neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers). The TACI-Fc proteins provided may comprise a transmembrane activator and a variant domain of a CAML interaction factor (TACI). The methods and uses provide therapeutic utility for a variety of immune diseases, disorders or conditions, such as B-cell mediated diseases, disorders or conditions.

Description

This application is a divisional application of the invention application with an application date of May 6, 2022, Chinese application number 202280047313.5, and invention name “Administration and treatment methods using TACI-Fc fusion immunomodulatory protein”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to the following patent applications: U.S. Provisional Application No. 63 / 186,027 filed on May 7, 2021; U.S. Provisional Application No. 63 / 239,899 filed on September 1, 2021; U.S. Provisional Application No. 63 / 256,505 filed on October 15, 2021; U.S. Provisional Application No. 63 / 278,072 filed on November 10, 2021; and U.S. Provisional Application No. 63 / 329,325 filed on April 8, 2022, the contents of each of which are hereby incorporated by reference in their entirety. Incorporation by Reference into the Sequence Listing

[0002] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named 761612003940SeqList.TXT created on May 4, 2022, which is 284,174 bytes in size. The information in the sequence listing in electronic format is incorporated by reference in its entirety. Technical Field

[0003] The present disclosure provides methods and uses of immunomodulatory TACI-Fc fusion proteins that exhibit neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers). The provided TACI-Fc fusion proteins may comprise variant domains of a transmembrane activator and a CAML interactor (TACI). The methods and uses provide therapeutic utility for a variety of immune diseases, disorders or conditions, such as B cell-mediated diseases, disorders or conditions. Background Art

[0004] Modulating immune responses by intervening in processes involving interactions between soluble ligands and their receptors is of increasing medical interest. Currently, biological products used to enhance or inhibit immune responses are generally limited to antibodies (e.g., anti-PD-1 antibodies) or soluble receptors for single cell surface molecules (e.g., CTLA-4-Fc). Improved therapeutic agents are needed that can modulate immune responses, particularly B cell immune responses. Embodiments that meet this need are provided. Summary of the invention

[0005] Provided herein are methods of treating an inflammatory or autoimmune disease or disorder in a subject in need thereof by administering to the subject any of the provided TACI-Fc fusion proteins as described herein, wherein the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide as described herein, and wherein the TACI-Fc fusion protein is administered once a week up to once every three months at a dose of from at or about 2.4 mg to at or about 960 mg.

[0006] Also provided herein are uses, including uses of pharmaceutical compositions containing any of the provided TACI-Fc fusion proteins as described herein, wherein the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide as described herein; and uses in the preparation of a medicament to perform such therapeutic methods for treating inflammatory or autoimmune diseases or disorders. In some embodiments, also provided herein are pharmaceutical compositions for treating inflammatory or autoimmune diseases or disorders in a subject, wherein the pharmaceutical compositions contain any of the provided TACI-Fc fusion proteins as described herein, wherein the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide as described herein. In some embodiments, the use or pharmaceutical composition for the use is used to administer the TACI-Fc fusion protein to a subject once a week up to once every three months at a dose of from about 2.4 mg to about 960 mg.

[0007] In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 8 mg to 960 mg. In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 80 mg to 960 mg.

[0008] In some embodiments, the variant TACI polypeptide of the TACI-Fc fusion is a portion of the extracellular domain consisting of the CRD2 TNF receptor domain shown in SEQ ID NO: 13, wherein there are amino acid substitutions K77E, F78Y and Y102D. In some embodiments, the variant TACI is shown in SEQ ID NO: 26. In any of the embodiments of the TACI-Fc fusion protein, the variant TACI is connected to the Fc domain via the linker.

[0009] Provided herein are methods of treating an inflammatory or autoimmune disease or disorder in a subject in need thereof by administering to the subject a TACI-Fc fusion protein, which is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y and Y102D in the amino acid sequence shown in SEQ ID NO:13, wherein the TACI-Fc fusion protein is administered once a week up to once every three months at a dose of from at or about 2.4 mg to at or about 960 mg.

[0010] Also provided herein are uses, including uses of pharmaceutical compositions containing any of the provided TACI-Fc fusion proteins as described herein, wherein the TACI-Fc fusion protein is a TACI-Fc fusion protein that is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13; and uses in the preparation of a medicament to perform such therapeutic methods for treating an inflammatory or autoimmune disease or disorder. In some embodiments, also provided herein are pharmaceutical compositions for treating an inflammatory or autoimmune disease or disorder in a subject, wherein the pharmaceutical composition contains a TACI-Fc fusion protein that is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the use or the pharmaceutical composition for use is for administering the TACI-Fc fusion protein to a subject once a week up to once every three months at a dose of from about 2.4 mg to about 960 mg.

[0011] In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 8 mg to 960 mg. In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 80 mg to 960 mg. Provided herein is a method of treating an inflammatory or autoimmune disease or disorder in a subject by administering a TACI-Fc fusion protein to a subject in need thereof, wherein the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein the TACI fusion protein is administered once a week to once every three months at a dose of from or about 2.4 mg to or about 960 mg. In some embodiments, the TACI is any TACI polypeptide described herein, such as any variant TACI polypeptide described herein, the linker is any linker as described herein, and the Fc is any Fc region described herein.

[0012] Provided herein are methods of treating an inflammatory or autoimmune disease or disorder in a subject by administering to a subject in need thereof a TACI-Fc fusion protein that is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein the TACI fusion protein is administered once a week up to once every three months at a dose of from at or about 8 mg to at or about 960 mg. In some embodiments, the TACI is any TACI polypeptide described herein, such as any variant TACI polypeptide described herein, the linker is any linker as described herein, and the Fc is any Fc region described herein.

[0013] In one aspect, provided herein is a method of treating an inflammatory or autoimmune disease or disorder in a subject in need thereof, the method comprising administering to the subject a TACI-Fc fusion protein, which is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y and Y102D in the amino acid sequence shown in SEQ ID NO:13, wherein the TACI-Fc fusion protein is administered once a week up to once every three months at a dose of from at or about 8 mg to at or about 960 mg.

[0014] Also provided herein are uses of any of the provided TACI-Fc fusion proteins as described. Uses include pharmaceutical compositions comprising the TACI-Fc fusion proteins for use in any such provided methods, and uses in the preparation of medicaments to perform any such provided methods.

[0015] In any of the provided methods or uses, the dose of the TACI-Fc fusion protein is administered once every three months. In any of the provided methods or uses, the dose of the TACI-Fc fusion protein is administered once a month (Q4W). In any of the provided methods or uses, the dose of the TACI-Fc fusion protein is administered once every other week (Q2W). In any of the provided methods or uses, the dose of the TACI-Fc fusion protein is administered once a week (Q1W).

[0016] In any of the provided methods or uses, the TACI-Fc fusion protein is administered at a dose of from about 80 mg to about 720 mg, from about 160 mg to about 560 mg, or from about 240 mg to about 480 mg. In any of the provided methods or uses, the TACI-Fc fusion protein is administered at a dose of from about 40 mg to about 480 mg, from about 80 mg to about 320 mg, or from about 80 mg to about 120 mg.

[0017] In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 80 mg to or about 720 mg. In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 160 mg to or about 560 mg. In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 240 mg to or about 480 mg.

[0018] In some embodiments, the dose of the TACI-Fc fusion protein is from at or about 24 mg to at or about 480 mg.

[0019] In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 40 mg to or about 480 mg. In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 80 mg to or about 320 mg. In some embodiments, the dosage of the TACI-Fc fusion protein is from or about 80 mg to or about 120 mg.

[0020] In any of the provided methods or uses, the TACI-Fc fusion protein is administered at a dose of from about 240 mg to about 480 mg once. In any of the provided methods or uses, the TACI-Fc fusion is administered at a dose of from about 80 mg to about 120 mg.

[0021] In embodiments of any of the provided methods or uses, the administering is via intravenous administration.

[0022] In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 2.4 mg. In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 8 mg. In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 24 mg. In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 80 mg. In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 240 mg. In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 480 mg. In some embodiments, the dose of the TACI-Fc fusion protein for intravenous administration is or is about 960 mg.

[0023] In embodiments of any of the provided methods or uses, the administering is via subcutaneous administration.

[0024] In some embodiments, the dose of the TACI-Fc fusion protein for subcutaneous administration is at or about 80 mg. In some embodiments, the dose of the TACI-Fc fusion protein for subcutaneous administration is at or about 240 mg. In some embodiments, the dose of the TACI-Fc fusion protein for subcutaneous administration is at or about 480 mg. In some embodiments, the dose of the TACI-Fc fusion protein for subcutaneous administration is at or about 960 mg.

[0025] In embodiments of any of the provided methods or uses, the variant TACI polypeptide in the TACI-Fc fusion protein is shown in SEQ ID NO:26.

[0026] In embodiments of any of the methods or uses provided, the linker in the TACI-Fc fusion protein is selected from GSGGS (SEQ ID NO:76), GGGGS (G4S; SEQ ID NO:77), GSGGGGS (SEQ ID NO:74), GGGGSGGGGS (2xGGGGS; SEQ ID NO:78), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO:79), GGGGSGGGGSGGGGSGGGGS (4xGGGGS; SEQ ID NO:84), GGGGSGGGGSGGGGSGGGGSGGGGS (5XGGGGS; SEQ ID NO:91), GGGGSSA (SEQ ID NO:80), or GSGGGGSGGGGS (SEQ ID NO:194), or a combination thereof. In embodiments of any of the methods or uses provided, the linker is shown in SEQ ID NO:74.

[0027] In any embodiment of the provided methods or uses, the Fc in the TACI-Fc fusion protein is an IgG1 Fc domain. In any embodiment of the provided methods or uses, the Fc is a variant IgG1 Fc that exhibits reduced binding affinity to Fc receptors and / or reduced effector functions compared to a wild-type IgG1 Fc domain. In some embodiments, the variant IgG1 Fc domain comprises one or more amino acid substitutions selected from the group consisting of L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C, according to EU numbering. In some embodiments, the variant IgG1 Fc comprises amino acid substitutions L234A, L235E, and G237A, according to EU numbering.

[0028] In any of the provided methods or uses, the Fc comprises an amino acid substitution C220S, wherein the residues are numbered according to the EU index of Kabat. In any of the provided methods or uses, the Fc lacks the hinge sequence EPKSS or EPKSC. The Fc region comprises K447del, wherein the residues are numbered according to the EU index of Kabat.

[0029] In embodiments of any of the provided methods or uses, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 73. In some of any of the provided methods or uses, the TACI-Fc fusion protein is set forth in SEQ ID NO:167.

[0030] In embodiments of any of the provided methods or uses, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 81. In embodiments of any of the provided methods or uses, the TACI-Fc fusion protein is set forth in SEQ ID NO:168.

[0031] In embodiments of any of the provided methods or uses, the administration is via intravenous administration. In embodiments of any of the provided methods or uses, the administration is via subcutaneous administration.

[0032] In any embodiment of the methods or uses provided, the B cell immune response or activity in the subject is reduced. In any embodiment of the methods or uses provided, the number of mature B cells and total circulating B cells in the subject is reduced. In any embodiment of the methods or uses provided, the circulating serum immunoglobulin (IgG) in the subject is reduced. In any embodiment of the methods or uses provided, one or more of B cell maturation, differentiation and / or proliferation is reduced or inhibited. In any embodiment of the methods or uses provided, the circulating levels of APRIL or BAFF proteins in the subject are reduced, optionally wherein the APRIL or BAFF proteins are APRIL homotrimers, BAFF homotrimers, APRIL / BAFF heterotrimers or BAFF 60 polymers.

[0033] In any of the provided methods or embodiments of the use, the disease or disorder is a B cell mediated disease or disorder. In any of the provided methods or embodiments of the use, the disease or disorder is an autoimmune disease and inflammatory condition, a B cell cancer, an antibody mediated pathology, a kidney disease, a transplant rejection, a graft versus host disease, or a viral infection.

[0034] In embodiments of any of the provided methods or uses, the disease or disorder is selected from systemic lupus erythematosus (SLE), lupus nephritis, cutaneous lupus erythematosus, Sjögren's syndrome, scleroderma (systemic sclerosis), multiple sclerosis, diabetes (e.g., type I diabetes), polymyositis, primary biliary cirrhosis, IgG4-related disease, IgA nephropathy, IgA vasculitis, ANCA vasculitis (microscopic polyangiitis, granulomatosis with polyangiitis [Wegener's granulomatosis], eosinophilic granulomatosis with polyangiitis [Zhag-Strauss]), cryoglobulinemia, cold agglutinin or warm agglutinin disease, immune thrombocytopenic purpura, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, graft-versus-host disease (GVHD), transplantation, rheumatoid arthritis, acute lupus nephritis, amyotrophic lateral sclerosis, neuromyelitis optica, transverse myelitis, Rasmussen encephalitis, CNS autoimmunity, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, neurocysticercosis, sarcoidosis, antiphospholipid antibody syndrome, IgG4-related disease, Hashimoto's thyroiditis, immune thrombocytopenia, Addison's disease, and dermatomyositis.

[0035] In any embodiment of the method or use provided, the disease or disorder is an autoantibody-related glomerular disease. In some embodiments, the autoantibody-related glomerular disease is immunoglobulin (Ig) A nephropathy (IgAN), lupus nephritis (LN), primary membranous nephropathy (pMN) or renal anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV). In some embodiments, the disease or disorder is an autoantibody-related glomerular disease, and the autoantibody-related glomerular disease is immunoglobulin (Ig) A nephropathy (IgAN). In some embodiments, the disease or disorder is lupus nephritis (LN). In some embodiments, the disease or disorder is primary membranous nephropathy (pMN). In some embodiments, the disease or disorder is renal anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV).

[0036] In any embodiment of the method or use provided, the disease or disorder is a B cell cancer. In some embodiments, the B cell cancer is a myeloma, a B cell chronic lymphocytic leukemia, a Waldenstrom's macroglobulinemia, or a non-Hodgkin's lymphoma. In some any embodiments, the type of myeloma includes multiple myeloma, plasmacytoma, multiple solitary plasmacytoma, and / or extramedullary myeloma. In some any embodiments, the type of myeloma includes light chain myeloma, non-secretory myeloma, and / or IgD or IgE myeloma.

[0037] In embodiments of any of the provided methods or uses, the subject is a human.

[0038] In any embodiment of the provided methods or uses, the TACI-Fc fusion protein is provided in a formulation comprising an acetate buffer having a pH from about 4.0 to about 6.0, a proline concentration from about 1% to about 10%, and a surfactant concentration from about 0.005% to about 0.05% (w / v). In some embodiments, the pH of the formulation is about 5.2. In some embodiments, the acetate buffer comprises acetate at a concentration from about 5mM to about 15mM. In some embodiments, the acetate buffer comprises acetate at a concentration of about 10mM. In some embodiments, the proline concentration is about 2% to about 5%. In some embodiments, the proline concentration is about 3%. In some embodiments, the surfactant concentration is about 0.015% (w / v). In some embodiments, the surfactant is polysorbate 80.

[0039] In some any embodiments, the amount of TACI-Fc fusion protein in the formulation is from about 50 mg to about 100 mg. In some any embodiments, the amount of TACI-Fc fusion protein in the formulation is at or about 80 mg. In some any embodiments, the concentration of the TACI-Fc fusion protein is between about 50 mg / mL and about 200 mg / mL. In some any embodiments, the concentration of the TACI-Fc fusion protein is at or about 100 mg / mL.

[0040] Provided herein is a formulation comprising a TACI-Fc fusion protein, an acetate buffer having a pH of from about 4.0 to about 6.0, a proline concentration of from or about 1% to about 10%, and a surfactant concentration of from about 0.005% to about 0.05% (w / v), wherein the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y and Y102D in the amino acid sequence shown in SEQ ID NO:13.

[0041] In some any embodiments, the variant TACI polypeptide of the TACI-Fc fusion protein in the formulation is shown in SEQ ID NO: 26. In some any embodiments, the linker of the TACI-Fc fusion protein is selected from GSGGS (SEQ ID NO: 76), GGGGS (G4S; SEQ ID NO: 77), GSGGGGS (SEQ ID NO: 74), GGGGSGGGGS (2xGGGGS; SEQ ID NO: 78), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO: 79), GGGGSGGGGSGGGGSGGGGS (4xGGGGS; SEQ ID NO: 84), GGGGSGGGGSGGGGSGGGGSGGGGS (5XGGGGS; SEQ ID NO: 91), GGGGSSA (SEQ ID NO: 80), or GSGGGGSGGGGS (SEQ ID NO: 194), or a combination thereof. In some any embodiments, the linker is shown in SEQ ID NO:74.

[0042] In some any embodiments, the Fc of the TACI-Fc fusion protein in the formulation is an IgG1 Fc domain. In some any embodiments, the Fc is a variant IgG1 Fc that exhibits reduced binding affinity to Fc receptors and / or reduced effector functions compared to a wild-type IgG1 Fc domain. In some any embodiments, the variant IgG1 Fc domain comprises one or more amino acid substitutions selected from the following: L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C, according to EU numbering. In some any embodiments, the variant IgG1 Fc comprises amino acid substitutions L234A, L235E, and G237A, according to EU numbering.

[0043] In some any embodiments, the Fc comprises the amino acid substitution C220S, wherein the residues are numbered according to the EU index of Kabat. In some any embodiments, the Fc lacks the hinge sequence EPKSS or EPKSC. In some any embodiments, the Fc region comprises K447del, wherein the residues are numbered according to the EU index of Kabat.

[0044] In some any embodiments, the Fc of the TACI-Fc fusion protein in the formulation comprises the amino acid sequence shown in SEQ ID NO: 73. In some any embodiments, the TACI-Fc fusion protein in the formulation has the sequence shown in SEQ ID NO: 167. In some any embodiments, the Fc of the TACI-Fc fusion protein in the formulation comprises the amino acid sequence shown in SEQ ID NO: 81. In some any embodiments, the TACI-Fc fusion protein in the formulation has the sequence shown in SEQ ID NO: 168.

[0045] In some any embodiments, the pH of the formulation is about 5.2. In some any embodiments, the acetate buffer comprises acetate at a concentration of from or about 5 mM to or about 15 mM. In some any embodiments, the acetate buffer comprises acetate at a concentration of or about 10 mM. In some any embodiments, the concentration of proline is from about 2% to about 5%. In some any embodiments, the concentration of proline is or about 3%. In some any embodiments, the concentration of the surfactant is from about 0.01 to about 0.025% (w / v). In some any embodiments, the concentration of the surfactant is or about 0.015% (w / v). In some embodiments, the surfactant is polysorbate 80.

[0046] In some any embodiments, the amount of TACI-Fc fusion protein in the formulation is from about 50 mg to about 100 mg. In some any embodiments, the amount of TACI-Fc fusion protein in the formulation is at or about 80 mg. In some any embodiments, the concentration of the TACI-Fc fusion protein is between about 50 mg / mL and about 200 mg / mL. In some any embodiments, the concentration of the TACI-Fc fusion protein is at or about 100 mg / mL.

[0047] In some any embodiments, the formulation is a liquid. In some any embodiments, the volume of the formulation is 0.5 mL to 2.0 mL. In some any embodiments, the volume of the formulation is or is about 0.8 mL.

[0048] A container is also provided, the container comprising any provided preparation, such as a preparation with any of the above features. In some any embodiments, the container is a bottle or a prefilled syringe. In some any embodiments, the container is a glass vial. In some any embodiments, the container holds up to a volume of or about 5 mL. In some any embodiments, the container holds up to a volume of or about 2 mL. In some embodiments, the container is a 2 mL glass vial.

[0049] In some any embodiments, a method of reducing an immune response in a subject is provided, the method comprising administering a therapeutically effective amount of the formulation to a subject in need thereof.

[0050] In some any embodiments, the B cell immune response in the subject is reduced, whereby B cell maturation, differentiation and / or proliferation is reduced or inhibited. In some any embodiments, the circulating levels of APRIL, BAFF or APRIL / BAFF heterotrimers in the subject are reduced. In some any embodiments, reducing the immune response treats a disease, disorder or condition in the subject. In some any embodiments, a method for reducing the circulating levels of APRIL, BAFF or APRIL / BAFF heterotrimers in a subject is provided, the method comprising administering a therapeutically effective amount of the formulation.

[0051] Also provided is a method for treating a disease, disorder or condition of a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the provided formulations, including any formulations having the above characteristics. In some any embodiments, the disease, disorder or condition is an autoimmune disease, and an inflammatory disorder, B cell cancer, an antibody-mediated condition, kidney disease, transplant rejection, graft-versus-host disease or a viral infection. In some any embodiments, the disease, disorder or condition is selected from: systemic lupus erythematosus (SLE); Sjogren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis and pemphigus vulgaris.

[0052] In any embodiment of the method or use provided, the disease or disorder is an autoantibody-related glomerular disease. In some embodiments, the autoantibody-related glomerular disease is immunoglobulin (Ig) A nephropathy (IgAN), lupus nephritis (LN), primary membranous nephropathy (pMN) or renal anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV). In some embodiments, the disease or disorder is an autoantibody-related glomerular disease, and the autoantibody-related glomerular disease is immunoglobulin (Ig) A nephropathy (IgAN). In some embodiments, the disease or disorder is lupus nephritis (LN). In some embodiments, the disease or disorder is primary membranous nephropathy (pMN). In some embodiments, the disease or disorder is renal anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV).

[0053] In some any embodiments, the disease, disorder or condition is a B cell cancer and the cancer is myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic representation of a functional inhibition assay involving recombinant APRIL and BAFF performed by TACI is shown. In the assay, Jurkat cells are transduced with a luciferase-based NF-κB reporter and used to stably express mouse or human TACI based on cell surface expression. Upon activation by recombinant APRIL or BAFF, endogenous NF-KB transcription factors bind to a DNA response element that controls transcription of the firefly luciferase gene. Luciferase expression can be monitored, such as by using Bio-Glo TM Reagents were detected and measured using a Cytation 3 reader.

[0055] Figure 2 Exemplary human TACI TD Fc fusion molecules for blocking human APRIL (upper panel) and BAFF (lower panel) mediated signaling are shown. TACI TD Fc fusions were incubated with APRIL or BAFF for 20 min (room temperature and shaking) and then added to wells containing 150,000 Jurkat / TACI / NFκB-luciferase cells for 5 hours.

[0056] Figure 3A Exemplary TACI TD Fc fusion molecules are shown for blocking the function of APRIL (upper panel of the figure) or BAFF (lower panel of the figure).

[0057] Figure 3B Shown are human TACI TDFc fusion molecules used to block mouse APRIL (left panel) and BAFF (right panel) mediated signaling.

[0058] Figure 4 Human TACI TD Fc fusion molecules are shown for blocking human APRIL (upper panel) and BAFF (lower panel) mediated signaling relative to TACI 13-118-Fc, TACI 30-110-Fc, and belimumab.

[0059] Figure 5A-Figure 5C Shown are the results of the studies on belimumab, BION-1301, and WT TACI-Fc molecules, including WT TACI30-110 (atacicept) and WT TACI 13-118-Fc (tacicept), for blocking BAFF ( Figure 5A )、APRIL( Figure 5B ) and the combination of BAFF+APRIL( Figure 5C )-mediated signal transduction is an exemplary human TACI TD Fc fusion molecule 26TACI CRD2-Fc.

[0060] Figure 6A-6J Analysis of parameters evaluated in the NZB / NZW mouse model of human SLE is shown. Proteinuria scores were evaluated starting at 20 weeks of age ( Fig. 6A ), average percentage change in body weight ( Figure 6B ) and the survival rate percentage ( Figure 6C ). Anti-double-stranded DNA IgG titer ( Fig.6D ) and blood urea nitrogen (BUN) ( Fig. 6E ) serum was analyzed (****p<0.0001 for anti-dsDNA IgG by Student's t-test compared to Fc; ***p=0.0008 for BUN-4 by Student's t-test compared to Fc). Kidneys were processed and analyzed by histology in repeated periodic acid Schiff (PAS)-stained sections, and the individual components and total histological scores are depicted in Fig. 6F Frozen kidneys were also sectioned and stained for immunohistochemical analysis of glomerular deposition of mouse IgG and complement C3, respectively. Figure 6G and Figure 6H as shown in . Fig.6I Histological scores ± SEM are shown. Sialadenitis as measured by submandibular gland histological scores is shown in Figure 6J middle.

[0061] Figure 7 Shown is the ability of TACI mutations (K77E / F78Y / Y102D) to inhibit APRIL (left panel) and BAFF (right panel) mediated signaling, quantified by luciferase production in Jurkat / NF-κB / TACI cells.

[0062] Fig. 8A and Figure 8B Schematic representation depicting an exemplary TACI-Fc fusion protein. Fig. 8A An exemplary TACI-Fc fusion protein containing two cysteine-rich pseudo-repeat sequences (CRDs) is depicted. Figure 8B An exemplary TACI-Fc fusion protein containing a cysteine-rich pseudo-repeat sequence (CRD, such as CRD2) is depicted.

[0063] Fig. 9 Depicts an exemplary sequence alignment for identifying corresponding residues in a sequence compared to a reference sequence. The symbol "*" between two aligned amino acids indicates that the aligned amino acids are identical. The symbol "-" indicates a gap in the alignment. Exemplary non-limiting positions for amino acid substitutions described herein are indicated with bold text. Based on the alignment of two similar sequences with shared identical residues, a skilled artisan can identify "corresponding" positions in a sequence by comparing to a reference sequence using conserved and identical amino acid residues as a guide. Fig. 9 An exemplary alignment of the reference TACI extracellular domain sequence shown in SEQ ID NO:122 (containing a complete extracellular domain with CRD1 and CRD2 and an initial methionine residue) and the TACI extracellular domain sequence shown in SEQ ID NO:13 (containing only a single CRD, i.e., CRD2) is provided; alignment of identical residues shows that, for example, amino acid residue E7 in SEQ ID NO:13 corresponds to residue E74 in SEQ ID NO:122, amino acid residue K10 in SEQ ID NO:13 corresponds to residue K77 in SEQ ID NO:122, amino acid residue Y12 in SEQ ID NO:13 corresponds to Y79 in SEQ ID NO:122, amino acid residue L15 in SEQ ID NO:13 corresponds to L82 in SEQ ID NO:122, amino acid residue R17 in SEQ ID NO:13 corresponds to R84 in SEQ ID NO:122; and amino acid residue D16 in SEQ ID NO:13 corresponds to D85 in SEQ ID NO:122. A skilled artisan can readily perform similar alignments between two similar protein sequences to identify corresponding residues, including based on the exemplification and description herein.

[0064] Figures 10A-10D Analysis of parameters evaluated in the mouse keyhole limpet hemocyanin (KLH) model is shown. Serum-KLHIgM OD levels were evaluated as primary response ( Fig. 10A ) and answer again ( Fig. 10B Similarly, serum anti-KLH IgG1 OD levels were evaluated as primary responses ( Fig. 10C ) and answer again ( Fig. 10D )both.

[0065] Figure 11A-11B Shown are analyses of harvested spleens evaluated in the mouse keyhole limpet hemocyanin (KLH) immunization model. Spleens were processed and measured by weight ( Fig.11A ) and total cell number ( Fig. 11B ) for analysis.

[0066] Fig.12 Depicted are analyses of spleens assessed for cell subtype population composition from the murine keyhole limpet hemocyanin (KLH) model, and results are shown for B cell subset numbers relative to group means.

[0067] Fig.13 Depicts analysis of spleens from the murine keyhole limpet hemocyanin (KLH) model assessed for cell subtype phenotypic composition and shows results for the number of germinal center B cells and plasma cells ( Fig.13 ).

[0068] Figures 14A-14D Figure 3. T cell populations in the mouse keyhole limpet hemocyanin (KLH) model. Splenic CD3+, CD8+, CD4+, and follicular helper T cells are depicted in Figure 3. Fig.14A , Fig. 14B , Fig. 14C and Fig.14D middle.

[0069] Fig.15 Delineation of Tcm and TEM cell populations in the mouse keyhole limpet hemocyanin (KLH) model.

[0070] Figure 16A-16B and Figure 17A-17B Depicts sialadenitis in diabetes-prone mice after treatment with the tested molecules ( Figure 16A-16B ) and insulitis ( Figure 17A-17B )’s overall incidence and extent.

[0071] Fig.18 and Fig.19 Depicted are serum immunoglobulin (IgM, IgA, and IgG) concentrations of exemplary test molecules following a single intravenous infusion in male Sprague Dawley rats in a pharmacokinetic / pharmacodynamic study.

[0072] Fig. 20A and Fig. 20B Individual animal serum concentration versus time profiles are depicted for an exemplary test molecule administered to cynomolgus monkeys in a PK / PD model. Fig. 20B The results for atacicept depicted in are based on published data (Carbonatto et al. (2008) Toxicol Sci 105:200-210).

[0073] Fig.21 Depicted are the levels of serum IgM, IgA, and IgG in animals receiving exemplary test molecules in the cynomolgus monkey PK / PD model.

[0074] Fig. 22 Depicted are absolute cell counts of animals receiving exemplary test molecules in the cynomolgus monkey PK / PD model.

[0075] Fig.23 Depicted are the % of cells compared to baseline for animals receiving exemplary test molecules in the cynomolgus monkey PK / PD model.

[0076] Fig.24 Depicted are the absolute counts or relative percentages of proliferating T cells in animals receiving exemplary test molecules in the cynomolgus monkey PK / PD model.

[0077] Figure 25A-25B Depicted in a two-compartment PK model every four weeks ( Fig.25A ) or every two weeks ( Fig.25B ) Predicted human PK characteristics after repeated IV dosing.

[0078] Fig.25C Depicted are serum IgA, IgG, IgM levels and their corresponding changes from baseline in a human cohort administered an exemplary TACI CRD2-Fc.

[0079] Figure 26A-Figure 26E Delineating class-switched memory B cells ( Fig.26A ), plasma cells ( Fig.26B ) and immunoglobulin secretion ( Figure 26C-Figure 26E ) inhibition.

[0080] Figure 27A-27C Depicts bone marrow in a mouse model of CIA that received the test molecule ( Fig.27A ),spleen( Fig.27B ) and lymph nodes ( Fig.27C ) levels of plasma cells.

[0081] Fig.28 Depicted are the number of plasma cells in bone marrow smears of cynomolgus monkeys that received an exemplary TACI-Fc fusion protein.

[0082] Figure 29A-29B Depicts the dose-dependent serum concentration versus time curve for animals receiving an exemplary TACI-Fc fusion protein in a 1-month GLP toxicology study in cynomolgus monkeys ( Fig.29A ) and % cells compared to baseline ( Fig.29B ).

[0083] Fig.30Depicted are levels of serum IgA, IgG, and IgM in animals receiving exemplary TACI-Fc fusions in a 1-month GLP toxicology study in cynomolgus monkeys.

[0084] Fig.31 Analysis of harvested spleens evaluated from the murine chronic graft versus host disease (cGVHD) model. Spleens were processed and analyzed based on weight and total cell number.

[0085] Fig.32 Depicts analysis of spleens from a murine chronic graft-versus-host disease model for evaluation of cell population composition and shows CD45 + Cells and B220 + Results of B cell numbers.

[0086] Fig.33 Depicts analysis of spleen assessed for cell subtype population composition and shows CD4 + and CD8 + Results for T cell subset populations.

[0087] Fig.34 Delineating CD4 in cGVHD Models + T cell subset population.

[0088] Fig.35A Characterizing B220 in cGVHD Model + B cells and CD1d 高 CD5 + B-1 cell number. Fig.35B Depicting transition-1 (T1) and transition-2 (T2) B cell populations in the cGVHD model.

[0089] Figure 36A-36B Delineation of follicular and marginal zone (MZ) B cells in the cGVHD model ( Fig.36A ), germinal center (GC) B cells and plasma cells ( Fig.36B )quantity.

[0090] Fig.37 To characterize the number of early plasma cells, plasmablasts, and long-lived plasma cells (LL-PCs) in the cGVHD model.

[0091] Fig.38 Depicted are renal IgG immune complex deposition in the kidney as measured by immunohistochemical staining with a fluorescently labeled antibody specific for mouse IgG.

[0092] Fig.39 Analysis of anti-dsDNA autoantibody serum titers at weeks 8 and 13 is shown.

[0093] Fig.40 H-2 in autoantibody-associated glomerulonephritis bm12 Analysis of anti-dsDNA autoantibody serum titers in mouse models.

[0094] Fig.41 Depicted are renal IgG immune complex deposition in the kidney as measured by immunohistochemical staining with a fluorescently labeled antibody specific for mouse IgG.

[0095] Fig.42 Levels of serum IgA, IgM, and IgG (IgG1, IgG2b, and IgG3) in animals receiving exemplary TACI-Fc fusions in a mouse model of autoantibody-associated glomerulonephritis. DETAILED DESCRIPTION

[0096] Provided herein are immunomodulatory proteins that are engaged with one or more ligands (e.g., produced as soluble factors) to inhibit or reduce B cell responses or activity. The immunomodulatory proteins provided include proteins that bind to BAFF or APRIL ligands to neutralize their activity and block or antagonize the activity of B cell stimulatory receptors (e.g., TACI or BCMA). The immunomodulatory protein provided can be a fusion protein of the TACI extracellular domain or its binding portion (hereinafter TACI ECD) and a multimerization domain, such as an immunoglobulin Fc. For example, TACI-Fc fusion proteins are provided herein. In some embodiments, the immunomodulatory proteins provided herein can be used to treat diseases, disorders or conditions associated with dysregulated immune responses, such as those associated with inflammatory or autoimmune symptoms, including inflammatory diseases or autoimmune diseases.

[0097] The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and prevent tissues from being excessively damaged during immune responses (e.g., during attacks against pathogen infections). However, in some cases, the immune system may become unregulated and may initiate abnormal immune responses against normal body parts or tissues, leading to autoimmune diseases or illnesses or autoimmune symptoms. In other cases, an undesirable immune response may be initiated against foreign tissues such as transplants, leading to transplant rejection.

[0098] In some respects, immunotherapy that changes immune cell activity (such as B cell activity) can treat certain diseases, disorders and conditions of immune response disorders. Specifically, the suppression or weakening of immune response (such as B cell response) may be desirable for reducing or preventing undesirable inflammation, autoimmune symptoms and / or transplant rejection. However, the therapeutic method of pursuing the interaction between the ligand and its receptor to mediate immune response is not completely satisfactory. In some cases, the therapy for intervening and changing the immunomodulatory effect of immune cell (such as or B cell) activation is subject to the size restrictions imposed by the spatial orientation requirements and the immune synapse boundaries. In some respects, existing therapeutic drugs (including antibody drugs) may not be able to interact with the multiple target proteins involved in regulating these interactions at the same time. For example, soluble receptors and antibodies are usually competitively bound (for example, no more than one target species are bound at a time), and therefore lack the ability to bind multiple targets simultaneously. In addition, the pharmacokinetic differences between the drugs that independently target one of these receptors may cause difficulties in the whole treatment process in terms of the required blood concentration of the drug combination that appropriately maintains two different targets.

[0099] BAFF and APRIL are TNF superfamily members that bind to both TACI and BCMA receptors on B cells; BAFF also binds to a third receptor, namely BAFF receptor (BAFF-R). Both BAFF and APRIL can bind to and activate BCMA and TACI; BAFF also binds to and activates BAFF-R (Xu et al. 2020Cancers (Basel) 12 (4): 1045). BAFF and APRIL jointly support the development, differentiation and survival of B cells, particularly plasmablasts and plasma cells, and play a role in the pathogenesis of B cell-related autoimmune diseases. BAFF and APRIL were originally expressed as transmembrane proteins, mainly expressed on stromal cells and bone marrow-derived cells (Smulski et al. Front. Immunol. 2018 9: 2285), and can be cut to release soluble cytokines. BAFF circulates in the form of homotrimers, 60 polymers, or heterotrimers containing 2 APRILs and 1 BAFF or 2 BAFFs and 1 APRIL protomers. APRIL circulates as homotrimers or heterotrimers and can be localized to the intracellular matrix or the cell surface through interactions with heparan sulfate proteoglycans.

[0100] Under proinflammatory conditions, the expression of BAFF and APRIL increases (Smulski et al. 2018), and the elevated serum levels of these cytokines are associated with disease severity in patients with B cell-related autoimmune diseases including systemic lupus erythematosus (SLE) (Samy et al. Int. Rev. Immunol. 2017 36: 3-19). The binding of BAFF / APRIL to its receptor triggers events in the development, differentiation and activation of B cells and plasma cells. For example, the activation of BAFF-R contributes to the survival and maturation of transitional and immature B cells, while TACI participates in T cell-independent B cell responses to certain antigens, B cell regulation and immunoglobulin (Ig) class switching recombination. BCMA, which is upregulated in activated B cells, is important for the long-term survival of plasma cells.

[0101] Inhibitors of BAFF and / or APRIL have been studied in clinical trials for the treatment of a variety of autoimmune diseases or other B cell-related diseases. It has been approved for the treatment of SLE (Benlysta Product Information, 2020), and single-pathway inhibitors of APRIL (e.g., BION1301 and VIS649) are currently being evaluated in phase 2 studies [NCT04684745; NCT04287985].

[0102] Co-neutralization of BAFF and APRIL significantly reduces B cell function, including antibody production, while inhibition of either BAFF or APRIL alone mediates relatively small effects. Fc fusions of the wild-type (WT) extracellular domain of TACI and the Fc domain of IgG1 (e.g., atacicept and tacicept) are in clinical development and target both BAFF and APRIL. These dual BAFF / APRIL antagonists have been shown to inhibit the survival of immature and mature B cells and plasma cells while retaining B cell progenitors and memory B cells (Cogollo et al. 2015 Drug Des Devel Ther. 9: 1331-9; Samy et al. 2017; Zhao et al. 2016 J Clin Pharmacol. 56: 948-959). Both reduce serum IgG, IgM and IgA levels and the number of mature and total circulating B cells (Coggollo et al. 2015; Chen et al. 2014 Clin Pharmacokinet. 53: 1033-44; Chen et al. 2016 Br J Clin Pharmacol. 82: 41-52; Zhao et al. 2016). When directly compared to inhibition of BAFF or APRIL alone in nonclinical studies, dual inhibitors have shown more pronounced pharmacodynamic (PD) effects and greater modification of disease models (Ramanujam et al. 2006 J Clin Invest. 116: 724-34; Benson et al. 2008 J Immunol. 180: 3655-3659; Haselmeyer et al. 2017 Ur J. Immunol. 47: 1075-1085; Samy et al. 2017). Atacicept and tacicept have shown promising clinical potential in certain autoimmune diseases, such as systemic lupus erythematosus (SLE) and IgA nephropathy, but have not yet clearly demonstrated long-term and / or complete disease remission. Although B cell-targeted therapies have shown promising therapeutic potential, they are not completely satisfactory. For example, soluble recombinant TACI (e.g., atacicept or tacicept) shows great promise as a therapeutic drug, but its usefulness appears to be hampered by low to moderate affinity for APRIL.

[0103] The embodiments provided include those that provide improved neutralization activity and inhibition or reduction of B cell responses. In some embodiments, the improved activity is mediated by the increased or improved binding or interaction of the provided immunomodulatory protein (e.g., TACI-Fc fusion protein) with BAFF and / or APRIL. The provided immunomodulatory protein blocks or antagonizes the interaction of BAFF or APRIL (e.g., BAFF or APRIL homotrimers, BAFF / APRIL heterotrimers, or BAFF 60 polymers) with homologous B cell stimulatory receptors, thereby neutralizing the activity of BAFF and / or APRIL ligands. In some embodiments, the provided immunomodulatory protein reduces one or more B cell responses or activities, including the ability of B cells to produce immunoglobulins. In some embodiments, the provided immunomodulatory protein (e.g., TACI-Fc fusion protein) reduces circulating serum immunoglobulins when administered to a subject. In some embodiments, the provided immunomodulatory protein reduces one or more of B cell maturation, differentiation, and proliferation. In the aspects provided, such activity is improved or superior to the activity achieved by WT TACI-Fc fusion protein (e.g., tacicept or atacicept). In some embodiments, the immunomodulatory proteins (TACI-Fc fusion proteins) provided are candidate therapeutic drugs for the treatment of various autoimmune and inflammatory diseases, particularly B cell-related diseases (such as SLE, SjS and other connective tissue diseases).

[0104] The embodiments provided include methods and uses of specific Fc fusion proteins of TACI variant TNF receptor domains (TD, i.e., CRD2) that simultaneously inhibit BAFF and APRIL cytokines. The embodiments provided relate to the identification of variant TACI polypeptides, which are engineered to have improved affinity for APRIL and / or BAFF after random mutagenesis and directed evolution of the second cysteine-rich domain (CRD2) of TACI (spanning residues 68-110). As shown herein, affinity maturation includes five selections alternating between APRIL and BAFF, wherein the concentration of the selection agent is reduced in parallel to maintain the selection pressure. The results show that, as compared to wild-type TACI, the variant TACI polypeptide exhibits significantly enhanced affinity for BAFF and APRIL. For example, variant TACI polypeptides containing one or more amino acid substitutions (substitutions or mutations) are provided herein, which give the protein improved binding affinity for BAFF and / or APRIL. Specifically, the embodiments provided include those that provide improved combined BAFF and APRIL inhibition. Thus, provided immunomodulatory proteins provide effective and durable disease suppression in the treatment of autoimmune or inflammatory diseases, including severe B cell-related autoimmune diseases such as SLE.

[0105] For example, the provided embodiments are based on the discovery that directed evolution through affinity modification of the TNFR domain (TD) of the extracellular domain of TACI facilitates the development of molecules with improved affinity for APRIL and / or BAFF. Thus, the affinity modification produces variant TACI containing a variant TNFR domain (vTD). Fusion of such a molecule to an immunoglobulin Fc yields an immunomodulatory protein that inhibits B cell activity and response. For example, reformatted as a soluble Fc fusion protein, the affinity-matured TACI variant output exhibits inhibition of APRIL and BAFF, as shown herein in a TACI-dependent reporter assay, and its IC 50 The values ​​were lower than those of wild-type TACI-Fc and belimumab comparators. In addition, the results in the evaluated animal models showed rapid and significant reductions in key lymphocyte subsets, including plasma cells, germinal center B cells, and follicular T helper cells. In addition, the tested variant molecules exhibited improved activity in mouse models, including significantly reduced autoantibodies and sialadenitis in the spontaneous SjS model, suppressed glomerular IgG deposition in the bm12-induced lupus model, and effectively suppressed anti-dsDNA self-Abs, blood urea nitrogen levels, proteinuria, sialadenitis, kidney lesions, and renal immune complex deposition in the NZB / W lupus model. In addition, as compared to wild-type TACI-Fc, the tested TACI-Fc fusions exhibited significant and sustained reductions in serum IgM, IgG, and IgA antibody titers in mice. The findings herein demonstrate that these immunomodulatory proteins consistently exhibit potent immunosuppressive activity and efficacy in vitro and in vivo, outperforming existing and / or approved immunomodulators such as belimumab, abatacept, atacicept or tacicept. Such biologics may therefore be attractive development candidates for the treatment of severe autoimmune and / or inflammatory diseases, including B cell-related diseases such as SLE, Sjögren's syndrome and other connective tissue diseases.

[0106] In addition, the observations herein demonstrate that the TACI-Fc fusion protein exhibits high serum exposure when administered to mice and cynomolgus monkeys. The favorable and higher serum exposure and more effective immunosuppressive activity achieved by the provided TACI-Fc fusion protein support their use at lower clinical doses and / or reduced dosing frequency (or longer dosing intervals) than existing WT TACI-Fc therapeutics. For example, existing WT TACI-Fc therapeutics (such as tacicept and atacicept) must be administered at least once a week. Reducing the dosing frequency can provide better symptom control for the treated subjects, improve compliance with the dosing regimen, improve patient quality of life or patient satisfaction, and / or reduce the cost of receiving treatment in general. In addition, reducing the dose, even at a more regular frequency, such as once a week, can also alleviate certain adverse effects.

[0107] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If the definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0108] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Definitions

[0109] Unless otherwise defined, all art terms, symbols and other technical and scientific terms or nomenclature used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions contained herein should not be construed as representing a substantial difference from what is generally understood in the art.

[0110] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0111] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments for the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0112] The term "affinity modified" as used in the context of a domain of a protein means a mammalian protein having an altered amino acid sequence (relative to a corresponding wild-type parent or unmodified domain) in an extracellular domain or its specific binding portion such that it has an increased or decreased binding activity (such as binding affinity) with at least one of its binding partners (alternatively "anti-structures") compared to a parent wild-type or unmodified (i.e., non-affinity modified domain) protein. In some embodiments, an affinity modified domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences (such as amino acid substitutions) in a wild-type or unmodified domain. An increase or decrease in binding activity (e.g., binding affinity) may be determined using a well-known binding assay (including flow cytometry). Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005). See also Linsley et al., Immunity, 1: 7930801 (1994). An increase in the binding activity (e.g., affinity) of a protein to one or more of its binding partners is an increase to a value that is at least 10% greater than a wild-type control, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type control value. A decrease in the binding activity (e.g., affinity) of a protein to at least one of its binding partners is a decrease to a value that is no greater than 90% of the control but no less than 10% of the wild-type control value, and in some embodiments, no greater than 80%, 70%, 60%, 50%, 40%, 30%, or 20% but no less than 10% of the wild-type control value. Affinity-modified proteins are altered by substitution, addition or deletion of amino acid residues in the primary amino acid sequence of the extracellular domain or its specific binding portion. The term "affinity-modified" is not to be construed as imposing any conditions on any particular starting composition or method by which affinity-modified proteins are produced. Therefore, affinity-modified proteins are not limited to wild-type protein domains that are subsequently converted into affinity-modified domains by any particular affinity-modification process. Affinity-modified domain polypeptides can be generated, for example, starting from wild-type mammalian domain sequence information, then modeled in silico for binding to its binding partner, and finally recombined or chemically synthesized to produce affinity-modified domain compositions of the substance. In only one alternative example, affinity-modified domains can be produced by site-directed mutagenesis of wild-type domains.Thus, the affinity modified TD domain represents a product, and not necessarily one produced by any given method. A variety of techniques may be employed, including recombinant methods, chemical synthesis, or a combination thereof.

[0113] The term "affinity-modified TD domain" refers to an affinity-modified domain of a member of the tumor necrosis receptor superfamily (TNFRSF) protein or its TNF ligand, which has therein an altered amino acid sequence of a TNFR domain or a TNF domain, respectively. For example, the affinity-modified TD domain of a TNFRSF protein has an altered amino acid sequence of a TNFR domain, which is composed of at least one cysteine-rich domain (CRD) within the extracellular domain of the TNFRSF protein or a specific binding portion thereof (relative to the corresponding wild-type parent or unmodified domain), such that it has an increased or decreased binding activity (e.g., binding affinity) to at least one of its binding partners (alternatively "anti-structures") compared to a parent wild-type or unmodified protein containing a non-affinity-modified or unmodified TD domain.

[0114] "Affinity-modified TACI" (also referred to as variant TACI) refers to a TACI protein molecule that antagonizes or blocks the activity of a B cell stimulatory receptor. For example, TACI binds to APRIL and / or BAFF, which are ligands of B cell stimulatory receptors, namely B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), and transmembrane activator and calcium regulator and cyclophilin ligand interactor (TACI). In a specific embodiment, BIM includes the extracellular domain of TACI, or a portion of the extracellular domain of TACI that contains a TNF receptor family domain (e.g., TD, e.g., CRD) that binds to the cognate ligands APRIL and / or BAFF and heterotrimers of APRIL and BAFF. Affinity-modified variants of the extracellular domain of TACI or a portion thereof may include one or more amino acid modifications (e.g., amino acid substitutions) in the TD that increase the binding affinity to the cognate ligands (e.g., APRIL and / or BAFF, and heterotrimers of APRIL and BAFF).

[0115] As used herein, "B cell stimulatory receptor" refers to one or more of the following: B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), and transmembrane activator and calcium regulator and cyclophilin ligand interactor (TACI), which are related tumor necrosis factor (TNFR) superfamily receptors expressed on B cells. The engagement or connection of these related receptors with their cognate ligands BAFF and / or APRIL or heterotrimers of APRIL and BAFF regulates B cell homeostasis, including B cell survival, B cell maturation and differentiation, and immunoglobulin class switching. B cell stimulatory receptors generally contain an extracellular portion, a transmembrane domain, and a cytoplasmic region, wherein the cytoplasmic region contains one or more TNF receptor-associated factor (TRAF) binding sites. Recruiting various TRAF molecules to the cytoplasmic domain can activate various transcription factors, such as NF-κB (e.g., NF-κB1 or NF-κB2), mediating B cell signaling pathways that regulate B cell homeostasis.

[0116] As used herein, "bind", "bound" or grammatical variations thereof refers to any attractive interaction that a molecule participates in with another molecule, resulting in a stable association in which the two molecules are in close proximity to each other. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as chemical compounds including drugs.

[0117] As used herein, binding activity refers to a characteristic of a molecule (e.g., a polypeptide) related to whether and how it binds to one or more binding partners. Binding activity can include any measure of the binding of a molecule to a binding partner. Binding activity includes the ability to bind to one or more binding partners, the affinity (e.g., high affinity) with which it binds to a binding partner, the avidity with which it binds to a binding partner, the strength of the bond to a binding partner, and / or the specificity or selectivity with which it binds to a binding partner.

[0118] As used herein, the term "binding affinity" means the specific binding affinity of a protein to its binding partner (i.e., its anti-structure) under specific binding conditions. Binding affinity refers to the intensity of the interaction between two or more molecules (such as binding partners), usually the intensity of the non-covalent interaction between two binding partners. The increase or decrease in the binding affinity of an affinity-modified domain or an immunomodulatory protein containing an affinity-modified domain to a binding partner is determined relative to the binding affinity of an unmodified domain (e.g., a natural or wild-type TD domain). Methods for determining binding affinity or relative binding affinity are known in the art, solid phase ELISA immunoassays, ForteBio Octet, Biacore measurements, or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol. 1 (9): 793-801 (1994). In some embodiments, binding affinity can be measured by flow cytometry, such as based on mean fluorescence intensity (MFI) in a flow binding assay.

[0119] As used herein, the term "binding affinity" means the specific binding affinity of a protein for its binding partner (i.e., its inverse structure) under specific binding conditions. In biochemical kinetics, avidity refers to the cumulative strength of multiple affinities of separate non-covalent binding interactions, such as between a protein and its binding partner (i.e., its inverse structure). Therefore, avidity is different from affinity, which describes the strength of a single interaction.

[0120] The term "biological half-life" refers to the length of time it takes for a substance (such as an immunomodulatory protein) to lose half of its pharmacological or physiological activity or concentration. Biological half-life may be affected by the elimination, excretion, degradation (e.g., enzymatic degradation / digestion) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derive and increase the biological half-life of proteins are known in the art and include, but are not limited to, multimerization domains (e.g., Fc immunoglobulin domains), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0121] As used herein, the term "cell surface anti-structure" (alternatively "cell surface binding partner") is an anti-structure (alternatively a binding partner) expressed on a mammalian cell. Typically, the cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor.

[0122] The term "binding partner" or "anti-structure" with respect to a protein (e.g., a receptor, a soluble ligand) or with respect to an extracellular domain or portion thereof or an affinity-modified variant thereof refers to at least one molecule (typically a native mammalian protein) to which a reference protein specifically binds under specific binding conditions. In some aspects, an affinity-modified domain or an immunomodulatory protein containing an affinity-modified domain specifically binds to a binding partner of the corresponding domain of a native or wild-type protein, but with increased or decreased affinity. A "cell surface binding partner" is a binding partner expressed on a mammalian cell. Typically, the cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor or a ligand of a receptor expressed on or by a cell (e.g., a mammalian cell, e.g., an immune cell) that forms an immune synapse.

[0123] The term "cis" with respect to binding to a cell surface molecule refers to binding to two or more different cell surface molecules, each of which is present on the surface of the same cell. In some embodiments, cis means that the two or more cell surface molecules are present only on one of the two mammalian cells forming the IS or only on the other (but not both).

[0124] As used herein, the term "conservative amino acid substitution" means an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of amino acid groups with side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0125] The term "corresponding to" with respect to a protein position, such as the recitation that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence, as described in the sequence listing, refers to the nucleotide or amino acid position identified after alignment with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm (such as the GAP algorithm). By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Fig. 9 Identification of corresponding residues by alignment of two sequences is exemplified.

[0126] As used herein, a "domain" (usually three or more, generally 5 or 7 or more amino acids, such as 10 to 200 amino acid residues) refers to a portion of a molecule (e.g., a protein or encoding nucleic acid) that is structurally and / or functionally different from the rest of the molecule and is identifiable. For example, domains include those portions of a polypeptide chain that can form an independently folded structure within a protein consisting of one or more structural motifs and / or be recognized by functional activity (e.g., binding activity). A protein can have one or more unique domains. For example, a domain can be identified, defined, or distinguished based on homology of primary sequence or structure to related family members (e.g., homology to a motif). In another example, a domain can be distinguished by its function, such as the ability to interact with a biological molecule (e.g., a cognate binding partner). A domain can independently exhibit a biological function or activity, such that the domain can be active, such as binding, independently or after fusion with another molecule. A domain can be a linear amino acid sequence or a nonlinear amino acid sequence. A variety of polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For the examples herein, definitions are provided, but it should be understood that it is within the technical scope of the art to identify specific domains by name. If necessary, appropriate software can be used to identify domains. It should be understood that the reference to amino acids, including reference to specific sequences shown as SEQ ID NOs for describing domain organization (e.g., domain organization of TD domains), is for illustrative purposes and is not intended to limit the scope of the embodiments provided. It should be understood that the description of polypeptides and their domains is theoretically derived based on homology analysis and comparison with similar molecules. Similarly, in some cases, the adjacent N and / or C-terminal amino acids of a given domain (e.g., TD) may also be included in the sequence, for example, to ensure that the domain is properly folded when expressed. Therefore, the exact locus is variable and not necessarily the same for each protein. For example, a specific TD domain (e.g., a specific CRD domain) may be several amino acids long or short (1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).

[0127] The terms "extracellular domain", "extracellular domain" or "ECD" used interchangeably herein refer to a region of a membrane protein located outside the vacuole membrane (e.g., the space outside the cell) when the full-length form of the membrane protein (e.g., a transmembrane protein) is expressed from a cell. For the purposes of this article, it is understood that reference to ECD refers to the sequences and domains that constitute this region, and there is no requirement that the protein containing the ECD is a membrane protein or that the domain is present outside the cell. For example, a soluble immunomodulatory protein may contain an ECD sequence of a membrane protein fused to another portion (e.g., a multimerization domain, such as an Fc region). The extracellular domain typically interacts with a specific ligand or a specific cell surface receptor, for example, by specifically binding to a binding domain of a ligand or a cell surface receptor. Examples of binding domains include cysteine-rich domains (CRDs). The extracellular domains of members of the TNFR superfamily contain TD domains (e.g., CRD domains). Therefore, reference to ECD herein includes the full-length sequence of the ECD of a membrane protein, as well as specific binding fragments containing CRDs that bind to a ligand or a cognate binding partner.

[0128] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition (such as containing an immunomodulatory protein or Fc fusion protein) that produces a statistically significant inhibition of disease progression (such as, for example, by improving or eliminating the symptoms and / or causes of the disease) when administered ex vivo (by contacting with cells from the patient) or in vivo (by administering to the patient) alone (i.e., as a monotherapy) or in combination with another therapeutic agent. The effective amount for treating a disease, condition or disorder (such as an immune system disease, condition or disorder) can be an amount that alleviates, reduces or relieves at least one symptom or biological response or effect associated with the disease, condition or disorder, prevents the progression of the disease, condition or disorder, or improves the patient's physical function. In the case of cell therapy, the effective amount is the effective dose or amount of cells administered to the patient. In some embodiments, the patient is a human patient.

[0129] As used herein, fusion protein refers to a polypeptide encoded by a nucleic acid sequence containing a coding sequence of two or more proteins (in some cases, 2, 3, 4, 5 or more proteins), wherein the coding sequence is located in the same reading frame, so that when the fusion construct is transcribed and translated in a host cell, a protein containing the two or more proteins is produced. Each of the two or more proteins can be adjacent to another protein in the construct, or separated by a linker polypeptide, which contains 1, 2, 3 or more amino acids, but is generally less than 20, 15, 10, 9, 8, 7 or 6 amino acids. The protein product encoded by the fusion construct is referred to as a fusion polypeptide. An example of a fusion protein according to the embodiment provided is an Fc fusion protein containing an affinity-modified domain connected to an immunoglobulin Fc domain (e.g., a variant of a TACI extracellular domain or a portion thereof containing a CRD).

[0130] The term "half-life extension moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of a protein not so conjugated to the moiety. In some embodiments, the half-life is extended by greater than or about 1.2 times, about 1.5 times, about 2.0 times, about 3.0 times, about 4.0 times, about 5.0 times, or about 6.0 times. In some embodiments, after in vivo administration, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week compared to a protein without a half-life extension moiety. Half-life refers to the length of time it takes for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extending moieties include Fc domains, multimerization domains, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see, WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS) and polyglutamic acid (glutamylated).

[0131] The Fc (crystallizable fragment) region or domain (also referred to as Fc polypeptide) of an immunoglobulin molecule corresponds primarily to the constant region of an immunoglobulin heavy chain, and is responsible for a variety of functions in some cases, including one or more effector functions of an antibody. The Fc domain contains part or all of the hinge domain of an immunoglobulin molecule plus the CH2 and CH3 domains. In some cases for inclusion in the fusion protein provided, all or part of the Fc hinge sequence may be missing. The Fc domain can form a dimer of two polypeptide chains joined by one or more disulfide bonds. In some embodiments, Fc is a variant Fc that exhibits reduced (e.g., reduced by greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) activity to promote effector function. In some embodiments, unless described with reference to a specific SEQ ID NO, reference to amino acid substitutions in the Fc region is by the EU numbering system. EU numbering is known and is in accordance with the most recently updated IMGTScientific Chart ( International ImMunoGeneTics http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: May 17, 2001, last updated: January 10, 2013) and the EU index as reported in Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0132] Immunoglobulin Fc fusions ("Fc fusions") (such as immunomodulatory Fc fusion proteins) are molecules comprising one or more polypeptides operably linked to the Fc region of an immunoglobulin. The Fc fusion may comprise, for example, an Fc region operably linked to the TACI extracellular domain or a portion thereof containing a CRD (including any provided affinity-modified variants thereof). The immunoglobulin Fc region may be indirectly or directly linked to one or more polypeptides. Various linkers are known in the art and may optionally be used to connect the Fc to a fusion partner to produce an Fc fusion. Fc fusions of the same type may dimerize to form Fc fusion homodimers. Fc fusions of different types (e.g., knob-and-hole structure engineering) may be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a mouse or human Fc.

[0133] The term "host cell" refers to any cell that can be used to express a protein encoded by a recombinant expression vector. The host cell can be a prokaryotic organism, such as Escherichia coli (E. coli), or it can be a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium, or CHO strain DX-B11 lacking DHFR.

[0134] As used herein, the term "immunological synapse" or "immunesynapse" (abbreviated as "IS") refers to the interface between a mammalian cell (e.g., an antigen presenting cell or a tumor cell) expressing MHC I (major histocompatibility complex) or MHC II and a mammalian lymphocyte (e.g., an effector T cell or a natural killer (NK) cell).

[0135] As used herein, the term "immunoglobulin" (abbreviated as "Ig") is synonymous with the term "antibody" (abbreviated as "Ab") and refers to mammalian immunoglobulins, including any of the five human classes: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. The term also includes less than full-length immunoglobulins, whether fully or partially synthetic (e.g., recombinant or chemically synthesized) or naturally occurring, including any fragment thereof containing at least a portion of the variable heavy (VH) chain and / or variable light (VL) chain region of the immunoglobulin molecule, the fragment being sufficient to form an antigen binding site and sufficient to specifically bind to an antigen when assembled. Antibodies may also include all or a portion of a constant region. Such fragments include antigen binding fragments (Fab), variable fragments (Fv) containing VH and VL, single-chain variable fragments (scFv) containing VH and VL connected in one chain, and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc and Fd polypeptide fragments. Therefore, it should be understood that when referring to antibodies herein, full-length antibodies and antigen binding fragments are included. The term antibody also includes antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules. Bispecific antibodies of the same bispecificity and heterobispecificity are included in the meaning of the term. Antibodies include polyclonal antibodies or monoclonal antibodies. Antibodies also include synthetic antibodies or recombinantly produced antibodies. For the structure and properties of different antibody classes, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0136] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially complete form, as opposed to an antibody fragment. A full-length antibody is an antibody that typically has two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies produced by secretory B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.) and antibodies produced synthetically with the same domains. Specifically, whole antibodies include those with heavy and light chains (including Fc regions). The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, a complete antibody may have one or more effector functions.

[0137] "Antibody fragments" include a portion of an intact antibody, the antigen binding region and / or the variable region of the intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules, including single-chain Fv (scFv) or single-chain Fab (scFab); antigen-binding fragments of any of the above items and multispecific antibodies derived from antibody fragments.

[0138] "Fv" is composed of a heavy chain variable region domain and a light chain variable region domain connected by non-covalent association. Six complementary determining regions (CDRs) (3 each for the heavy chain and light chain) emanate from the folding of these two domains, which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even if a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, in some cases, its affinity is lower than the entire binding site.

[0139] “dsFv” refers to a stable V H -V L Fv with engineered intermolecular disulfide bonds.

[0140] The "Fd fragment" is a fragment containing the variable domain of the antibody heavy chain (V H ) and a constant region domain (C H 1).

[0141] A "Fab fragment" is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment of the same structure produced synthetically (e.g., by recombinant methods). A Fab fragment contains the light chain (containing the V L and C L ) and containing the heavy chain variable domain (V H ) and a constant region domain of the heavy chain (C H 1) Another chain.

[0142] “F(ab') 2 "Fragments" are antibody fragments obtained by digestion of immunoglobulin with pepsin at pH 4.0-4.5, or fragments of the same structure produced synthetically (e.g., by recombinant methods). F(ab') 2The fragments essentially contain two Fab fragments, wherein each heavy chain portion contains an additional few amino acids including a cysteine ​​residue that forms a disulfide bond joining the two fragments.

[0143] "Fab' fragment" is a fragment containing F(ab') 2 A fragment of half of the fragment (one heavy chain and one light chain).

[0144] The "Fd' fragment" is a fragment containing F(ab') 2 A fragment of an antibody that is a heavy chain portion of the antibody.

[0145] "Fv' fragment" is a V fragment containing only an antibody molecule H and V L Fragments of the domain.

[0146] A "scFv fragment" refers to a fragment comprising variable light chains (V L ) and variable heavy chain (V H ). The length of the linker allows the two variable domains to be bridged without being seriously disturbed. An exemplary linker is (Gly-Ser) n residues, some of which are Glu or Lys residues scattered throughout to increase solubility.

[0147] "Diabodies" are dimeric scFvs; diabodies typically have a shorter peptide linker than scFvs and preferentially dimerize.

[0148] As used herein, the term "immune activity" refers to one or more activities of immune cells (such as T cells or B cells), including, for example, activation, cell survival, cell proliferation, cytokine production (such as interferon-γ), cytotoxic activity, or the ability to activate the NF-κB pathway or other signal transduction cascades leading to activation of transcription factors in immune cells. The assay for evaluating the immunological activity of an immunomodulatory protein can be compared with a control protein with inhibitory activity.

[0149] "Immunomodulatory protein" or "immunomodulatory polypeptide" is a protein that regulates immune activity. "Modulation" or "modulating" immune response means an immune activity that is enhanced or suppressed. Such regulation includes any induction, or change in degree or scope, or inhibition of the immune activity of immune cells (such as B cells or T cells). For example, the soluble Fc fusion protein herein can inhibit the immune activity of B cells. The immunomodulatory protein can be a single polypeptide chain or a polymer (dimer or higher polymer) of at least two polypeptide chains that are covalently bonded to each other by, for example, interchain disulfide bonds. Therefore, monomers, dimers and higher polymeric proteins are within the scope of the defined terms. Multimeric proteins can be homopolymeric (having the same polypeptide chains) or heteropolymeric (having different polypeptide chains).

[0150] As used herein, modification refers to the modification of the amino acid sequence of a polypeptide or the nucleotide sequence in a nucleic acid molecule, and includes changes in the amino acids or nucleotides of the sequence, respectively. Amino acid modifications or changes can be amino acid or nucleotide deletions, insertions or substitutions (replacements), respectively. Methods for modifying polypeptides are conventional for those skilled in the art, such as by using recombinant DNA methods.

[0151] The term "multimerization domain" refers to an amino acid sequence that promotes the formation of a polymer of two or more polypeptides. The multimerization domain includes a sequence that promotes the stable interaction of a polypeptide molecule with one or more other polypeptide molecules, and each other polypeptide molecule contains a complementary multimerization domain (e.g., a first multimerization domain and a second multimerization domain), which can be the same or different multimerization domains. The interaction between the complementary multimerization domains (e.g., the interaction between the first multimerization domain and the second multimerization domain) forms a stable protein-protein interaction to produce a multimer of a polypeptide molecule and another polypeptide molecule. In some cases, the multimerization domain is identical and interacts with itself to form a stable protein-protein interaction between two polypeptide chains. Typically, a polypeptide is engaged with a multimerization domain directly or indirectly. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. For example, the multimerization domain may be an immunoglobulin constant region or domain, such as, for example, an Fc domain or portion thereof from IgG (including IgGl, IgG2, IgG3 or IgG4 subtypes), IgA, IgE, IgD and IgM, and modified forms thereof.

[0152] The term "nucleic acid" is used interchangeably with "polynucleotide" and refers to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form. Unless explicitly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, and the analogs have binding properties similar to natural nucleotides and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implies that conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences are encompassed as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0153] As used herein, the terms "in an operable combination," "in an operable order," and "operably linked" refer to the connection or orientation of nucleic acid sequences such that the arrangement of the segments allows them to act synergistically for their intended purpose. In some embodiments, the terms refer to the connection of nucleic acids to produce a nucleic acid molecule capable of directing transcription of a given gene, and / or to produce a desired protein molecule that is functional. For example, segments of a DNA sequence (e.g., a coding sequence and one or more regulatory sequences) are linked in such a way as to allow gene expression when an appropriate molecule (e.g., a transcriptional activator) binds to the regulatory sequence.

[0154] The term "pharmaceutical composition" refers to a composition suitable for medical use in a mammalian subject (usually a human). A pharmaceutical composition generally comprises an effective amount of an active agent (e.g., an immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is generally a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0155] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked by peptide bonds. The terms do not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The term includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. The term also includes molecules in which one or more amino acid analogs or non-classical or non-natural amino acids are included, such as those that can be synthesized or recombinantly expressed using known protein engineering techniques. In addition, proteins can be derived by well-known organic chemistry techniques as described herein.

[0156] The term "purified" as applied to nucleic acids, such as nucleic acids encoding immunomodulatory proteins or proteins (e.g., immunomodulatory proteins), generally refers to nucleic acids or polypeptides that are substantially free of other components as determined by analytical techniques well known in the art (e.g., purified polypeptides or polynucleotides form discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or protein is at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., by weight or on a molar basis).

[0157] The term "recombinant" indicates that a material (e.g., a nucleic acid or a polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The material in its natural environment or state can be altered or the material taken out from its natural environment or state can be altered. For example, "recombinant nucleic acid" is prepared by recombining nucleic acids in, for example, cloning, affinity modification, DNA shuffling, or other well-known molecular biological processes. "Recombinant DNA molecules" are composed of DNA segments joined together by such molecular biological techniques. As used herein, the terms "recombinant protein" or "recombinant polypeptide" refer to protein molecules (e.g., immunomodulatory proteins) expressed using recombinant DNA molecules. "Recombinant host cells" are cells containing and / or expressing recombinant nucleic acids or otherwise altered by genetic engineering, for example, by introducing nucleic acid molecules encoding recombinant proteins (e.g., immunomodulatory proteins provided herein) into cells. Transcription control signals in eukaryotic organisms include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (similar control elements, i.e., promoters, are also found in prokaryotes.) The choice of a particular promoter and enhancer depends on the cell type used to express the protein of interest.

[0158] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing a desired coding sequence (e.g., encoding an immunomodulatory protein) and a suitable nucleic acid sequence required for expressing an operably connected coding sequence in a specific cell. The nucleic acid sequence required for expression in prokaryotes includes a promoter, optionally an operator sequence, a ribosome binding site, and possible other sequences. Known eukaryotic cells utilize promoters, enhancers, and termination and polyadenylation signals. The secretion signal peptide sequence can also be optionally encoded by a recombinant expression vector, which is operably connected to the coding sequence so that the expressed protein can be secreted by the recombinant host cell, such as for its expression as a secretable protein or for more easily separating or purifying the immunomodulatory protein from the cell (if necessary). The term includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the host cell genome into which it has been introduced. In the vector there is a viral vector, such as a lentiviral vector.

[0159] As used herein, the term "sequence identity" refers to the sequence identity between a gene or protein at the nucleotide or amino acid level, respectively." sequence identity" is a measure of the identity between proteins at the amino acid level and a measure of the identity between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence when the alignment sequence is compared. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the alignment sequence is compared. Methods for comparing sequences are well known in the art, and such methods include GAP, BESTFIT, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software, FASTA and TFASTA. The BLAST algorithm calculates sequence identity percentages and performs statistical analysis on the similarity between two sequences. Software for performing BLAST analysis can be publicly obtained through the National Center for Biotechnology Information (NCBI) website. In some cases, the percent sequence identity can be determined as the percentage of amino acid residues (or nucleotide residues) in the candidate sequence that are identical to the amino acid residues (or nucleotide residues) in the reference sequence, after aligning the sequences and introducing spaces (if necessary) to achieve the maximum percent sequence identity. Reference to sequence identity includes sequence identity across the full length of each sequence compared. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0160] The term "soluble" as used herein for proteins means that the protein is not a membrane protein or is not anchored in a cell membrane. Proteins can be constructed as soluble proteins by including only an extracellular domain or a portion thereof and without a transmembrane domain. In some cases, the solubility of the protein can be improved by directly or indirectly connecting or attaching to an Fc domain or other half-life extending molecule via a linker, which in some cases can also improve the stability and / or half-life of the protein. In some aspects, the soluble protein is an Fc fusion protein.

[0161] As used herein, the term "specific binding" means that the protein binds to the target protein under specific binding conditions, so that its affinity or avidity is at least 10 times the average affinity or avidity of the same protein and a sufficient statistical random peptide or polypeptide set, but optionally 50, 100, 250 or 500 times, or even at least 1000 times the ability. Specific binding protein need not be combined with only a single target molecule, but can be specifically bound to more than one target molecule. In some cases, the specific binding protein can be combined with a protein (e.g., a paralog or an ortholog) with a structural conformation similar to the target protein. Technicians will recognize that it is possible to specifically bind to molecules (i.e., orthologs) with the same function in animals of different species or molecules (e.g., paralogs) with substantially similar epitopes to the target molecule, and will not weaken the binding specificity determined by a statistically effective set relative to a unique non-target (e.g., random polypeptide). Therefore, the immunomodulatory protein of the present invention can be specifically bound to more than one different type of target molecule due to cross-reactivity. Solid phase ELISA immunoassays, ForteBio Octet or Biacore measurements can be used to determine the specific binding between two proteins. Typically, the dissociation constant (Kd) for the interaction between two binding proteins is less than about 1 x 10 - 5 M, and usually as low as about 1x 10 -12 In certain aspects of the present disclosure, the dissociation constant of the interaction between the two binding proteins is less than about 1 x 10 -6 M, 1x 10 -7 M, 1x 10 -8 M, 1x 10 -9 M, 1x 10 -10 M or 1x 10 -11 M or smaller.

[0162] As used herein with respect to proteins, the term "specific binding fragment" or "fragment" means a polypeptide that is shorter than the full-length protein or a specific domain or region thereof and specifically binds to a binding partner of the full-length protein or specific domain or region in vitro and / or in vivo. A specific binding fragment is a fragment of a full-length extracellular domain of a polypeptide or a binding domain of a polypeptide, but still binds to a binding partner of the binding domain. For example, a specific binding fragment is a fragment of an extracellular domain of a full-length TNFR family member or its full-length TNFR domain (TD) (e.g., CRD), but still binds to a binding partner of the TNFR family member or the CRD of a TNFR family member. In some embodiments, the specific binding fragment has at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence length of the full-length sequence of the extracellular domain or a domain or region of the extracellular domain. In some embodiments, the specific binding fragment may have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific binding fragment includes a CRD1 and / or CRD2 domain. In some embodiments, the specific binding fragment includes a CRD2 domain.

[0163] As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. The subject can be male or female, and can be of any suitable age, including infants, young children, adolescents, adults, and elderly subjects.

[0164] As used herein, "synthetic" with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or a polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0165] As used herein, the term "TNF receptor superfamily" or "TNFRSF" refers to a group of cell surface cytokine receptors, which are all type I (N-terminal extracellular) transmembrane glycoproteins containing one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on shared structural features, including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular regions, which generally play a role in the binding of proteins to their cognate binding partners or ligands. TNFRSF proteins may have only one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or extracellular domain of TNFRSF members contains between 1 and 6 CRD pseudo-repeats. For example, the BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are typically trimerized or multimerized complexes that are stabilized by disulfide bonds within their cysteine ​​residues. Binding of TNFRSF proteins to their ligands promotes a variety of biological activities in cells, such as induction of apoptotic cell death or cell survival and proliferation.

[0166] The term "TD" refers to one or more structural domains of a TNFRSF protein or a TNF family ligand. For example, the TD of a TNFRSF protein is a cysteine-rich domain (CRD) module of about 40 amino acids that contains six (6) conserved cysteines. Therefore, reference to the CRD may also be used interchangeably with the term TD for the TD of a TNFRSF protein. The six cysteines participate in the formation of intrachain disulfide bonds. The extracellular domain (ECD) of a TNFRSF member contains one or more CRD domains; therefore, the term TD is also used with respect to the ECD of such a protein molecule. Reference to a variant TD (vTD) refers to a variant or modified sequence of a TD.

[0167] The term "trans" with respect to binding to a cell surface molecule refers to binding to two different cell surface molecules, each of which is present on the surface of a different cell. In some embodiments, trans means that with respect to the two different cell surface molecules, the first is present only on one of the two mammalian cells that form the IS, and the second is present only on the other of the two mammalian cells that form the IS.

[0168] The term "transmembrane protein" as used herein means a membrane protein that substantially or completely spans a lipid bilayer, such as those found in biological membranes (such as mammalian cells) or in artificial constructs (such as liposomes). Transmembrane proteins include a transmembrane domain ("transmembrane domain"), which is integrated into a lipid bilayer by the transmembrane domain, and the integration is thermodynamically stable under physiological conditions by the transmembrane domain. Transmembrane domains can generally be applied by any number of commercially available bioinformatics software, based on the hydrophobicity that is improved relative to the protein region that interacts with an aqueous environment (such as cytosol, extracellular fluid), predicted from its amino acid sequence. Transmembrane domains are generally hydrophobic alpha helices that span membranes. Transmembrane proteins can pass through two layers of lipid bilayers one or more times.

[0169] As used herein, the terms "treating", "treatment" or "therapy" of a disease or disorder means slowing, stopping or reversing the progression of a disease, disorder or disorder, as evidenced by a reduction, cessation or elimination of clinical or diagnostic symptoms, by administering an immunomodulatory protein or engineered cell of the invention alone or in combination with another compound as described herein. "Treating", "treatment" or "therapy" also means a reduction in the severity of symptoms of an acute or chronic disease, disorder or disorder, or a reduction in the recurrence rate, such as in the case of a relapsing or remitting autoimmune disease course or inflammatory disorder, or a reduction in inflammation in the case of the inflammatory aspects of an autoimmune disease or inflammatory disorder. "Preventing", "prophylaxis" or "prevention" of a disease, disorder or disorder as used in the context of the present invention refers to administering an immunomodulatory protein of the invention alone or in combination with another compound to prevent the occurrence or onset of a disease, disorder or disorder or some or all symptoms of a disease, disorder or disorder, or to reduce the likelihood of the onset of a disease, disorder or disorder.

[0170] The term "variant" (also referred to as "modified" or "mutant", which can be used interchangeably) as used in reference to variant proteins or polypeptides means a protein produced by human intervention, such as a mammalian (e.g., human or mouse) protein. A variant is a polypeptide having an altered or modified amino acid sequence relative to an unmodified or wild-type protein or relative to a domain thereof, such as by one or more amino acid substitutions, deletions, additions, or a combination thereof. A variant polypeptide can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences (e.g., amino acid substitutions). Variant polypeptides typically exhibit at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the corresponding form of the wild-type or unmodified protein (such as its mature sequence (lacking a signal sequence) or its portion containing the extracellular domain or its binding domain). Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions. Variant proteins are not limited to any particular method of preparation, and include, for example, chemical synthesis, recombinant DNA technology, or a combination thereof. Variant proteins of the present invention specifically bind to at least one or more binding partners. In some embodiments, the altered amino acid sequence results in altered (i.e., increased or decreased) binding activity, such as binding affinity or avidity, to one or more binding partners. Variant proteins may therefore be "affinity-modified" proteins as described herein.

[0171] As used herein, the terms "wild type" or "natural" or "native" are used interchangeably in connection with biological materials (such as nucleic acid molecules, proteins, host cells, etc.) that are found in nature and have not been modified by human intervention. II. TACI Immunomodulatory Proteins and Variant TACI Polypeptides

[0172] Provided herein are TACI immunomodulatory proteins containing the extracellular domain (ECD) of the TACI receptor or a portion thereof that binds to at least one TACI cognate binding partner. Also provided herein are variant TACI polypeptides that exhibit altered (e.g., increased) binding activity or affinity to one or more TACI cognate binding partners. In some embodiments, the TACI cognate binding partner is one or more of BAFF or APRIL or is a BAFF / APRIL heterotrimer. Provided are TACI immunomodulatory proteins and polypeptides including soluble fusion proteins thereof, wherein the TACI portion of the extracellular domain or a portion thereof is linked to another portion (e.g., an immunoglobulin Fc or other multimerization domain or half-life extension portion). Thus, in some embodiments, the immunomodulatory protein is a TACI-Fc fusion protein. In some embodiments, a TACI-Fc fusion protein is provided that contains (1) a TACI polypeptide, the TACI polypeptide being composed of the extracellular domain of the TACI receptor or a portion thereof that binds to at least one TACI cognate binding partner or a variant TACI polypeptide thereof, and (2) an Fc domain. The TACI polypeptide or variant TACI polypeptide may be linked directly or indirectly (eg, via a peptide linker) to the Fc domain.

[0173] TACI is a member of the tumor necrosis factor receptor family, characterized by having an extracellular domain (ECD) containing a cysteine-rich pseudo-repeat domain (CRD). TACI is a membrane-bound receptor having an extracellular domain containing two cysteine-rich pseudo-repeat sequences (CRD1 and CRD2), a transmembrane domain, and a cytoplasmic domain that interacts with CAML (calcium regulator and cyclophilin ligand), which is an integral membrane protein located at intracellular vesicles and is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with a subset of B cells and T cells. The TACI receptor binds two members of the tumor necrosis factor (TNF) ligand family. One ligand is named BAFF (B cell activating factor of the TNF family), and is also variously named ZTNF4, "neutrokine-α," "BLyS," "TALL-1," and "THANK" (Yu et al., International Publication No. WO 98 / 18921 (1998); Moore et al., Science 285:269 (1999); Mukhopadhyay et al., J. Biol. Chem. 274:15978 (1999); Schneider et al., J. Exp. Med. 189:1747 (1999); Shu et al., J. Leukoc. Biol. 65:680 (1999)). Another ligand has been named APRIL, and has also been variously named "ZTNF2" and "TNRF death ligand-1" (Hahne et al., J. Exp. Med. 188: 1185 (1998); Kelly et al., Cancer Res. 60: 1021 (2000)). Both ligands are also bound by the B cell maturation receptor (BCMA) (Gross et al., Nature 404: 995 (2000)). Binding of the TACI receptor to its ligands BAFF or APRIL stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.

[0174] The amino acid sequence of full-length TACI is shown in SEQ ID NO:88. The protein is a type III membrane protein and lacks a signal peptide; after expression in eukaryotic cells, the N-terminal methionine is removed. In some embodiments, the mature TACI protein does not contain the N-terminal methionine as shown in SEQ ID NO:88. The extracellular domain of TACI (amino acid residues 1-166 of SEQ ID NO:88; ECD shown in SEQ ID NO:122) contains two cysteine-rich domains (CRDs, also referred to as tumor necrosis family receptor domains or TDs below), each of which exhibits affinity for binding to BAFF and APRIL. The first cysteine-rich domain (CRD1) contains amino acid residues 34-66 of the sequence shown in SEQ ID NO:122. The second cysteine-rich domain (CRD2) corresponds to amino acids 71-104 of the sequence shown in SEQ ID NO:122. TACI also contains a stem region of approximately 60 amino acids following the second cysteine ​​repeat in the extracellular domain, corresponding to amino acid residues 105-165 of the sequence shown in SEQ ID NO:122.

[0175] In some embodiments, the variant TACI polypeptides provided herein contain one or more amino acid modifications, such as one or more substitutions (alternatively "mutations" or "substitutions"), deletions or additions, in the extracellular domain of a reference TACI polypeptide, such as a wild-type or unmodified TACI polypeptide containing a CRD (hereinafter also referred to as a TD). Thus, the variant TACI polypeptides provided are or comprise a variant TD ("vTD"), wherein the one or more amino acid modifications (e.g., substitutions) are located in the CRD. In some embodiments, the one or more amino acid modifications (such as one or more substitutions (alternatively "mutations" or "substitutions"), deletions or additions) are located in the CRD1 region. In some embodiments, the one or more amino acid modifications (such as one or more substitutions (alternatively "mutations" or "substitutions"), deletions or additions) are located in the CRD2 region. In some embodiments, the one or more amino acid modifications (such as one or more substitutions (alternatively "mutations" or "substitutions"), deletions or additions) are located in amino acids within both the CRD1 region and the CRD2 region.

[0176] In some embodiments, the reference (e.g., unmodified) TACI sequence is a wild-type TACI sequence or a portion thereof containing one or two CRDs. In some embodiments, the reference (e.g., unmodified) TACI is or comprises the extracellular domain (ECD) of TACI or a portion thereof containing one or two CRD domains. In some embodiments, the extracellular domain of the reference (e.g., unmodified) TACI polypeptide comprises CRD1 and CRD2. However, the variant TACI polypeptide need not comprise both CRD1 and CRD2. In some embodiments, the variant TACI polypeptide comprises CRD1 or its specific binding fragment or consists essentially of it. In some embodiments, the variant TACI polypeptide comprises CRD2 or its specific binding fragment or consists essentially of it. In some embodiments, the variant TACI is a soluble polypeptide and lacks a transmembrane domain. In some embodiments, the variant TACI polypeptide also comprises a transmembrane domain, and in some cases also comprises a cytoplasmic domain.

[0177] In some embodiments, the reference (e.g., unmodified) TACI sequence is a mammalian TACI sequence. In some embodiments, the reference (e.g., unmodified) TACI sequence can be a mammalian TACI, including but not limited to human, mouse, cynomolgus monkey or rat TACI. In some embodiments, the reference (e.g., unmodified) TACI sequence is human. The extracellular domain of an exemplary human TACI sequence is shown in SEQ ID NO: 122.

[0178] In some embodiments, the reference (e.g., unmodified) TACI sequence has (i) the amino acid sequence shown in SEQ ID NO: 122 or a sequence lacking the N-terminal methionine, (ii) an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 122 and binds to APRIL, BAFF or APRIL / BAFF heterotrimer, or (iii) is a fragment or portion of (i) or (ii) containing CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF or APRIL / BAFF heterotrimer. In some embodiments, the reference (e.g., unmodified) TACI sequence lacks the N-terminal methionine as shown in SEQ ID NO: 122. TACI extracellular domain (ECD): SEQ ID NO: 122

[0179] In some embodiments, the reference (e.g., unmodified) TACI sequence is an extracellular domain sequence of TACI, which is a portion of the ECD containing an N-terminal deletion relative to the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-28, which residues correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-29, which residues correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-30, which residues correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-31, which residues correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-32, which residues correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-33, which residues correspond to the residues shown in SEQ ID NO:122.

[0180] In embodiments of any of the provided embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion containing a deletion of one or more residues of the stem portion of the TACI extracellular domain. In some embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion lacking one or more consecutive C-terminal amino acid residues, starting at residue 105 and up to or including amino acid residue 166, which residues correspond to the residues of the ECD sequence shown in SEQ ID NO: 122. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 of the ECD sequence is deleted.

[0181] In some embodiments, a reference (e.g., unmodified) TACI sequence contains an ECD portion having a contiguous amino acid sequence that includes CRD1 and / or CRD2 (e.g., CRD1 and CRD2 or only CRD2) and only a segment or portion of a stem sequence. A suitable stem segment includes one or more amino acids from amino acid residues 105 to 154 of SEQ ID NO: 122. For example, the stem segment can consist of the following with respect to SEQ ID NO: 122: amino acid residue 105, amino acid residues 105 to 106, amino acid residues 105 to 107, amino acid residues 105 to 108, amino acid residues 105 to 109, amino acid residues 105 to 110, amino acid residues 105 to 111, amino acid residues 105 to 112, amino acid residues 105 to 113, amino acid residues 105 to 114, amino acid residues 105 to 115, amino acid residues 105 to 116, Amino acid residues 105 to 117, amino acid residues 105 to 118, amino acid residues 105 to 119, amino acid residues 105 to 120, amino acid residues 105 to 121, amino acid residues 105 to 122, amino acid residues 105 to 123, amino acid residues 105 to 124, amino acid residues 105 to 125, amino acid residues 105 to 126, amino acid residues 105 to 127, amino acid residues 105 to 128, amino acid residues 105 to 129, amino acid residues 105 to 130, amino acid residues 105 to 131, amino acid residues 105 to 132, amino acid residues 105 to 133, amino acid residues 105 to 134, amino acid residues 105 to 135, amino acid residues 105 to 136, amino acid residues 105 to 137, amino acid residues 105 to 138, amino acid residues 105 to 139, amino acid residues 105 to 140, amino acid residues 105 to 141, amino acid residues 105 to 142, amino acid residues 105 to 143, amino acid residues 105 to 144, amino acid residues 105 to 145, amino acid residues 105 to 146, amino acid residues 105 to 147, amino acid residues 105 to 148, amino acid residues 105 to 149, amino acid residues 105 to 150, amino acid residues 105 to 151, amino acid residues 105 to 152, amino acid residues 105 to 153 and amino acid residues 105 to 154.

[0182] In some embodiments, the reference (e.g., unmodified) TACI sequence lacks one or more potential furin cleavage sites or is mutated therein. In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or portion in which the arginine residue at position 119 is mutated, e.g., R119G. In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or portion in which the glutamine residue at position 121 is mutated, e.g., Q121P. In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or portion in which the arginine residue at position 122 is mutated, e.g., R122Q.

[0183] In some embodiments, the reference TACI sequence is a TACI ECD sequence as set forth in International PCT Publication Nos. WO 2000 / 067034, WO 2002 / 094852, or WO 2008 / 154814.

[0184] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence shown in SEQ ID NO:131. TACI ECD(CRD1 / CRD2):SEQ ID NO:131

[0185] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence shown in SEQ ID NO:130. TACI ECD(CRD1 / CRD2):SEQ ID NO:130

[0186] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence shown in SEQ ID NO: 1 (encoded by the nucleotide sequence shown in SEQ ID NO: 36). TACI ECD(CRD1 / CRD2):SEQ ID NO:1

[0187] In some embodiments, the reference TACI sequence is an extracellular domain region of TACI that consists essentially of only the CRD2 sequence and is missing or lacking the entire CRD1 sequence and essentially all of the stem region. Although previous studies have shown that residues in the stem region may contain proteolytic cleavage sites, it is believed that sufficient expression of TACI and / or binding activity to its cognate ligand requires at least CRD1 and CRD2. For example, International PCT Publication No. WO 2002 / 094852 demonstrates that TACI molecules containing CRD1 and CRD2, but in which the entire amino-terminal region and part of the stem region sequence are deleted, exhibit reduced protein degradation when expressed. Other studies have shown that at least a portion of the N-terminal region preceding CRD1 is required for sufficient binding activity of TACI to its cognate ligand, see, for example, International Publication No. WO 2008 / 154814, in which residues 13-118 or 13-108 of the extracellular region of TACI were determined to be required for biological activity while minimizing degradation of TACI during expression. Surprisingly, it is found herein (eg, Example 3) that the extracellular region of TACI consisting essentially only of CRD2 with a small portion of the stem region exhibits significantly improved cognate binding activity compared to longer TACI molecules containing both CRD1 and CRD2.

[0188] Provided herein is an immunomodulatory protein (e.g., a TACI-Fc fusion protein) comprising a TACI polypeptide, which is a CRD2-containing portion of the TACI extracellular domain (ECD) region and lacks the N-terminal region and CRD1 and one or more residues of the stem portion of the TACI extracellular domain, for example, relative to the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the CRD2-containing portion of the TACI extracellular domain includes amino acid residues 71-104, which correspond to the residues set forth in SEQ ID NO: 122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein contains a deletion of N-terminal amino acid residues 1-66 corresponding to the residues set forth in SEQ ID NO: 122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein contains a deletion of N-terminal amino acid residues 1-67 corresponding to the residues set forth in SEQ ID NO: 122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein contains a deletion of N-terminal amino acid residues 1-68 corresponding to the residues set forth in SEQ ID NO: 122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein contains a deletion of N-terminal amino acid residues 1-69 corresponding to the residues set forth in SEQ ID NO: 122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein contains a deletion of N-terminal amino acid residues 1-70 corresponding to the residues set forth in SEQ ID NO: 122. In an embodiment of any such embodiments, the TACI polypeptide of the immunomodulatory protein lacks one or more consecutive C-terminal amino acid residues starting at residue 105 and up to or including amino acid residue 166 corresponding to the residues of the ECD sequence set forth in SEQ ID NO: 122. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 of the ECD sequence is deleted.

[0189] In some embodiments, the immunomodulatory proteins provided herein (e.g., TACI-Fc fusion proteins) have a TACI polypeptide having a sequence containing an ECD portion having a contiguous amino acid sequence of the TACI ECD including CRD2 (e.g., residues 71-104 with respect to SEQ ID NO: 122), but lacking the N-terminal region and CRD1 and one or more residues of the stem portion of the TACI extracellular domain, e.g., relative to the amino acid sequence shown in SEQ ID NO: 122. For example, the TACI ECD portion can consist of, with respect to the amino acid residues set forth in SEQ ID NO: 122, amino acid residues 67 to 118, amino acid residues 67 to 117, amino acid residues 67 to 116, amino acid residues 67 to 115, amino acid residues 67 to 114, amino acid residues 67 to 113, amino acid residues 67 to 112, amino acid residues 67 to 111, amino acid residues 67 to 110, amino acid residues 67 to 109, amino acid residues 67 to 108, amino acid residues 67 to 107, amino acid residues 67 to 106, amino acid residues 67 to 105, or amino acid residues 67 to 104. In some examples, the TACI ECD portion can consist of the following, with respect to the residues shown in SEQ ID NO:122: amino acid residues 68 to 118, amino acid residues 68 to 117, amino acid residues 68 to 116, amino acid residues 68 to 115, amino acid residues 68 to 114, amino acid residues 68 to 113, amino acid residues 68 to 112, amino acid residues 68 to 111, amino acid residues 68 to 110, amino acid residues 68 to 109, amino acid residues 68 to 108, amino acid residues 68 to 107, amino acid residues 68 to 106, amino acid residues 68 to 105, or amino acid residues 68 to 104. In some examples, the TACI ECD portion can consist of the following, with respect to the residues set forth in SEQ ID NO: 122: amino acid residues 69 to 118, amino acid residues 69 to 117, amino acid residues 69 to 116, amino acid residues 69 to 115, amino acid residues 69 to 114, amino acid residues 69 to 113, amino acid residues 69 to 112, amino acid residues 69 to 111, amino acid residues 69 to 110, amino acid residues 69 to 109, amino acid residues 69 to 108, amino acid residues 69 to 107, amino acid residues 69 to 106, amino acid residues 69 to 105, or amino acid residues 69 to 104.In some examples, the TACI ECD portion can consist of the following, with respect to the residues shown in SEQ ID NO:122: amino acid residues 70 to 118, amino acid residues 70 to 117, amino acid residues 70 to 116, amino acid residues 70 to 115, amino acid residues 70 to 114, amino acid residues 70 to 113, amino acid residues 70 to 112, amino acid residues 70 to 111, amino acid residues 70 to 110, amino acid residues 70 to 109, amino acid residues 70 to 108, amino acid residues 70 to 107, amino acid residues 70 to 106, amino acid residues 70 to 105, or amino acid residues 70 to 104. In some examples, the TACI ECD portion can consist of the following, with respect to the residues shown in SEQ ID NO: 122: amino acid residues 71 to 118, amino acid residues 71 to 117, amino acid residues 71 to 116, amino acid residues 71 to 115, amino acid residues 71 to 114, amino acid residues 71 to 113, amino acid residues 71 to 112, amino acid residues 71 to 111, amino acid residues 71 to 110, amino acid residues 71 to 109, amino acid residues 71 to 108, amino acid residues 71 to 107, amino acid residues 71 to 106, amino acid residues 71 to 105, or amino acid residues 71 to 104. Any of the above TACI ECD sequences can also be a TACI reference sequence according to an immunomodulatory protein provided herein, wherein such immunomodulatory protein contains a variant TACI polypeptide modified by one or more amino acid modifications (e.g., substitutions) as described herein compared to such TACI reference sequence.

[0190] Specifically, the TACI polypeptides provided herein include a TACI ECD sequence having or consisting of the sequence shown in SEQ ID NO: 13 (encoded by the nucleotide sequence shown in SEQ ID NO: 48). In some embodiments, the reference TACI sequence has or consists of the sequence shown in SEQ ID NO: 13, wherein the variant TACI polypeptides provided are modified by one or more amino acid modifications (e.g., substitutions) as described herein compared to such a reference TACI sequence. TACI ECD sequence (CRD2): SEQ ID NO: 13

[0191] The TACI polypeptides provided include variant TACI polypeptides. Immunomodulatory proteins, such as TACI-Fc fusion proteins, are also provided, which contain variant TACI polypeptides provided. In embodiments of any of the embodiments provided, the variant TACI sequence has the sequence of a reference (e.g., unmodified) TACI sequence (such as any of those described above), but additionally contains one or more amino acid modifications, such as one or more amino acid substitutions. Specifically, variant TACI polypeptides are provided herein, which contain at least one affinity-modified TD domain (e.g., CRD1 and / or CRD2) or a specific binding fragment thereof, wherein the domain or its specific binding fragment contains one or more amino acid substitutions in the TD domain of a reference (e.g., unmodified or wild-type) TACI polypeptide, such that the variant TACI polypeptide exhibits an altered (e.g., increased) binding activity or affinity to one or both of APRIL or BAFF compared to a reference (e.g., unmodified or wild-type) TACI polypeptide. In some embodiments, the binding affinity of the variant TACI polypeptide to APRIL and / or BAFF differs from that of a reference (e.g., unmodified or wild-type) TACI polypeptide control sequence, as determined by, for example, solid phase ELISA immunoassay, flow cytometry, or Biacore assay. The binding affinity to each cognate binding partner is independent; that is, in some embodiments, the variant TACI polypeptide has an increased binding affinity to one or both of APRIL and BAFF, and a decreased or unchanged binding affinity to the other of APRIL or BAFF, relative to a reference (e.g., unmodified or wild-type) TACI polypeptide.

[0192] In some embodiments, the variant TACI polypeptide has an increased binding affinity for BAFF relative to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has an increased binding affinity for APRIL relative to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has an increased binding affinity for APRIL and BAFF relative to a reference (unmodified or wild-type) TACI polypeptide. The cognate ligands BAFF and / or APRIL can be mammalian proteins, such as human proteins or mouse proteins. In specific embodiments, the cognate ligands BAFF and / or APRIL are human. In some embodiments, the variant TACI polypeptide having an increased or greater binding affinity for APRIL and / or BAFF will have an increase in binding affinity of at least about 5% (e.g., at least about 10%, 15%, 20%, 25%, 35%, or 50%) relative to a reference (e.g., unmodified or wild-type) TACI polypeptide control. In some embodiments, the binding affinity is increased by more than about 1.2-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold relative to a reference (e.g., unmodified, wild-type) TACI polypeptide. In any example, the reference (e.g., unmodified or wild-type) TACI polypeptide has the same sequence as the variant TACI polypeptide, but it does not contain one or more amino acid modifications (e.g., substitutions).

[0193] In some embodiments, the equilibrium dissociation constant (K) of any of the preceding embodiments with BAFF d ) can be less than 1x 10 -5 M, 1x 10 -6 M, 1x 10 -7 M, 1x 10 -8 M, 1x 10 -9 M, 1x 10 -10 M or 1x 10 -11 M or 1x10 -12 M. In some embodiments, any of the preceding embodiments and the K of BAFF d Less than or less than about 1x 10 -9 M, 1x 10 -10 M or 1x10 -11 M or 1x 10 -12 M. In some embodiments, any of the preceding embodiments and the K of BAFF d In 1x 10 -9 M is about 1x 10 -12In some embodiments, any of the preceding embodiments and the K of BAFF d is or about 1x 10 -9 M, is or about 2x 10 -9 M, about 4x 10 -9 M, about 6x 10 -9 M, about 8x 10 -9 M, is or about 1x 10 -10 M, is or about 2x 10 -10 M, about 4x 10 -10 M, about 6x 10 -10 M, about 8x 10 -10 M, is or about 1x 10 -11 M, is or about 2x 10 -11 M, about 4x 10 -11 M, about 6x 10 -11 M, about 8x 10 -11 M is either or about 1x 10 -12 M, or any value between any of the foregoing terms. In some embodiments, the embodiments provided include variant TACI polypeptides as described above, and with K of BAFF d The decrease (higher binding affinity) is greater than or about 1.5-fold, such as greater than or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.

[0194] In some embodiments, the equilibrium dissociation constant (K) of any of the preceding embodiments with APRIL d ) can be less than 1x 10 -5 M, 1x 10 -6 M, 1x 10 -7 M, 1x 10 -8 M, 1x 10 -9 M, 1x 10 -10 M or 1x 10 -11 M or 1x 10 -12 M. In some embodiments, any of the preceding embodiments and APRIL K d Less than or less than about 1x10 -9 M, 1x 10 -10 M or 1x10 -11 M or 1x 10 -12 M. In some embodiments, any of the preceding embodiments and APRIL K d In 1x 10 -9 M is about 1x 10 -12In some embodiments, any of the preceding embodiments and APRIL have a K d is about 1x10 -9 M, is or about 2x 10 -9 M, about 4x 10 -9 M, about 6x 10 -9 M, about 8x 10 -9 M, is or about 1x 10 - 10 M, is or about 2x 10 -10 M, about 4x 10 -10 M, about 6x 10 -10 M, about 8x 10 -10 M, is or about 1x 10 - 11 M, is or about 2x 10 -11 M, about 4x10 -11 M, about 6x 10 -11 M, about 8x 10 -11 M is either or about 1x 10 -12 M, or any value between any of the foregoing terms. In some embodiments, the embodiments provided include variant TACI polypeptides as described above, and with a K of APRIL d The decrease (higher binding affinity) is greater than or about 1.5-fold, such as greater than or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.

[0195] A reference (e.g., unmodified or wild-type) TACI sequence does not necessarily have to be used as a starting composition to generate a variant TACI polypeptide described herein. Thus, the use of the term "modification" (e.g., "substitution") does not mean that the embodiments of the present invention are limited to a particular method of preparing a variant TACI polypeptide or an immunomodulatory protein containing a variant TACI polypeptide. Variant TACI polypeptides can be prepared, for example, by de novo peptide synthesis, and thus do not necessarily require modification, such as "substitution", in the sense of changing codons to encode a modification (e.g., substitution). This principle also extends to the terms "addition" and "deletion" of amino acid residues, which likewise do not imply a particular method of preparation. Methods for designing or generating variant TACI polypeptides are not limited to any particular method. In some embodiments, however, a reference (e.g., unmodified or wild-type) TACI encoding nucleic acid is mutagenized from a reference (e.g., unmodified or wild-type) TACI genetic material and screened for a desired specific binding affinity or other functional activity. In some embodiments, variant TACI polypeptides are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases and then screened. The National Center for Biotechnology Information provides this information and its website is publicly accessible via the Internet, as is the UniProtKB database mentioned above.

[0196] Unless otherwise stated, as indicated throughout this disclosure, one or more amino acid modifications in a variant TACI polypeptide are named according to the amino acid position number corresponding to the position number of the reference ECD sequence shown in SEQ ID NO: 122. The skilled artisan can readily identify the corresponding position of the modification (e.g., amino acid substitution) in a TACI polypeptide (including the portion thereof containing the TD (e.g., CRD1 and / or CRD2)), such as by aligning a reference sequence (e.g., SEQ ID NO: 1 or 13) with SEQ ID NO: 122. An example of an alignment to identify corresponding residues is shown in Fig. 9 In the modification list throughout this disclosure, the amino acid position is indicated in the middle, the corresponding reference (e.g., unmodified wild-type) amino acid is listed before the number, and the identified variant amino acid substitution is listed after the number. If the modification is a deletion of the position, "del" is indicated, and if the modification is an insertion of the position, "ins" is indicated. In some cases, the insertion is listed with the amino acid position indicated in the middle, and the corresponding reference amino acid is listed before and after the number, and the identified variant amino acid insertion is listed after the unmodified (e.g., wild-type) amino acid.

[0197] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) in a reference (e.g., unmodified or wild-type) TACI sequence, such as any of those described. The one or more amino acid modifications (e.g., substitutions) may be located in the extracellular domain (extracellular domain) of the reference (e.g., unmodified or wild-type) TACI sequence. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the CRD1 domain or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the CRD2 domain or a specific binding fragment thereof. In some embodiments of the variant TACI polypeptide, some of the one or more amino acid modifications (e.g., substitutions) are located in the CRD1 domain or a specific binding fragment thereof, and some of the one or more amino acid modifications (e.g., substitutions) are located in the CRD2 domain or a specific binding fragment thereof.

[0198] In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications (e.g., substitutions) in the reference TACI sequence. The modifications (e.g., substitutions) may be in the CRD1 domain or the CRD2 domain. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions in the CRD1 domain of the reference TACI sequence or a specific binding fragment thereof. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in the CRD2 domain of a reference TACI sequence, or a specific binding fragment thereof.

[0199] In some embodiments, a variant TACI polypeptide having one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference (e.g., unmodified or wild-type) TACI polypeptide or a specific binding fragment thereof containing a CRD1 and / or CRD2 domain as shown in SEQ ID NO: 122. In some embodiments, the specific binding fragment contains a CRD1 domain, e.g., the specific binding fragment contains a sequence of amino acids 34-66 shown as SEQ ID NO: 122. In some cases, the CRD1 domain is the only complete CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is or contains a CRD2 domain, e.g., the specific binding fragment contains a sequence of amino acids 71-104 shown as SEQ ID NO: 122. In some cases, the CRD2 domain is the only complete CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is or contains a CRD1 domain and a CRD2 domain, for example, the specific binding fragment contains amino acids 34-104 of SEQ ID NO: 122. In some embodiments, the specific binding fragment contains a continuous portion of the stem domain, for example, the specific binding fragment contains a continuous portion of amino acids 105-165 of SEQ ID NO: 122. In an embodiment of any embodiment, the specific binding fragment of SEQID NO: 122 is less than the full-length ECD shown in SEQ ID NO: 122. In some embodiments, the specific binding fragment is shown in SEQ ID NO: 1. In some embodiments, the specific binding fragment is shown in SEQ ID NO: 13. In some embodiments, the specific binding fragment is shown in SEQ ID NO: 130. In some embodiments, the specific binding fragment is shown in SEQ ID NO: 131.

[0200] In some embodiments, a variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a reference (e.g., unmodified or wild-type) TACI polypeptide or a specific binding fragment thereof (e.g., to the amino acid sequence of SEQ ID NO: 1, 13 or 122).

[0201] In some embodiments, a variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:122.

[0202] In some embodiments, a variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0203] In some embodiments, a variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:13.

[0204] In some embodiments, a variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:130.

[0205] In some embodiments, a variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:131.

[0206] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) in a reference TACI polypeptide or a specific binding fragment thereof, which amino acid modifications correspond to one or more positions 40, 59, 60, 61, 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102 and 103 with respect to the numbering of SEQ ID NO: 122. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) selected from the group consisting of W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or conservative amino acid substitutions thereof. In some embodiments, the reference TACI polypeptide comprises a CRD1 domain or a CRD2 domain, e.g., the reference TACI polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 122.

[0207] In some embodiments, the amino acid substitutions are located only in the CRD2 domain. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) in a reference TACI polypeptide or a specific binding fragment thereof corresponding to one or more positions 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103 with respect to the numbering of SEQ ID NO: 122. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) selected from the group consisting of E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or conservative amino acid substitutions thereof. In some embodiments, in the CRD domain, the reference TACI polypeptide includes only the CRD2 domain but lacks the CRD1 domain, e.g., the reference TACI polypeptide is shown in SEQ ID NO: 13. Thus, in some embodiments, a variant TACI polypeptide comprises a portion of the ECD sequence of a TACI polypeptide that contains the CRD2 domain but lacks the CRD1 domain.

[0208] A conservative amino acid modification (e.g., substitution) is any amino acid other than a reference (e.g., unmodified) or wild-type amino acid that falls into the same amino acid class as the substituted amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxy or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine).

[0209] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 75 with respect to numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 75 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid is an acidic amino acid or amide, such as a substitution to a different acidic amino acid or amide, compared to a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 75 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 75 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 75 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 75 is glutamine (Gln, Q).

[0210] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 77 with respect to numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 77 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 77 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 77 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 77 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 77 is glutamine (Gln, Q).

[0211] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 78 with respect to numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 78 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 78 is an aromatic amino acid, such as a substitution to a different aromatic amino acid, compared to a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 78 is phenylalanine (Phe, F). In some embodiments, the substituted amino acid at position 78 is tyrosine (Tyr, Y). In some embodiments, the substituted amino acid at position 78 is tryptophan (Trp, W).

[0212] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 84 with respect to numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 84 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 84 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 84 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 84 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 84 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 84 is glutamine (Gln, Q).

[0213] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 101 with respect to numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 101 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 101 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 101 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 101 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 101 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 101 is glutamine (Gln, Q).

[0214] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 102 with respect to numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 102 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 102 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 102 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 102 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 102 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 102 is glutamine (Gln, Q).

[0215] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution E74V. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution K77E. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution L82H. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution L82P. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution R84G. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution R84L. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution D85V. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution A101D. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide contains two or more amino acid substitutions of any two or more of the foregoing. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions that are conservative amino acid substitutions of any of the foregoing. In the embodiments provided, the variant TACI polypeptide comprises at least one amino acid substitution in any of the reference TACI polypeptide sequences as described. In some embodiments, the at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO: 13. In some embodiments, the at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO: 130. In some embodiments, the at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO: 131.

[0216] In some embodiments, the variant TACI polypeptide comprises an amino acid substitution E74V. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution K77E. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution L82H. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution L82P. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution R84G. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution R84L. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution D85V. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution A102D. In some embodiments, the variant TACI polypeptide comprises an amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide contains two or more amino acid substitutions of any two or more of the foregoing. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions that are conservative amino acid substitutions of any of the foregoing. In the embodiments provided, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitutions are in the reference TACI sequence shown in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence shown in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence shown in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence shown in SEQ ID NO: 131.

[0217] In some embodiments, the amino acid substitution is D85E / K98T. In some embodiments, the amino acid substitution is I87L / K98T. In some embodiments, the amino acid substitution is R60G / Q75E / L82P. In some embodiments, the amino acid substitution is R60G / C86Y. In some embodiments, the amino acid substitution is W40R / L82P / F103Y. In some embodiments, the amino acid substitution is W40R / Q59R / T61P / K98T. In some embodiments, the amino acid substitution is L82P / I87L. In some embodiments, the amino acid substitution is G76S / P97S. In some embodiments, the amino acid substitution is K77E / R84L / F103Y. In some embodiments, the amino acid substitution is Y79F / Q99E. In some embodiments, the amino acid substitution is L83S / F103S. In some embodiments, the amino acid substitution is K77E / R84Q. In some embodiments, the amino acid substitution is K77E / A101D. In some embodiments, the amino acid substitution is K77E / F78Y / Y102D. In some embodiments, the amino acid substitution is Q75E / R84Q. In some embodiments, the amino acid substitution is Q75R / R84G / I92V. In some embodiments, the amino acid substitution is K77E / A101D / Y102D. In some embodiments, the amino acid substitution is R84Q / S88N / A101D. In some embodiments, the amino acid substitution is R84Q / F103V. In some embodiments, the amino acid substitution is K77E / Q95R / A101D. In some embodiments, the amino acid substitution is I87M / A101D. In the embodiments provided, the variant TACI polypeptide comprises an amino acid substitution in any reference TACI polypeptide sequence as described. In some embodiments, the amino acid substitution is in the reference TACI sequence shown in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 131.

[0218] In an embodiment of any embodiment, the variant TACI polypeptide comprises one or more amino acid substitutions from Q75E, K77E, F78Y, R84G, R84Q, A101D or Y102D or any combination thereof. In some embodiments, the variant TACI polypeptide comprises any 1, 2, 3, 4, 5 or 6 of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains one of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains two of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains three of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains four of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains five of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains six of the above amino acid substitutions.

[0219] In an embodiment of any embodiment, the one or more amino acid substitutions include Q75E / R84Q. In an embodiment of any embodiment, the one or more amino acid substitutions include Q75E / K77E. In an embodiment of any embodiment, the one or more amino acid substitutions include Q75E / F78Y. In an embodiment of any embodiment, the one or more amino acid substitutions include Q75E / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions include Q75E / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions include F77E / F78Y. In an embodiment of any embodiment, the one or more amino acid substitutions include K77E / R84Q. In an embodiment of any embodiment, the one or more amino acid substitutions include K77E / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions include K77E / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions include F78Y / R84Q. In an embodiment of any embodiment, the one or more amino acid substitutions include F78Y / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions include F78Y / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions include R84Q / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions include R84Q / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions include A101D / Y102D. In the embodiments provided, the variant TACI polypeptide comprises an amino acid substitution as described in any reference TACI polypeptide sequence (such as the sequence shown in SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 130 or SEQ ID NO: 131).

[0220] In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions R84G, A101D, K77E / R84Q, K77E / A101D, K77E / F78Y, K77E / F78Y / Y102D, Q75E / R84Q, K77E / A101D / Y102D, R84Q, K77E, A101D, Q75E, K77E / F78Y / R84Q, F78Y, F78Y / R84Q, F78Y / A101D, F78Y / Y102D, or K77E / Y102D. In the embodiments provided, the variant TACI polypeptide comprises an amino acid substitution in any reference TACI polypeptide sequence as described, such as the sequence shown in SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 130, or SEQ ID NO: 131.

[0221] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E and F78Y (K77E / F78Y). In the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 131.

[0222] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E and Y102D (K77E / Y102D). In the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 131.

[0223] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions F78Y and Y102D (F78Y / Y012D). In the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 131.

[0224] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions K77E, F78Y, and Y102D (K77E / F78Y / Y102D). In the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 131.

[0225] In some embodiments, the variant TACI polypeptide contains the amino acid substitution Q75E / R84Q. In the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitutions are in the reference TACI sequence set forth in SEQ ID NO: 131.

[0226] In some embodiments, the variant TACI polypeptide comprises any of the mutations listed in Table 1. Table 1 also provides exemplary sequences with reference to the SEQ ID NOs of reference (e.g., unmodified) TACI polypeptides and exemplary variant TACI polypeptides. As noted, the exact locus or residue corresponding to a given domain may vary, such as depending on the method used to identify or classify the domain. Similarly, in some cases, the adjacent N- and / or C-terminal amino acids of a given domain (e.g., CRD) may also be included in the sequence of the variant TACI polypeptide, for example to ensure proper folding of the domain upon expression. Therefore, it should be understood that the examples of SEQ ID NOs in Table 1 should not be considered limiting. For example, a particular domain of a variant TACI polypeptide (e.g., an ECD domain or a portion thereof containing CRD1 / CRD2 or only CRD2) may be several amino acids longer or shorter than the amino acid sequence shown in the corresponding SEQ ID NO, such as 1-10 (e.g., 1, 2, 3, 4, 5, 6, or 7) amino acids longer or shorter.

[0227] In some embodiments, the variant TACI polypeptide comprises any of the mutations (amino acid substitutions) listed in Table 1. In some examples, the mutations (amino acid substitutions) are made in a reference TACI comprising the amino acid sequence set forth in SEQ ID NO: 122. In some examples, the mutations (amino acid substitutions) are made in a reference TACI comprising the CRD1 and CRD2 domains of TACI (e.g., as set forth in SEQ ID NO: 1). In some examples, the mutations (amino acid substitutions) are made in a reference TACI further truncated by deleting the N-terminal and C-terminal amino acid residues to retain CRD2 (e.g., as set forth in SEQ ID NO: 13).

[0228] The use of the term "modification" (such as "substitution" or "mutation") does not mean that the embodiments of the present invention are limited to a particular method of preparing an immunomodulatory protein. Variant TACI polypeptides can be prepared, for example, by de novo peptide synthesis, and therefore the modification (such as "substitution") is not necessarily required in the sense that the codons are changed to encode the modification (such as substitution). This principle also extends to the terms "addition" and "deletion" of amino acid residues, which likewise do not imply a particular method of preparation. The means used to design or produce vTD are not limited to any particular method. However, in some embodiments, a wild-type or unmodified TD encoding nucleic acid is mutagenized from wild-type or unmodified TD genetic material and screened for changes in the desired specific binding activity (such as binding affinity) and / or NF-κB regulation or other functional activity. In some embodiments, vTD is synthesized de novo using protein or nucleic acid sequences available from any number of publicly available databases and then screened. The National Center for Biotechnology Information provides such information and its website is publicly accessible via the Internet, as is the UniProtKB database.

[0229] In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence comprising CRD1 and CRD2, such as a variant TACI polypeptide as set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120. In some embodiments, the variant TACI polypeptide comprises a polypeptide sequence exhibiting at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and wherein one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI are retained. In some embodiments, the variant TACI polypeptide comprises a specific binding fragment of any one of SEQ ID NOs: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds BAFF, APRIL or BAFF / APRIL heterotrimer and contains therein a contiguous sequence containing therein one or more amino acid modifications (e.g., one or more substitutions) that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0230] In some embodiments, the variant TACI polypeptide consists of or consists essentially of a variant TACI extracellular domain (ECD) sequence as set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120. In some embodiments, the variant TACI polypeptide consists of or consists essentially of a polypeptide sequence exhibiting at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and wherein one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI are retained. In some embodiments, the variant TACI polypeptide consists of or consists essentially of a specific binding fragment of any one of SEQ ID NOs: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds BAFF, APRIL or APRIL / BAFF heterotrimer and contains the following contiguous sequence, which contains one or more amino acid modifications (e.g., one or more substitutions) therein that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0231] In some embodiments, the variant TACI polypeptide comprises a reference TACI polypeptide containing CRD2 but lacking CRD1 extracellular domain (ECD) sequence, such as a variant TACI polypeptide as set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192. In some embodiments, the variant TACI polypeptide comprises a polypeptide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and in which one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI are retained. In some embodiments, the variant TACI polypeptide comprises a specific binding fragment of any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds BAFF, APRIL or BAFF / APRIL heterotrimer and contains therein a contiguous sequence containing therein one or more amino acid modifications (e.g., one or more substitutions) that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0232] In some embodiments, the variant TACI polypeptide consists of or consists essentially of the sequence shown in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192. In some embodiments, the variant TACI polypeptide consists of or consists essentially of a polypeptide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and in which one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI are retained. In some embodiments, the variant TACI polypeptide consists of or consists essentially of a specific binding fragment of any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds BAFF, APRIL or BAFF / APRIL heterotrimer and contains the following contiguous sequence, which contains one or more amino acid modifications (e.g., one or more substitutions) therein that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0233] In some embodiments, the variant TACI polypeptide comprises the sequence shown in SEQ ID NO: 20. In some embodiments, the variant TACI polypeptide consists essentially of the sequence shown in SEQ ID NO: 20. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO: 20.

[0234] In some embodiments, the variant TACI polypeptide comprises the sequence shown in SEQ ID NO: 26. In some embodiments, the variant TACI polypeptide consists essentially of the sequence shown in SEQ ID NO: 26. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO: 26.

[0235] In some embodiments, the variant TACI polypeptide comprises the sequence shown in SEQ ID NO: 27. In some embodiments, the variant TACI polypeptide consists essentially of the sequence shown in SEQ ID NO: 27. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO: 27.

[0236] In some embodiments, the variant TACI polypeptide comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, the variant TACI polypeptide consists essentially of the sequence set forth in SEQ ID NO: 107. In some embodiments, the variant TACI polypeptide consists of the sequence set forth in SEQ ID NO:107.

[0237] In some embodiments, the variant TACI polypeptide is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 37-47, 56, or 57. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 37-47, 56, or 57, and in which one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI are retained. Also provided herein is a nucleic acid containing a sequence as shown in any one of SEQ ID NOs: 37-47, 56 or 57, or a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity (e.g., at least 96% identity, 97% identity, 98% identity or 99% identity) to any one of SEQ ID NOs: 37-47, 56 or 57.

[0238] In some embodiments, the variant TACI polypeptide is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 49-55 or 58-70. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 49-55 or 58-70, and in which one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI are retained. Also provided herein is a nucleic acid containing a sequence as shown in any one of SEQ ID NOs: 49-55 or 58-70, or a sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity (e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity) to any one of SEQ ID NOs: 49-55 or 58-70.

[0239] In some embodiments, also provided herein are TACI ECD fusion sequences, wherein any of the above TACI ECD sequences are linked or fused to a multimerization domain (such as any of those described herein).

[0240] The interaction of two or more polypeptides of an immunomodulatory protein can be promoted by direct or indirect connection with any part or other polypeptide, which itself can interact to form a stable structure. For example, individually encoded polypeptide chains can be joined by multimerization, whereby the multimerization of the polypeptide is mediated by a multimerization domain. Typically, a multimerization domain provides the formation of a stable protein-protein interaction between a first polypeptide and a second polypeptide.

[0241] In some embodiments, two or more individual polypeptides of an immunomodulatory protein can be joined by multimerization, such as being joined into dimerization, trimerization, tetramerization or pentamerization molecules. In some cases, the individual polypeptides are identical. For example, a trimerization molecule can be formed from three copies of the same individual polypeptide. In other examples, a tetramerization molecule is generated from four copies of the same individual polypeptide. In other examples, a pentamerization molecule is generated from five copies of the same individual polypeptide. The multimerization domain can be a domain that promotes dimerization, trimerization, tetramerization or pentamerization of a polypeptide chain.

[0242] In some embodiments, the immunomodulatory protein forms a multimer, e.g., a dimer. In some embodiments, the dimer is a homodimer, wherein the two polypeptides of the immunomodulatory protein are identical. In some embodiments, the dimer is a heterodimer, wherein the two polypeptides of the immunomodulatory protein are different.

[0243] In some embodiments, the multimerization domain includes any domain capable of forming a stable protein-protein interaction. The multimerization domain can interact with each other by: an immunoglobulin sequence (e.g., an Fc domain; see, e.g., International Patent Publication Nos. WO 93 / 10151 and WO 2005 / 063816 US; U.S. Publication No. 2006 / 0024298; U.S. Patent No. 5,457,035); a leucine zipper (e.g., from nuclear transformation proteins fos and jun, or proto-oncogene c-myc, or from general nitrogen control (General Control of Nitrogen, GCN4)) (see, e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz ​​et al., (1989) Science, 243:1695-1699); a hydrophobic region; a hydrophilic region; or a free thiol forming an intermolecular disulfide bond between a homopolymer or heteropolymer chimeric molecule. In addition, the multimerization domain can include an amino acid sequence constituting a protrusion, which is complementary to the amino acid sequence constituting a mortise, as described in, for example, the following documents: U.S. Patent No. 5,731,168; International Patent Publication No. WO 98 / 50431 and WO2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9: 617-621. This multimerization region can be engineered so that spatial interactions not only promote stable interactions, but also promote the formation of heterodimers from a mixture of chimeric monomers to exceed homodimers. Generally, a protrusion is constructed by replacing the small amino acid side chains in the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Optionally, a compensatory cavity of the same or similar size of a protrusion is produced on the interface of the second polypeptide by replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0244] A TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) can be joined to the N-terminus or C-terminus of a multimerization domain at any position (but typically via its N-terminus or C-terminus) to form a chimeric polypeptide. The connection can be a direct connection or an indirect connection via a linker. Likewise, a chimeric polypeptide can be a fusion protein or can be formed by chemical connection, such as by covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerization domain, a nucleic acid encoding all or part of a TACI polypeptide sequence (such as any of the described TACI ECDs, including variant TACI polypeptide sequences) can be operably linked to a nucleic acid encoding the multimerization domain sequence, directly or indirectly or optionally via a linker domain. In some cases, the construct encodes a chimeric protein in which the C-terminus of the TACI polypeptide sequence is joined to the N-terminus of the multimerization domain. In some cases, the construct can encode a chimeric protein in which the N-terminus of the TACI polypeptide sequence is joined to the N-terminus or C-terminus of the multimerization domain.

[0245] The polypeptide multimer contains two chimeric proteins, which are produced by directly or indirectly linking two identical or different TACI polypeptide sequences (e.g., two identical or different variant TACI polypeptide sequences) to a multimerization domain. In some examples, where the multimerization domain is a polypeptide, a gene fusion encoding a TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) and a multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion protein can be expressed in a host cell transformed with a recombinant expression vector and allowed to assemble into a multimer, wherein the multimerization domains interact to form a multivalent polypeptide. Chemical linkage of the multimerization domain to a TACI polypeptide (e.g., a variant TACI polypeptide) can be achieved using a heterobifunctional linker.

[0246] The resulting chimeric polypeptide (e.g., fusion protein) and multimers formed therefrom can be purified by any suitable method, such as, for example, by affinity chromatography on a protein A or protein G column. In the case where two nucleic acid molecules encoding different polypeptides are transformed into cells, the formation of homodimers and heterodimers will occur. Expression conditions can be adjusted so that heterodimer formation is preferred over homodimer formation.

[0247] In some embodiments, the multimerization domain is the Fc region of an immunoglobulin.

[0248] In some embodiments, the multimerization domain is an immunoglobulin (e.g., IgG1) Fc region, wherein the fusion protein is a TACI-Fc containing: (1) a TACI sequence containing or consisting of any of the provided TACI ECD sequences; and (2) an immunoglobulin Fc region. Thus, the provided embodiments include TACI-Fc fusion proteins containing: (1) a TACI sequence containing or consisting of any of the above-described TACI ECD polypeptide sequences (e.g., variant TACI polypeptides); and (2) an immunoglobulin Fc region.

[0249] In some embodiments, provided herein is a TACI-Fc fusion sequence comprising (1) a TACI ECD sequence comprising the sequence shown in SEQ ID NO: 13, and (2) an immunoglobulin Fc region. In some embodiments, provided herein is a TACI-Fc fusion sequence comprising (1) a TACI ECD sequence consisting of or consisting essentially of the sequence shown in SEQ ID NO: 13, and (2) an immunoglobulin Fc region.

[0250] In some embodiments, the TACI-Fc fusion is a variant TACI-Fc fusion that contains or consists of any of the variant TACI polypeptides described above and an immunoglobulin Fc region.

[0251] In some embodiments, provided herein is a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence comprising CRD1 and CRD2, such as a TACI sequence comprising a sequence as shown in any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region. In some embodiments, provided herein is a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence comprising CRD1 and CRD2, such as a TACI sequence consisting of or consisting essentially of a sequence as shown in any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region.

[0252] In some embodiments, provided herein is a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence comprising a CRD2 domain but lacking a CRD1 domain, such as a TACI sequence comprising a sequence as shown in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and (2) an immunoglobulin Fc region. In some embodiments, provided herein is a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence comprising a CRD2 domain but lacking a CRD1 domain, such as a TACI sequence consisting of or consisting essentially of a sequence as shown in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and (2) an immunoglobulin Fc region.

[0253] In the provided embodiments of TACI-Fc, the immunoglobulin Fc region can be a wild-type Fc of an immunoglobulin, such as an IgG1 Fc. In some cases, the Fc region can be a variant Fc lacking effector function (also referred to as an "effector-free Fc"). Exemplary Fc regions and variants thereof in the provided TACI-Fc fusion proteins are described below.

[0254] In some embodiments, the Fc is a murine or human Fc. In some embodiments, the Fc is a mammalian or human IgG1, IgG2, IgG3 or IgG4 Fc region.

[0255] In some embodiments, the Fc region is or comprises a sequence as shown in any one of SEQ ID NOs: 71, 73, 75, 81, 82, 83, 134, 135, 136, 137, 138, 139, 140, 173, 174, 175, 176, 193, 218, 219, 220, or 221. In some embodiments, the Fc region is or is derived from an IgG1 as shown in any one of SEQ ID NOs: 71, 73, 75, 81, 82, 83, 134, 135, 136, 137, 139, 140, 173, 174, 175, 176, 193, 218, 220, or 221. In some embodiments, the Fc region is or is derived from an IgG2 as shown in any one of SEQ ID NOs: 138 or 219. In some embodiments, the Fc region is or is derived from IgG4, such as any one of those shown in SEQ ID NO: 139, 140, or 220. In some embodiments, the Fc region in the Fc fusion proteins provided herein may also include an Fc region that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of any of the above Fc regions.

[0256] In some embodiments, the Fc is derived from IgG1, such as human IgG1. In some embodiments, the Fc is an IgG1 Fc as shown in SEQ ID NO: 71, which has an allotype containing residues Glu (E) and Met (M) at positions 356 and 358 according to EU numbering. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 71 or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 71. In other embodiments, the Fc is an IgG1 Fc containing amino acids of a human G1m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, for example, as shown in SEQ ID NO: 81. Thus, in some cases, the Fc provided herein may contain amino acid substitutions E356D and M358L to reshape the residues of the allotype G1 m1. In some embodiments, the Fc comprises an amino acid sequence as set forth in SEQ ID NO: 81, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 81.

[0257] In some embodiments, the Fc region has the amino acid sequence shown in SEQ ID NO:81.

[0258] In some embodiments, the variant Fc comprises the sequence shown in SEQ ID NO: 173. In some embodiments, the variant Fc comprises the sequence shown in SEQ ID NO: 174. In some embodiments, the Fc region used in the constructs provided herein may further lack a C-terminal lysine residue.

[0259] In some embodiments, the Fc region is derived from IgG2, such as human IgG2. In some embodiments, the Fc region comprises an amino acid sequence as shown in SEQ ID NO: 138 or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 138. In some embodiments, the Fc region is an IgG2 Fc region having the sequence shown in SEQ ID NO: 138. In some embodiments, the Fc region is an IgG2 Fc region having the sequence shown in SEQ ID NO: 219.

[0260] In some embodiments, Fc is derived from IgG4, such as human IgG4. In some embodiments, Fc comprises the amino acid sequence shown in SEQ ID NO: 139 or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 139. In some embodiments, IgG4 Fc is a stable Fc in which the CH3 domain of human IgG4 is replaced by the CH3 domain of human IgG1, and it exhibits suppressed aggregate formation; an antibody in which the CH3 and CH2 domains of human IgG4 are replaced by the CH3 and CH2 domains of human IgG1, respectively; or an antibody in which the arginine at position 409 of human IgG4 indicated in the EU index proposed by Kabat et al. is substituted by lysine, and it exhibits suppressed aggregate formation (see, e.g., U.S. Pat. No. 8,911,726). In some embodiments, the Fc is an IgG4 containing an S228P mutation, which has been shown to prevent recombination between therapeutic antibodies and endogenous IgG4 by Fab-arm exchange (see, e.g., Labrijin et al. (2009) Nat. Biotechnol., 27(8):767-71). In some embodiments, the Fc comprises an amino acid sequence as set forth in SEQ ID NO: 140 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 140. In some embodiments, the Fc region is an IgG4 Fc region as set forth in SEQ ID NO: 140. In some embodiments, the Fc region is an IgG4 Fc region as set forth in SEQ ID NO: 220.

[0261] In some embodiments, the Fc region is a variant Fc region, wherein the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or render the Fc inert to Fc effector function. Exemplary effectorless or inert mutations include those described herein.

[0262] In some embodiments, the Fc region contains one or more modifications that change (e.g., reduce) one or more of its normal functions. Typically, in addition to the antigen binding ability that is the main function of an immunoglobulin, the Fc region is also responsible for effector functions, such as complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). In addition, the FcRn sequence present in the Fc region increases the half-life in vivo by conjugating with the FcRn receptor in vivo and plays a role in regulating IgG levels in serum. In some embodiments, such functions can be reduced or changed in Fc for use with the Fc fusion protein provided.

[0263] In some embodiments, one or more amino acid modifications may be introduced into the Fc region to generate an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of changes or mutations in Fc sequences that can change effector function. For example, WO 00 / 42072, WO 2006019447, WO 2012125850, WO 2015 / 107026, US2016 / 0017041 and Shields et al. J Biol. Chem. 9 (2): 6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to FcR. The contents of these publications are specifically incorporated herein by reference.

[0264] In some embodiments, the immunomodulatory protein provided comprises an Fc region that exhibits reduced effector functions, which makes it a desired candidate for certain applications, in which the in vivo half-life of the immunomodulatory protein is important, but some effector functions (such as CDC and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the immunomodulatory protein lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The primary cell NK cells that mediate ADCC only express FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991) Table 2 on page 464. Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in the following literature: U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (CellTechnology, Mountain View, CA); and CytoTox 96 TMNon-radioactive cytotoxicity assay (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo (e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that immunomodulatory protein n cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0265] Immunomodulatory proteins with reduced effector function include those with substitutions of one or more of the following Fc region residues according to EU numbering: 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0266] In some embodiments, the Fc region of the immunomodulatory protein has an Fc region in which any one or more amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are substituted with different amino acids compared to the native Fc region. Such changes in the Fc region include, for example, deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L233S, IgG1-C226S / C229S / E233P / L233S, IgG1- 34V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E; such as WO The changes of G236R / L328R, L235G / G236R, N325A / L328R and N325LL328R described in 2008 / 092117; amino acid insertions at positions 233, 234, 235 and 237 (indicated according to EU numbering); and changes at the sites described in WO 2000 / 042072.

[0267] Certain Fc variants with improved or reduced binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, WO 2006019447, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0268] In some embodiments, an immunomodulatory protein is provided, comprising a variant Fc region comprising one or more amino acid substitutions that extend half-life and / or improve binding to a neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO 2015107026. Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues, according to EU numbering: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0269] In some embodiments, the Fc region of the immunomodulatory protein comprises one or more amino acid substitutions C220S, C226S and / or C229S according to EU numbering. In some embodiments, the Fc region of the immunomodulatory protein comprises one or more amino acid substitutions R292C and V302C. For other examples of Fc region variants, see Duncan and Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0270] In some embodiments, alterations are made in the Fc region that result in reduced CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0271] In some embodiments, the variant Fc region comprising one or more amino acid modifications (e.g., amino acid substitutions) is derived from wild-type IgG1, such as wild-type human IgG1. In some embodiments, the wild-type IgG1 Fc can be the Fc shown in SEQ ID NO:71, which has an allotype containing residues Glu (E) and Met (M) at positions 356 and 358 according to EU numbering. In some embodiments, the variant Fc region is derived from the amino acid sequence shown in SEQ ID NO:71. In other embodiments, the wild-type IgG1 Fc contains amino acids of the human G1m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, for example, as shown in SEQ ID NO:81. Therefore, in some cases, the variant Fc is derived from the amino acid sequence shown in SEQ ID NO:81.

[0272] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO:71 or 81 (corresponding to K447del by EU numbering).

[0273] In some embodiments, the variant Fc region comprises a C5S amino acid modification (corresponding to C220S by EU numbering) of a wild-type or unmodified Fc region according to the numbering of SEQ ID NO:71.

[0274] In some embodiments, the Fc region is a variant Fc containing at least one amino acid substitution, the amino acid substitution is N82G according to SEQ ID NO: 71 (corresponding to N297G according to EU numbering). In some embodiments, the Fc further contains at least one amino acid substitution according to the numbering of SEQ ID NO: 71, i.e., R77C or V87C (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification according to the numbering of SEQ ID NO: 71 (corresponding to C220S according to EU numbering). For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: according to EU numbering, N297G and one or more of the following amino acid modifications C220S, R292C or V302C (with respect to SEQ ID NO: 71, corresponding to N82G and one or more of the following amino acid modifications C5S, R77C or V87C), for example, the Fc region comprises the sequence shown in SEQ ID NO: 82.

[0275] In some embodiments, according to EU numbering, the variant Fc contains the amino acid substitution L234A / L235E / G237A. In some embodiments, according to EU numbering, the variant Fc contains the amino acid substitution A330S / P331S. In some embodiments, the variant Fc contains the amino acid substitution L234A / L235E / G237A / A330S / P331S (Gross et al. (2001) Immunity 15:289). In some embodiments, the variant Fc comprises the sequence shown in SEQ ID NO:175. In some embodiments, the variant Fc comprises the sequence shown in SEQ ID NO:176. In some embodiments, the Fc region used in the construct provided herein may further lack a C-terminal lysine residue.

[0276] In some embodiments, the Fc region is a variant Fc, which includes mutations L234A, L235E and G237A according to EU numbering. In some embodiments, the wild-type Fc is further modified by removing one or more cysteine ​​residues, such as by replacing the cysteine ​​residue with a serine residue (C220S) at position 220 according to EU numbering. Exemplary inert Fc regions with reduced effector function are shown in SEQ ID NO: 83 and SEQ ID NO: 75, which are based on the allotypes shown in SEQ ID NO: 71 or SEQ ID NO: 81, respectively. In some embodiments, the Fc region may further lack a C-terminal lysine residue. In some embodiments, the variant Fc region comprises one or more of amino acid modifications C220S, L234A, L235E or G237A, for example, the Fc region comprises the sequence shown in SEQ ID NO: 73, 75, 83 or 136. In some embodiments, the variant Fc comprises a sequence shown in SEQ ID NO: 73. In some embodiments, the variant Fc comprises a sequence as shown in SEQ ID NO: 75. In some embodiments, the variant Fc comprises a sequence as shown in SEQ ID NO: 83. In some embodiments, the variant Fc comprises a sequence as shown in SEQ ID NO: 136.

[0277] In some embodiments, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:73.

[0278] In some embodiments, the Fc region is an IgG1 Fc, but does not contain a hinge sequence. In some embodiments, the IgG1 Fc region does not contain the hinge sequence EPKSC (SEQ ID NO: 239). In some embodiments, the IgG1 Fc region does not contain the hinge sequence EPKSS (SEQ ID NO: 238).

[0279] In some embodiments, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:221.

[0280] In some embodiments, the Fc region is a variant Fc region comprising one or more of amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, e.g., the Fc region comprises the sequence shown in SEQ ID NO: 134. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO: 71 (corresponding to K447del according to EU numbering).

[0281] In some embodiments, the Fc region is a variant Fc region comprising one or more of amino acid modifications C220S, R292C, N297G, V302C. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). An exemplary variant Fc region is shown in SEQ ID NO: 135.

[0282] In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S / E233P / L234V / L235A / G236del / S267K. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). An exemplary variant Fc region is shown in SEQ ID NO: 137.

[0283] Examples of such Fc regions for inclusion in immunomodulatory polypeptides are shown in Table 2.

[0284] In some embodiments, the Fc region is a variant Fc region containing any combination of the Fc mutations in Table 2. In some embodiments, the Fc region is a variant Fc region having a sequence as shown in any one of the SEQ ID NOs in Table 2.

[0285] For example, the variant Fc region can be an effector-less Fc that exhibits reduced effector activity compared to the wild-type IgG1 set forth in SEQ ID NO: 71 or SEQ ID NO: 81. In some embodiments, the variant Fc comprises an amino acid sequence set forth in any one of SEQ ID NOs: 75, 82, 83, 134, 73, 135, 136, or 137, or an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 75, 82, 83, 134, 73, 135, 136, or 137. In some embodiments, the variant Fc has the sequence set forth in SEQ ID NO: 73. In embodiments, when produced and expressed from a cell, the provided immunomodulatory protein (eg, TACI-Fc fusion) is a homodimer containing two identical polypeptide chains.

[0286] In some embodiments, the immunomodulatory protein contains a first immunomodulatory Fc fusion polypeptide and a second immunomodulatory Fc fusion polypeptide, wherein the first polypeptide and the second polypeptide are different. In some embodiments, the first Fc polypeptide fusion contains an Fc region and one or more variant TACI polypeptide sequences, and the second polypeptide fusion contains an Fc region and one or more TACI polypeptide sequences. In such embodiments, the Fc region can be a region that promotes or facilitates heterodimer formation.

[0287] In some embodiments, the Fc domain of one or both of the first and second immunomodulatory Fc fusion polypeptides comprises a modification (e.g., substitution) such that the interface of the Fc molecule is modified to facilitate and / or promote heterodimerization. Methods for promoting heterodimerization of the Fc chain include mutagenesis of the Fc region, such as by comprising a set of "knob-and-hole" mutations or comprising mutations to achieve electrostatic manipulation of the Fc to facilitate attractive interactions between different polypeptide chains. In some embodiments, the Fc region of the heterodimeric molecule may additionally contain one or more other Fc mutations, such as any of those described above. In some embodiments, the heterodimeric molecule contains an Fc region with a mutation that reduces effector function. In some embodiments, such an Fc region contains mutations C220S, L234A, L235E, and / or G237A according to EU numbering. In some embodiments, any of the above mutations in the Fc skeleton may be carried out in an allotype containing residues Glu (E) and Met (M) at positions 356 and 358 according to EU numbering. In other embodiments, any of the above mutations in the Fc framework may be made in an allotype containing residues Asp (D) and Leu (L) at positions 356 and 358 according to EU numbering.

[0288] In some embodiments, the modification comprises introducing a protrusion (knob) into a first Fc polypeptide and introducing a cavity (hole) into a second Fc polypeptide such that the protrusion can be positioned in the cavity to facilitate complexing of the first and second Fc-containing polypeptides. Amino acids targeted for substitution and / or modification to create a protrusion or cavity in a polypeptide are typically interface amino acids that interact or contact one or more amino acids in the interface of the second polypeptide.

[0289] In some embodiments, the first polypeptide modified to contain a protrusion (knob) amino acid comprises replacing a natural or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide and can therefore be located in a compensatory cavity (hole) in the adjacent interface of the second polypeptide. Most commonly, the replacement amino acid is an amino acid with a larger side chain volume than the original amino acid residue. Those skilled in the art understand how to determine and / or evaluate the properties of amino acid residues to identify ideal replacement amino acids that produce protrusions. In some embodiments, the replacement residues used to form the protrusion are naturally occurring amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W). In some examples, the original residue identified for replacement is an amino acid residue with a small side chain, such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0290] In some embodiments, the second polypeptide modified to contain a cavity (hole) is a polypeptide comprising replacing a natural or original amino acid with an amino acid having at least one side chain, and the at least one side chain is recessed from the interface of the second polypeptide, so that the corresponding protrusion can be accommodated from the interface of the first polypeptide. Most commonly, the replacement amino acid is an amino acid with a side chain volume smaller than the original amino acid residue. Those skilled in the art understand how to measure and / or evaluate the properties of amino acid residues to identify the ideal replacement residue for forming a cavity. Generally, the replacement residue for forming a cavity is a naturally occurring amino acid, and includes, for example, alanine (A), serine (S), threonine (T) and valine (V). In some examples, the original amino acid identified for replacement is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine or tryptophan.

[0291] For example, the CH3 interface of human IgG1 involves sixteen residues on each domain located on four antiparallel β-strands that bury 2 (see, e.g., Deisenhofer et al. (1981) Biochemistry, 20: 2361-2370; Miller et al. (1990) J Mol. Biol., 216, 965-973; Ridgway et al. (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). Modification of the CH3 domain to create a protrusion or cavity is described in, e.g., U.S. Pat. No. 5,731,168; International Patent Applications WO 98 / 50431 and WO 2005 / 063816; and Ridgway et al. (1996) Prot. Engin., 9: 617-621. In some examples, modifications to the CH3 domain to create a protrusion or cavity are typically targeted to residues located on the two central antiparallel β strands. The aim is to minimise the risk that the resulting protrusion may be accommodated by protruding into the surrounding solvent rather than being accommodated by a compensating cavity in the partner CH3 domain.

[0292] In some embodiments, the heterodimer molecule contains a T366W mutation in the CH3 domain of the "knob chain", and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain". In some cases, for example, by introducing a Y349C mutation into the CH3 domain of the "knob" or "hole" chain, and introducing an E356C mutation or an S354C mutation into the CH3 domain of the other chain, an additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681). In some embodiments, the heterodimer molecule contains an S354C, T366W mutation in one of the two CH3 domains, and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. For example, the knob Fc may contain the sequence shown in SEQ ID NO: 89, which contains S354C and T366W, and the hole Fc shown in SEQ ID NO: 90, which contains mutations Y349C, T366S, L368A, and Y407V). In some embodiments, the heterodimeric molecule comprises E356C, T366W mutations in one of the two CH3 domains, and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises Y349C, T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. Other examples of knob-in-hole technology are known in the art, for example as described in EP 1 870 459 A1.

[0293] In some embodiments, the Fc variants containing CH3 protrusion (knob) or cavity (hole) modifications can be joined to the multi-domain immunomodulatory polypeptide at any position, but typically via its N-terminus or C-terminus, to the N-terminus or C-terminus of one or more TACI polypeptide sequences (e.g., variant TACI polypeptide sequences), such as to form a fusion polypeptide. The connection can be a direct connection or an indirect connection via a linker. Typically, the knob and hole molecules are produced by co-expression of a first immunomodulatory polypeptide linked to an Fc variant containing one or more CH3 protrusion modifications and a second immunomodulatory polypeptide linked to an Fc variant containing one or more CH3 cavity modifications.

[0294] Exemplary sequences of knob and hole Fc polypeptides are shown in SEQ ID NOs: 128 and 129, respectively. In some embodiments, the knob or hole Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). Exemplary sequences of knob and hole Fc polypeptides are shown in SEQ ID NOs: 89 and 90, respectively.

[0295] In some embodiments, a single polypeptide of a multidomain polypeptide or a single polypeptide of a single domain polypeptide is connected to a multimerization domain that forms an immunomodulatory protein and is a trimer, tetramer, or pentamer. In some embodiments, the single polypeptides of such molecules are identical. In some embodiments, such a multimerization domain is a cartilage oligomeric matrix protein (COMP) assembly domain, a vasodilator-stimulated phosphoprotein (VASP) tetramerization domain, or a ZymoZipper (ZZ) 12.6 domain.

[0296] In some embodiments, the multimerization domain is part of the cartilage oligomeric matrix protein (COMP) assembly domain (Voulgaraki et al., Immunology (2005) 115 (3): 337-346). In some examples, COMP is or contains the amino acid sequence as shown in SEQ ID NO: 146 (e.g., amino acids 29-72 of full-length COMP, Uniprot accession number P49747) or a sequence having about 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 146.

[0297] In some embodiments, the multimerization domain is a vasodilator-stimulated phosphoprotein (VASP) tetramerization domain (Bachmann et al., J Biol Chem (1999) 274(33):23549-23557). In some embodiments, VASP is or contains the amino acid sequence as shown in SEQ ID NO: 147 (e.g., amino acids 343-375 of full-length VASP; Uniprot Accession No. P50552) or a sequence having about 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 147.

[0298] In some embodiments, the TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) is joined to a multimerization domain (e.g., an Fc region) via a linker (e.g., a peptide linker). In some embodiments, the peptide linker can be a single amino acid residue in length or longer. In some embodiments, the peptide linker is at least one amino acid residue in length but not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length.

[0299] In some embodiments, the linker is (by single letter amino acid code): GGGGS ("4GS"; SEQ ID NO:77) or a multimer of a 4GS linker, such as 2, 3, 4, or 5 repeats of a 4GS linker. In some embodiments, the peptide linker is (GGGGS) 2 (SEQ ID NO:78), (GGGGS) 3 (SEQ ID NO:79), (GGGGS) 4 (SEQ ID NO:84) or (GGGGS) 5 (SEQ ID NO:91). In some embodiments, the linker can also include a series of alanine residues alone or also include another peptide linker (such as a 4GS linker or a polymer thereof). In some embodiments, the linker (in single-letter amino acid code) is GSGGGGS (SEQ ID NO:74) or GGGGSSA (SEQ ID NO:80). In some examples, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO:133). In some examples, the linker is shown in SEQ ID NO:194 or 195.

[0300] In some embodiments, the TACI polypeptide (e.g., variant TACI polypeptide) is directly linked to the Fc sequence. In some embodiments, the TACI polypeptide (e.g., variant TACI polypeptide) is indirectly linked to the Fc sequence, such as via a linker. In some embodiments, one or more "peptide linkers" connect the TACI polypeptide (e.g., variant TACI polypeptide) to the Fc region. In some embodiments, the length of the peptide linker can be a single amino acid residue or longer. In some embodiments, the length of the peptide linker is at least one amino acid residue but not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. Exemplary linkers include any linker as described herein.

[0301] In some embodiments, the TACI-Fc fusion protein has the structure TACI polypeptide (TACI)-linker-Fc region. In some embodiments, the immunomodulatory protein is a homodimer of two identical copies of the TACI-Fc fusion protein. For example, the interaction between the Fc regions of the two identical polypeptide fusions forms a covalent disulfide bond to obtain a dimeric molecule containing two TACI polypeptides (e.g., two variant TACI polypeptides).

[0302] In some embodiments, a TACI-Fc fusion protein is provided, which contains a TACI polypeptide (e.g., any of the ones described above), a linker, and an Fc region in order. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a truncated wild-type TACI polypeptide, such as any of the ones described. In some embodiments, the TACI polypeptide of the TACI Fc fusion is shown in SEQ ID NO: 13. The linker can be any of the ones described. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). In some embodiments, the linker is GS (G4S) 2 (SEQ ID NO: 194). The Fc region can be any Fc region as described. In some embodiments, the Fc region is a wild-type IgG1 Fc as shown in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc as shown in SEQ ID NO: 73.

[0303] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 171. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 197. In some embodiments, the TACI-Fc fusion is encoded by the sequence shown in SEQ ID NO: 208.

[0304] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:172.

[0305] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:196 and is encoded by the sequence shown in SEQ ID NO:207.

[0306] In some embodiments, the TACI polypeptide is a variant TACI polypeptide. In some embodiments, a variant TACI-Fc fusion protein is provided, which contains a variant TACI polypeptide (e.g., any one as described above), a linker, and an Fc region in order. In some embodiments, the TACI polypeptide of the TACI Fc fusion is a variant TACI polypeptide, such as any one as described. In some embodiments, the variant TACI of the variant TACI Fc fusion is shown in any one of SEQ ID NOs: 2-12, 21, 22, or 101-120. In some embodiments, the variant TACI of the variant TACI Fc fusion is shown in any one of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). In some embodiments, the linker is GS (G4S) 2 (SEQ ID NO: 194). In some embodiments, the Fc region is a wild-type IgG1 Fc as shown in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc as shown in SEQ ID NO: 73.

[0307] In some embodiments, the TACI-Fc fusion protein has the amino acid sequence set forth in any one of SEQ ID NOs: 167-170, 200, or 222-237.

[0308] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:167.

[0309] In some embodiments, the TACI-Fc fusion is encoded by the sequence shown in SEQ ID NO:211.

[0310] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:168.

[0311] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:169.

[0312] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:170.

[0313] In some embodiments, the TACI-Fc fusion protein contains multiple copies of a TACI polypeptide sequence (e.g., a variant TACI-polypeptide sequence), such as 2, 3 or 4 TACI polypeptide sequences. In some embodiments, the TACI-Fc fusion protein contains two TACI polypeptide sequences (e.g., two variant TACI polypeptide sequences). In some cases, the TACI polypeptide sequences may be directly connected or may be indirectly connected via a linker (e.g., a peptide linker, including any of the ones described). In such examples, one of the TACI polypeptide sequences is joined or connected to the Fc region, such as joined or connected to the N-terminus or C-terminus of the Fc region. In other cases, the TACI polypeptide sequences may be separated from each other by the Fc region and each is separately joined to the N-terminus or C-terminus of the Fc region. The connection to the Fc region may be a direct connection or may be an indirect connection via a linker (e.g., a peptide linker, including any of the ones described).

[0314] In some embodiments, the TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) can be arranged in series in the fusion protein (hereinafter referred to as "tandem" Fc fusion constructs). In some embodiments, the TACI-Fc fusion protein has the following structure: (TACI)-linker-(TACI)-linker-Fc region. In some embodiments, the immunomodulatory protein is a tetravalent molecule that is a homodimer of two identical copies of the TACI-Fc fusion protein. For example, the interaction between the Fc regions of two identical polypeptide fusions forms a covalent disulfide bond to obtain a dimeric molecule containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0315] In some embodiments, a TACI-Fc fusion protein is provided, which contains, in order, a TACI polypeptide (e.g., any one as described above); a linker; another TACI polypeptide, e.g., any one as described; and an Fc region. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a truncated wild-type TACI polypeptide, e.g., any one as described. In some embodiments, each TACI polypeptide in the TACI Fc fusion is shown in SEQ ID NO: 13. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a variant TACI polypeptide, e.g., any one as described. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a variant TACI as shown in any one of SEQ ID NOs: 2-12, 21, 22, or 101-120. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a variant TACI as shown in any one of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linker can be any one as described. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). The Fc region can be any Fc region as described. In some embodiments, the Fc region is the wild-type IgG1 Fc shown in SEQ ID NO: 81. In some embodiments, the Fc region is the variant Fc shown in SEQ ID NO: 73. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 198 and is encoded by the sequence shown in SEQ ID NO: 209.

[0316] In some embodiments, the TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) can be separated by an Fc region in the fusion protein, wherein the Fc region is located between two TACI polypeptide sequences (hereinafter referred to as a "barbell" Fc fusion construct). In some embodiments, the TACI-Fc fusion protein has the following structure: (TACI)-linker-Fc region-linker-(TACI). In some embodiments, the linkers can be the same or different. In some embodiments, the immunomodulatory protein is a tetravalent molecule that is a homodimer of two identical copies of the TACI-Fc fusion protein. For example, the interaction between the Fc regions of two identical polypeptide fusions forms a covalent disulfide bond to obtain a dimeric molecule containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0317] In some embodiments, a TACI-Fc fusion protein is provided, which contains, in order, a TACI polypeptide (e.g., any one of the above); a linker; an Fc region; a linker; and another TACI polypeptide, e.g., any one of the above. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a truncated wild-type TACI polypeptide, e.g., any one of the above. In some embodiments, each TACI polypeptide in the TACI Fc fusion is shown in SEQ ID NO: 13. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a variant TACI polypeptide, e.g., any one of the above. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a variant TACI as shown in any one of SEQ ID NOs: 2-12, 21, 22, or 101-120. In some embodiments, each TACI polypeptide in the TACI Fc fusion is a variant TACI as shown in any one of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linkers may be any of those described, and may be the same or different. In some embodiments, the first linker is GSGGGGS (SEQ ID NO: 74) and the second linker is (GGGGS) 4 (SEQ ID NO:84). The Fc region can be any Fc region as described. In some embodiments, the Fc region is a wild-type IgG1 Fc as shown in SEQ ID NO:81. In some embodiments, the Fc region is a variant Fc as shown in SEQ ID NO:73. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:201 and is encoded by the sequence shown in SEQ ID NO:212. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:202 and is encoded by the sequence shown in SEQ ID NO:213.

[0318] In some embodiments, a TACI-Fc fusion protein is provided, which is a dimer formed by two identical TACI polypeptides (e.g., variant TACI polypeptides) connected to an Fc domain as described. In some embodiments, any provided TACI-Fc fusion polypeptides (e.g., variant TACI-Fc fusions) of the same species (also referred to as copies) will dimerize to produce homodimers. In some embodiments, the dimer is a homodimer in which the two TACI-Fc polypeptides (e.g., variant TACI-Fc polypeptides) are identical. For the generation of homodimeric Fc molecules, the Fc region is an Fc region capable of forming a homodimer with a matching Fc region by co-expression of a separate Fc region in a cell. In some embodiments, dimerization is mediated by one or more covalent disulfide bonds formed between the Fc regions of the polypeptide fusions.

[0319] Also provided are nucleic acid molecules encoding immunomodulatory proteins. In some embodiments, for the production of immunomodulatory proteins, nucleic acid molecules encoding the immunomodulatory proteins are inserted into appropriate expression vectors. The resulting immunomodulatory proteins can be expressed in host cells transformed with expression vectors, wherein the assembly between Fc domains is performed by the interchain disulfide bonds formed between the Fc portions to produce dimeric (e.g., divalent) immunomodulatory proteins.

[0320] Also provided are nucleic acid molecules encoding TACI-Fc fusion proteins (e.g., variant TACI-Fc fusion proteins). In some embodiments, for the production of Fc fusion proteins, nucleic acid molecules encoding TACI-Fc fusion proteins (e.g., variant TACI-Fc fusion proteins) are inserted into appropriate expression vectors. The resulting TACI-Fc fusion proteins (e.g., variant TACI-Fc fusion proteins) can be expressed in host cells transformed with expression, wherein the assembly between Fc domains is performed by the interchain disulfide bonds formed between the Fc parts to produce dimeric (e.g., divalent) TACI-Fc fusion proteins. The resulting Fc fusion proteins can be easily purified on protein A or protein G columns by affinity chromatography. In order to produce heterodimers, additional purification steps may be required. For example, in the case where two nucleic acids encoding different immunomodulatory proteins are transformed into cells, the formation of heterodimers must be achieved biochemically, because the immunomodulatory proteins carrying Fc domains will also be expressed as disulfide-linked homodimers. Therefore, homodimers can be reduced under conditions that are conducive to destroying interchain disulfide bonds but do not affect intrachain disulfide bonds. In some cases, different immunomodulatory protein monomers are mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of the mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically engineering and expressing immunomodulatory proteins comprising Fc fusion molecules containing one or more TACI variants using a knob-in-hole approach as described.

[0321] In embodiments, when produced and expressed from a cell, an immunomodulatory protein (eg, TACI-Fc (eg, variant TACI-Fc)) is provided as a homodimer containing two identical polypeptide chains. Fig. 8A and Figure 8B The structures of exemplary TACI-Fc fusion proteins provided herein are depicted.

[0322] Provided herein are TACI (26) -Fc_73 homodimers of two identical variant TACI-Fc fusion proteins containing a variant of the TACI cysteine-rich domain 2 (CRD2) as shown in SEQ ID NO: 26, which homodimers are designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI (26) -Fc_73 homodimer is a dimer composed of two identical receptor Fc-fusion protein chains, each chain having a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO: 167 linked by a covalent disulfide bond.

[0323] Provided herein are TACI (26) -Fc_81 homodimers of two identical variant TACI-Fc fusion proteins containing a variant of the TACI cysteine-rich domain 2 (CRD2) as shown in SEQ ID NO: 26, which homodimers are designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI (26) -Fc_81 homodimer is a dimer composed of two identical receptor Fc-fusion protein chains, each chain having a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO: 168 linked by a covalent disulfide bond.

[0324] Provided herein are TACI (27) -Fc_73 homodimers of two identical variant TACI-Fc fusion proteins containing a variant of the TACI cysteine-rich domain 2 (CRD2) as shown in SEQ ID NO: 27, which homodimers are designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI (27) -Fc_73 homodimer is a dimer composed of two identical receptor Fc-fusion protein chains, each chain having a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO: 169 linked by a covalent disulfide bond.

[0325] Provided herein are TACI(27)-Fc_81 homodimers of two identical variant TACI-Fc fusion proteins containing a variant of the TACI cysteine-rich domain 2 (CRD2) as shown in SEQ ID NO: 27, which homodimers are designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI(27)-Fc_81 homodimer is a dimer composed of two identical receptor Fc-fusion protein chains, each chain having a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO: 170 linked by a covalent disulfide bond.

[0326] In some embodiments, provided TACI-Fc (eg, variant TACI-Fc) fusion proteins (eg, homodimers thereof) exhibit an IC of less than 400 pM for neutralizing BAFF. 50 In some embodiments, the IC50 for neutralizing BAFF is between 1 pM and 400 pM, such as between 10 pM and 300 pM, between 10 pM and 200 pM, between 10 pM and 100 pM, between 10 pM and 50 pM, between 10 pM and 20 pM, between 20 pM and 400 pM, between 20 pM and 300 pM, between 20 pM and 200 pM, between 20 pM and 100 pM, between In some embodiments, the IC of neutralizing BAFF is between 20 pM and 50 pM, between 50 pM and 400 pM, between 50 pM and 300 pM, between 50 pM and 200 pM, between 50 pM and 100 pM, between 100 pM and 400 pM, between 100 pM and 300 pM, between 100 pM and 200 pM, between 200 pM and 400 pM, between 200 pM and 300 pM, or between 300 pM and 400 pM. 50 is at or about 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM or 100 pM, or any value therebetween.

[0327] In some embodiments, provided TACI-Fc (eg, variant TACI-Fc) fusion proteins (eg, homodimers thereof) exhibit an IC for neutralizing APRIL of less than 400 pM. 50In some embodiments, the IC50 for neutralizing APRIL is between 0.5 pM and 100 pM, such as between 0.5 pM and 50 pM, between 0.5 pM and 25 pM, between 0.5 pM and 10 pM, between 0.5 pM and 5 pM, between 0.5 pM and 1 pM, between 1 pM and 100 pM, between 1 pM and 50 pM, between 1 pM and 25 pM, between 1 pM and 1 In some embodiments, the IC of neutralizing APRIL is between 1 pM and 5 pM, between 5 pM and 100 pM, between 5 pM and 50 pM, between 5 pM and 25 pM, between 5 pM and 10 pM, between 10 pM and 100 pM, between 10 pM and 50 pM, between 10 pM and 25 pM, or between 25 pM and 100 pM, between 25 pM and 50 pM, or between 50 pM and 100 pM. In some embodiments, the IC of neutralizing APRIL is between 1 pM and 5 pM, between 5 pM and 100 pM, between 5 pM and 50 pM, between 5 pM and 25 pM, between 5 pM and 100 pM, between 25 pM and 50 pM, or between 50 pM and 100 pM. 50 is at or about 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 20 pM or 25 pM, or any value therebetween. III. Nucleic acids, vectors and methods for producing the polypeptides or cells

[0328] Provided herein are isolated or recombinant nucleic acids (collectively referred to as "nucleic acids") encoding any of the immunomodulatory proteins provided herein. In some embodiments, the nucleic acids provided herein (including all nucleic acids described below) can be used for the recombinant production (e.g., expression) of the immunomodulatory proteins provided herein. In some embodiments, the nucleic acids provided herein (including all nucleic acids described below) can be used for the expression of the immunomodulatory proteins provided herein (such as the TACI fusion proteins provided herein). The nucleic acids provided herein can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleic acids provided herein are typically DNA molecules, and are typically double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA / RNA nucleic acids or combinations thereof comprising any nucleotide sequence of the present invention are also provided.

[0329] In some cases, a heterologous (non-natural) signal peptide can be added to the nucleic acid encoding the immunomodulatory protein. This may be desirable, for example, in the case of expressing a TACI fusion protein without an amino terminal signal sequence. In some embodiments, the signal peptide is a signal peptide from: an immunoglobulin (such as an IgG heavy chain or an IgG-κ light chain), a cytokine (such as an interleukin-2 (IL-2) or CD33), a serum albumin protein (such as HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (such as a chymotrypsinogen or a trypsinogen), or other signal peptides that can be effectively expressed and secreted from a cell protein in some aspects. Exemplary signal peptides include any one described in Table 3.

[0330] In some embodiments, the immunomodulatory protein comprises a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the immunomodulatory protein when secreted.

[0331] Also provided herein are recombinant expression vectors and recombinant host cells that can be used to produce immunomodulatory proteins, such as the TACI fusion proteins provided herein.

[0332] In any of the embodiments provided above, nucleic acids encoding immunomodulatory polypeptides provided herein can be introduced into cells using recombinant DNA and cloning techniques. For this reason, recombinant DNA molecules encoding immunomodulatory polypeptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, suitable restriction enzymes can be used to excise the coding sequence of the peptide from DNA. Alternatively, chemical synthesis techniques (such as the phosphoramidite method) can be used to synthesize DNA molecules. Similarly, a combination of these techniques can be used. In some cases, recombinant or synthetic nucleic acids can be generated by polymerase chain reaction (PCR). As known to those skilled in the art, the DNA insert encoding the immunomodulatory protein can be cloned into a suitable transduction / transfection vector. An expression vector containing the nucleic acid molecule is also provided.

[0333] In some embodiments, the expression vector can express the immunomodulatory protein in a suitable cell under conditions suitable for expressing the protein. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding the immunomodulatory protein operably connected to a suitable expression control sequence. The method of realizing such operative connection before or after the DNA molecule is inserted into the vector is well known. The expression control sequence includes a promoter, an activator, an enhancer, an operator, a ribosome binding site, a start signal, a stop signal, a capping signal, a polyadenylation signal and other signals related to transcription or translation control.

[0334] In some embodiments, expression of the immunomodulatory protein is controlled by a promoter or enhancer to control or regulate expression. The promoter is operably linked to the portion of the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein.

[0335] Use the obtained recombinant expression vector with DNA molecule thereon to transform the appropriate host.This transformation can be carried out using methods well known in the art.In some embodiments, the nucleic acid provided herein also comprises a nucleotide sequence encoding a secretion or signal peptide operably connected to the nucleic acid encoding the immunomodulatory polypeptide, so that the obtained soluble immunomodulatory polypeptide is recovered from culture medium, host cell or host cell periplasm.In other embodiments, select appropriate expression control signal to allow membrane expression of immunomodulatory polypeptide.In addition, commercially available kits and contracted manufacturing companies can also be used to prepare engineered cells or recombinant host cells provided herein.

[0336] In some embodiments, the resulting expression vector with a DNA molecule thereon is used to transform (e.g., transduce) appropriate cells. Methods known in the art can be used to introduce. Exemplary methods include those methods for transferring nucleic acids encoding receptors, including via viruses (e.g., retroviruses or slow viruses), transduction, transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, nucleic acid is transferred to cells by slow virus or retrovirus transduction methods.

[0337] Any of a large number of publicly available and well-known mammalian host cells (including mammalian T cells or APCs) can be used to prepare polypeptides or engineered cells. The selection of cells depends on a variety of factors recognized in the art. These factors include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, conversion rate, ease of recovering the peptide, expression characteristics, biosafety, and cost. To balance these factors, it must be deeply understood that not all cells can be used equally effectively for the expression of a specific DNA sequence.

[0338] In some embodiments, the host cell is a mammalian cell. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40 transformed monkey kidney cells (COS-1; ATCC CRL 1650) and mouse embryonic cells (NIH-3T3; ATCC CRL 1658).

[0339] In some embodiments, the host cell can be a variety of eukaryotic cells, such as yeast cells, or mammalian cells, such as Chinese hamster ovary (CHO) or HEK293 cells. In some embodiments, the host cell is a suspension cell, and the polypeptide is engineered or produced in a culture suspension, such as in a cultured suspension CHO cell (e.g., CHO-S cell). In some examples, the cell line is a DHFR-deficient (DHFR-) CHO cell line, such as DG44 and DUXB11. In some embodiments, the cell lacks glutamine synthase (GS), for example, CHO-S cells, CHOK1 SV cells, and CHOZN ((R)) GS- / - cells. In some embodiments, the CHO cell (e.g., suspension CHO cell) can be a CHO-S-2H2 cell, a CHO-S-clone 14 cell, or an ExpiCHO-S cell.

[0340] In some embodiments, the host cell can also be a prokaryotic cell, such as Escherichia coli. The transformed recombinant host is cultured under polypeptide expression conditions and then purified to obtain soluble protein. The recombinant host cell can be cultured under conventional fermentation conditions so that the desired polypeptide is expressed. Such fermentation conditions are well known in the art. Finally, the polypeptide provided herein can be recovered and purified from recombinant cell culture by any of many methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography and affinity chromatography. When completing the configuration of mature protein, a protein refolding step can be used as needed. Finally, high performance liquid chromatography (HPLC) can be used in the final purification step.

[0341] In some embodiments, the recombinant vector is a viral vector. Exemplary recombinant viral vectors include lentiviral vector genomes, poxvirus vector genomes, vaccinia virus vector genomes, adenovirus vector genomes, adenovirus-associated virus vector genomes, herpes virus vector genomes, and alphavirus vector genomes. The viral vector can be live, attenuated, replication-conditional or replication-defective, non-pathogenic (defective), replicable viral vectors and / or modified to express heterologous gene products, such as variant immunomodulatory polypeptides provided herein. The vector used to generate the virus can also be modified to change the attenuation of the virus, including any method that increases or decreases the transcription or translation load.

[0342] Exemplary viral vectors that can be used include modified vaccinia virus vectors (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartaglia et al., AIDS Research and Human Retroviruses 8:1445-47 (1992); Gheradi et al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14 (1975); Hu et al., J. Virol. 75:10300-308 (2001); U.S. Pat. Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615 and 7,368,116); adenovirus vectors or adenovirus-associated virus vectors (see, e.g., Molin et al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93 (2004); U.S. Pat. Nos. 6,143,290; 6,596,535; 6,855,317; 6,936,257; 7,125,717; 7,378,087; 7,550,296); retroviral vectors, including those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations (see, e.g., Buchscher et al., J. Virol. 66:2731-39 (1992); Johann et al., J. Virol. 66:1635-40 (1992); Sommerfelt et al., Virology 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-78 (1989); Miller et al., J. Virol. 65:2220-24 (1991); Miller et al., Mol. Cell Biol. 10:4239 (1990); Kolberg, NIHRes. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215 (1991)); lentiviral vectors, including those based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV), and maedi / visna virus (see, e.g., Pfeifer et al., Annu. Rev. Genomics Hum.Genet.2:177-211 (2001); Zufferey et al., J.Virol.72:9873, 1998; Miyoshi et al., J.Virol.72:8150, 1998; Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al., J.Virol.69:2729, 1995; Nightingale et al., Mol.Therapy,13:1121, 2006; Brown et al., J.Virol.73:9011 (1999); WO 2009 / 076524; WO 2012 / 141984; WO 2016 / 011083; McWilliams et al., J. Virol. 77:11150, 2003; Powell et al., J. Virol. 70:5288, 1996) or any variant thereof, and / or a vector that can be used to produce any of the above viruses. In some embodiments, the recombinant vector can include a regulatory sequence, such as a promoter or enhancer sequence, which can regulate the expression of the viral genome (such as in the case of RNA viruses) in the packaging cell line (see, for example, U.S. Patent Nos. 5,385,839 and 5,168,062). .

[0343] In some aspects, nucleic acid or expression vector comprises the nucleic acid sequence of the coding immunomodulatory protein operably connected with suitable expression control sequence.It is well known that the method of realizing this operably connected before or after the nucleic acid sequence of coding immunomodulatory protein is inserted into the vector.Expression control sequence comprises promoter, activator, enhancer, operator, ribosome binding site, start signal, termination signal, capping signal, polyadenylation signal and other signals related to transcription or translation control.Described promoter can be operably connected with the part of coding immunomodulatory protein in nucleic acid sequence.

[0344] Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al., J. Molec. Appl Genet. 1:273 (1982)), the TK promoter of herpes virus (McKnight, Cell 31:355 (1982)), the SV40 early promoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor virus promoter (see generally Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," Protein Engineering: Principles and Practice, Cleland et al. (eds.), pp. 163-181 (John Wiley & Sons, Inc. 1996)). One useful combination of promoter and enhancer is provided by the myeloproliferative sarcoma virus promoter and the human cytomegalovirus enhancer.

[0345] Alternatively, prokaryotic promoters (such as the bacteriophage T3 RNA polymerase promoter) can be used to control the production of immunomodulatory proteins in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al., Mol Cell. Biol. 10:4529 (1990); and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

[0346] Expression vectors can be introduced into host cells using a variety of standard techniques, including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, etc. Transfected cells can be selected and bred to provide recombinant host cells comprising expression vectors stably integrated into the host cell genome. For example, Ausubel (1995) and Murray (editor) are used to describe the technology for introducing vectors into eukaryotic cells and for selecting such stable transformants using dominant selectable markers, Gene Transfer and Expression Protocols (Humana Press 1991).

[0347] For example, a suitable selectable marker is a gene that provides resistance to the antibiotic neomycin. In this case, select in the presence of neomycin-type drugs (such as G-418, etc.). The selection system can also be used to increase the expression level of the target gene, and the process is referred to as "amplification". Amplify in the following manner: culture transfectants in the presence of low-level selection agents, then increase the amount of the selection agent to select the cells that produce the product of the high-level introduced gene. Suitable amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (for example, hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used. Alternatively, the phenotypic markers (such as green fluorescent protein, or cell surface proteins (such as CD4, CD8, class I MHC, placental alkaline phosphatase)) of the phenotype introduced into the sorting of the cells and the non-transfected cells can be used by means such as FACS sorting or magnetic bead separation technology.

[0348] In some embodiments, polypeptide provided herein can also be prepared by synthetic method.Solid phase synthesis is the preferred technology for preparing independent peptides, because it is the most cost-effective method for preparing small peptides.For example, known solid phase synthesis technology comprises the use of blocking group, joint and solid support and specific protection and deprotection reaction conditions, joint cleavage conditions, use of scavenger and solid phase peptide synthesis other aspects.Then peptide can be assembled into polypeptide as provided in this paper. IV. Pharmaceutical Compositions

[0349] Provided herein are compositions containing any of the provided immunomodulatory proteins described herein (e.g., TACI-Fc fusion proteins). In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a TACI-Fc fusion protein as described, provided as a formulation with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Also provided are any of the provided pharmaceutical compositions, including any of the provided formulations, for use in treating an autoimmune or inflammatory disease in a patient in need thereof, such as any use for treating such a disease or condition as described in Section V. Also provided are methods for treating an autoimmune or inflammatory disease in a patient in need thereof by administering any such pharmaceutical composition or formulation, such as for treating any disease or condition as described in Section V.

[0350] The pharmaceutical composition may also include a pharmaceutically acceptable excipient. For example, the pharmaceutical composition may contain one or more excipients for changing, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Such a composition may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0351] In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition can be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.

[0352] In some embodiments, the pharmaceutical composition is a liquid, such as an immunomodulatory protein (e.g., TACI-Fc fusion protein) dissolved in an aqueous solution (e.g., saline or Ringer's solution). In some embodiments, the pH of the pharmaceutical composition is about 4.0 to about 8.5 (e.g., about 4.0 to about 5.0, about 4.5 to about 5.5, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, about 6.5 to about 7.5, about 7.0 to about 8.0, or about 7.5 to about 8.5).

[0353] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, a binder, a coating, a preservative, a lubricant, a flavoring agent, a sweetener, a colorant, a solvent, a buffer, a chelating agent or a stabilizer. Examples of pharmaceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch or potato starch. Examples of pharmaceutically acceptable binders include polyvinyl pyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose or cellulose. Examples of pharmaceutically acceptable coatings include hydroxypropyl methylcellulose (HPMC), shellac, corn protein zein or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinyl pyrrolidone, carboxymethyl cellulose or sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate or stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid or sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame or sorbitol. Examples of pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates or tartrates.

[0354] In certain embodiments, the property of the main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution or buffered saline. In some embodiments, the pharmaceutical composition comprises a Tris buffer of pH about 7.0-8.5. In some embodiments, the pharmaceutical composition comprises an acetate buffer of pH about 4.0-6.0. The preparation can contain a certain concentration of buffer, and the buffer has enough buffering capacity to maintain the selected pH of the preparation at a selected temperature. In various embodiments, the concentration of the buffer solution can be from about 1mM to about 100mM, from about 2mM to about 50mM, from about 3mM to about 30mM, from about 4mM to about 20mM, or from about 5mM to about 10mM, or from about 10mM to about 40mM, from about 15mM to about 35mM, from about 20mM to about 30mM, from about 25mM to about 35mM.

[0355] In some embodiments, the buffer solution contains acetate at a concentration of from about 1 mM to about 100 mM, from about 2 mM to about 50 mM, from about 3 mM to about 30 mM, from about 4 mM to about 20 mM, or from 5 mM to 15 mM, or from about 5 mM to about 10 mM, or from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, from about 20 mM to about 30 mM, from about 25 mM to about 35 mM. In some embodiments, the buffer solution contains acetate at a concentration of from 5 mM to 15 mM. In some embodiments, the buffer solution contains acetate at a concentration of or about 5 mM, or about 6 mM, or about 7 mM, or about 8 mM, or about 9 mM, or about 10 mM, or about 11 mM, or about 12 mM, or about 13 mM, or about 14 mM, or about 15 mM, or any value between any of the foregoing. In some embodiments, the buffer solution contains acetate at a concentration of or about 5 mM. In some embodiments, the buffer solution contains acetate at a concentration of or about 10 nM. In some embodiments, the buffer solution contains acetate at a concentration of or about 12 mM. In some embodiments, the buffer solution contains acetate at a concentration of or about 15 mM. Exemplary pH ranges of acetic acid (acetate) buffer and / or final formulations may include the following pH ranges: between about 4.0 and about 6.0, between about 4.5 and about 5.5, between about 4.8 and about 5.2, or between about 5.0. Therefore, the acetic acid (acetate) buffer and / or the final formulation can be prepared to have a pH of about 4.0, about 4.5, about 4.8, about 5.0, about 5.2, about 5.5, about 5.7, or about 6.0, or any value between any of the foregoing. In some embodiments, the pH of the buffer solution is at or about 5.0. In some embodiments, the pH of the buffer solution is at or about 5.2. In some embodiments, the pH of the buffer solution is at or about 5.5. Those skilled in the art can determine the pH of the acetic acid (acetate) buffer in the formulation.

[0356] In certain embodiments, acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions may contain formulation materials for improving, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., proline, glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., acetate, borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight the invention relates to a pharmaceutical composition comprising: a polypeptide having an ionic strength; a salt-forming counterion (such as sodium); a preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); a solvent (such as glycerol, propylene glycol or polyethylene glycol); a sugar alcohol (such as mannitol or sorbitol); a suspending agent; a surfactant or wetting agent (such as pluronics, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyloxapal); a stability enhancer (such as sucrose or sorbitol); a tonicity enhancer (such as alkali metal halides (preferably sodium chloride, potassium chloride), mannitol sorbitol); a delivery vehicle; a diluent; an excipient and / or a pharmaceutical adjuvant. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18" edition, (AR Genrmo ed.), 1990, Mack Publishing Company.

[0357] Free amino acids (such as, but not limited to, lysine, proline, serine, and alanine) can be used as bulking agents, stabilizers, and antioxidants to stabilize the proteins in the provided formulations, as well as for other standard uses. In some embodiments, the free amino acids are present in the formulation at a concentration of about 1% to about 10% or 2% to 5%.

[0358] In some embodiments, the formulation contains proline as a free amino acid. In some embodiments, the formulation provided contains proline at a concentration of about 1% to about 10%. In some embodiments, the formulation provided contains proline at a concentration of about 2% to about 5%. In some embodiments, the formulation provided contains proline at a concentration of or about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or any value between any of the foregoing. In some embodiments, the formulation provided contains proline at a concentration of or about 2%. In some embodiments, the formulation provided contains proline at a concentration of or about 3%. In some embodiments, the formulation provided contains proline at a concentration of or about 4%.

[0359] The formulation provided may further comprise a surfactant. Protein molecules may be easily adsorbed on the surface and are prone to denaturation and subsequent aggregation at gas-liquid, solid-liquid and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. In some cases, physical agitation (such as physical agitation generated during the transportation and handling of the product) may exacerbate this effect. Surfactants may be used to prevent, minimize or reduce surface adsorption. The surfactant contained in the formulation may be selected, for example, to enhance or promote the maintenance of protein molecule stability by preventing or reducing aggregation and / or adsorption. Sorbitan fatty acid esters such as polysorbates are surfactants that exhibit a wide range of hydrophilic and emulsifying characteristics. They may be used alone or in combination with other surfactants to cover a wide range of stable needs. Such features may be suitable for use with active protein agents because they can be customized to cover a wide range of biopharmaceutical hydrophobic and hydrophilic characteristics. Useful surfactants include, but are not limited to, polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates and poloxamer 188.

[0360] The surfactant concentration of the provided formulation can be less than about 1% (w / v). In this regard, the surfactant concentration can generally be used in the following ranges: between 0.001%-0.10% (w / v), between about 0.001%-0.05% (w / v), between about 0.001%-0.025% (w / v), between about 0.001%-0.01% (w / v), between about 0.005%-0.10%, between about 0.005%-0.05%, between about 0.005%-0.025%, between about 0.005%-0.01%, between about 0.01%-0.10%, between about 0.01%-0.05%, or between about 0.01% and 0.025%. Surfactant concentrations and / or amounts less than, greater than, or between these ranges can also be used. Thus, formulations can be produced containing essentially any desired concentration or amount of one or more surfactants, including, for example, about 0.001% (w / v), 0.002% (w / v), 0.003% (w / v), 0.004% (w / v), 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), 0.10% (w / v), 0.11% (w / v), 0.12% (w / v), 0.13% (w / v), 0.14% (w / v), 0.15% (w / v), 0.16% (w / v), 0.17% (w / v), 0.18% (w / v), 0.19% (w / v), 0.20% (w / v), 0.21% (w / v), 0.22% (w / v), 0.23% (w / v), 0.24% (w / v), 0.25% (w / v), 0.26% (w / v), 0.27% (w / v), 0.28% (w / v), 0.29% (w / v), 0.30% (w / v), 0.31% (w / v), 0.32% (w / v), 0.33% (w / v), 0.34% (w / v), / v), 0.010% (w / v), 0.015% (w / v), 0.02% (w / v), 0.025% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v) or 0.10% (w / v), or any value between any of the foregoing.

[0361] In some embodiments, the surfactant is polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the formulation is between about 0.001%-0.10% (w / v), between about 0.001%-0.05% (w / v), between about 0.001%-0.025% (w / v), between about 0.001%-0.01% (w / v), between about 0.005%-0.10%, between about 0.005%-0.05%, between about 0.005%-0.025%, between about 0.005%-0.01%, between about 0.01%-0.10%, between about 0.01%-0.05%, or between about 0.01% and 0.025%. In some embodiments, polysorbate 80 is present at a concentration of 0.001% (w / v), 0.002% (w / v), 0.003% (w / v), 0.004% (w / v), 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), 0.010% (w / v), In some embodiments, the concentration of polysorbate 80 is at or about 0.010% (w / v). In some embodiments, the concentration of polysorbate 80 is at or about 0.015% (w / v). In some embodiments, the concentration of polysorbate 80 is at or about 0.02% (w / v). In some embodiments, the concentration of polysorbate 80 is at or about 0.02% (w / v).

[0362] In some embodiments, the pharmaceutical composition further comprises an agent for controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads or liposomes.

[0363] In some embodiments, the pharmaceutical composition is sterile. Sterilization can be accomplished by filtering through a sterile filter membrane or irradiation. In the case of a lyophilized composition, the method can be used for sterilization before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution form. In addition, parenteral compositions are usually placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0364] A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition or vehicle. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients in the formulation. It must also be suitable for contact with any tissue, organ or part of the body it may encounter, meaning that it must not carry the risk of toxicity, irritation, allergic reaction, immunogenicity or any other complication that would be too much to outweigh its therapeutic benefit.

[0365] In some embodiments, the pharmaceutical composition is formulated to contain the following amounts of TACI-Fc fusion: from or about 1 mg to or about 100 mg, such as from or about 1 mg to or about 75 mg, from or about 1 mg to or about 50 mg, from or about 1 mg to or about 25 mg, from or about 1 mg to or about 10 mg, from or about 1 mg to or about 5 mg, from or about 5 mg to or about 100 mg, from or about 5 mg to or about 75 mg, from or about 5 mg to or about 50 mg, from or about 5 mg to or about 2 In some embodiments, the present invention relates to an agent that is at least about 5 mg to about 10 mg, from about 10 mg to about 100 mg, from about 10 mg to about 75 mg, from about 10 mg to about 50 mg, from about 10 mg to about 25 mg, from about 25 mg to about 100 mg, from about 25 mg to about 75 mg, from about 25 mg to about 50 mg, from about 50 mg to about 100 mg, from about 50 mg to about 75 mg, or from about 75 mg to about 100 mg. In some embodiments, the pharmaceutical composition is formulated to contain the following amount of TACI-Fc fusion protein: at or about 10 mg, at or about 20 mg, at or about 25 mg, at or about 30 mg, at or about 40 mg, at or about 50 mg, at or about 60 mg, at or about 70 mg, at or about 75 mg, at or about 80 mg, or at or about 100 mg, or any value between any of the foregoing. In some embodiments, the pharmaceutical composition is formulated to contain the TACI-Fc fusion protein in an amount of at or about 80 mg.

[0366] In some embodiments, the pharmaceutical composition is formulated in a volume of from or about 0.5 mL to or about 10 mL, such as from or about 0.5 mL to or about 5 mL, from or about 0.5 mL to or about 2 mL, from or about 0.5 mL to or about 1 mL, from or about 1 mL to or about 10 mL, from or about 1 mL to or about 5 mL, or from or about 5 mL to or about 10 mL. In some embodiments, the pharmaceutical composition is formulated in a volume of at or about 0.5 mL, at or about 1 mL, at or about 2 mL, at or about 2.5 mL, at or about 3 mL, at or about 4 mL, at or about 5 mL, at or about 6 mL, at or about 7 mL, at or about 8 mL, at or about 9 mL, or at or about 10 mL. In some embodiments, the composition is formulated in a volume of at or about 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.2 mL, 1.4 mL, 1.6 mL, 1.8 mL or 2.0 mL, or any value in between any of the foregoing.

[0367] In some embodiments, the concentration of the TACI-Fc fusion protein in the composition is from at or about 1 mg / mL to at or about 50 mg / mL, such as from at or about 1 mg / mL to at or about 25 mg / mL, from at or about 1 mg / mL to at or about 15 mg / mL, from at or about 1 mg mL to at or about 5 mg / mL, from at or about 5 mg / mL to at or about 50 mg / mL, from at or about 5 mg / mL to at or about 25 mg / mL, from at or about 5 mg / mL to at or about 15 mg / mL, from at or about 15 mg / mL to at or about 50 mg / mL, from at or about 15 mg / mL to at or about 25 mg / mL, or from at or about 25 mg / mL to at or about 50 mg / mL. In some embodiments, the concentration of the TACI-Fc fusion protein in the composition is at or about 1 mg / mL, at or about 5 mg / mL, at or about 10 mg / mL, at or about 15 mg / mL, at or about 20 mg / mL, at or about 25 mg / mL, at or about 30 mg / mL, at or about 40 mg / mL, or at or about 50 mg / mL. Provided herein are any such compositions contained in a container such as a vial. In particular aspects, the container such as a vial is sterile. The container can be any biocompatible container, such as a glass container. In some embodiments, the vial is a 2 mL glass vial.

[0368] In some embodiments, the concentration of the TACI-Fc fusion protein in the composition is greater than 50 mg / mL. In some embodiments, the concentration of the composition is between at or about 50 mg / mL and 200 mg / mL, such as between at or about 50 mg / mL and 150 mg / mL, between at or about 50 mg / mL and 100 mg / mL, between at or about 100 mg / mL and 200 mg / mL, between at or about 100 mg / mL and 150 mg / mL, or between at or about 150 mg / mL and 200 mg / mL. In some embodiments, the concentration of the TACI-Fc fusion protein in the composition is at or about 60 mg / mL, at or about 70 mg / mL, at or about 80 mg / mL, at or about 100 mg / mL, at or about 120 mg / mL, at or about 140 mg / mL, at or about 160 mg / mL, at or about 180 mg / mL, or at or about 200 mg / mL, or any value between any of the foregoing. In some embodiments, the concentration of the TACI-Fc fusion protein in the composition is at or about 100 mg / mL. Provided herein are any such compositions contained in a container such as a vial. In particular aspects, the container such as a vial is sterile. The container can be any biocompatible container, such as a glass container. In some embodiments, the vial is a 2 mL glass vial.

[0369] In some embodiments, the TACI-Fc fusion protein (such as any described) is formulated in a buffer solution containing 10 mM acetate, 3% proline, 0.015% polysorbate 80 at pH 5.2. In some embodiments, the TACI-Fc fusion protein is provided at 100 mg / mL as a liquid for injection (e.g., IV or SC). In some embodiments, the TACI-Fc fusion protein is provided in a volume of 1 mL or about 8 mL (e.g., 80 mg) in a container (e.g., a 2 mL glass vial).

[0370] In some embodiments of the provided formulations, the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI, the variant TACI being a portion of the extracellular domain consisting of the CRD2 TNF receptor domain shown in SEQ ID NO: 13, wherein there are amino acid substitutions K77E, F78Y and Y102D. In some embodiments, the variant TACI is shown in SEQ ID NO: 26. In any of the embodiments of the TACI-Fc fusion proteins, the variant TACI is linked to the Fc domain via the linker. In some of any of the provided methods or uses, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 167. In some of any of the embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 168.

[0371] In some any embodiments, when the TACI-Fc fusion protein or a formulation containing the TACI-Fc fusion protein is administered at a concentration of less than 100 mg / mL, it can be diluted in a physiologically acceptable buffer such as 0.9% sodium chloride (normal saline).

[0372] Once the pharmaceutical composition has been prepared, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or lyophilized powder. Such preparations can be stored in a ready-to-use form or in a form (e.g., lyophilized) reconstructed before administration. Also provided herein is a kit for producing a single-dose administration unit. In some aspects, the kit can each contain a first container with dried protein and a second container with an aqueous preparation. In certain embodiments, a kit containing a single chamber and a multi-chamber pre-filled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0373] In some embodiments, a pharmaceutical composition such as any provided formulation is stable at or about -20°C for up to 6 months or longer, such as up to 12 months or longer. In some embodiments, the pharmaceutical composition is stored at or about -20°C. In some embodiments, storage is performed under the following conditions: the formulation of the TACI-Fc fusion protein is protected from light.

[0374] In some embodiments, the pharmaceutical composition is administered to the subject. Generally, according to standard pharmaceutical practice, the dosage and administration route of the pharmaceutical composition are determined according to the subject's size and condition. For example, the therapeutically effective dose can be estimated initially in a cell culture assay or in an animal model such as a mouse, rat, rabbit, dog, pig or monkey. The animal model can also be used to determine an appropriate concentration range and administration route. The information can then be used to determine the available dose and administration route in people. The exact dose will be determined based on factors related to the subject in need of treatment. Adjust the dose and administration to provide a sufficient level of active compound or to maintain the desired effect. Factors that can be considered include the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, the time and frequency of administration, one or more drug combinations, reaction sensitivity and the response to therapy.

[0375] Long-acting pharmaceutical compositions can be administered once every 3 to 4 days, weekly or every two weeks according to the half-life and clearance rate of a particular formulation. The frequency of administration will depend on the pharmacokinetic parameters of the molecule in the formulation used. Typically, the composition is administered until the dosage for the desired effect is reached. Therefore, the composition can be administered in a single dose, or in multiple doses (with the same or different concentrations / dosages) over time, or in a continuous infusion. Further improvements are routinely made to suitable dosage. Suitable dosage can be determined by using appropriate dose-response data.

[0376] In some embodiments, the pharmaceutical composition is administered to a subject by any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, applied directly to a wound site, applied to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, percutaneously, by aerosolization, intrapleurally, intraventricularly, intraarticularly, intraocularly, or intraspinally.

[0377] In some embodiments, provided pharmaceutical formulations may be, for example, in a form suitable for intravenous infusion. In some embodiments, provided formulations may be in a form suitable for subcutaneous administration. V. Methods for Assessing the Activity of Immunomodulatory Proteins and Immunomodulation

[0378] In some embodiments, provided immunomodulatory proteins (such as TACI fusion proteins provided herein) exhibit immunomodulatory activity. Provided immunomodulatory proteins (such as TACI fusion proteins) can modulate B cell activity, such as one or more of B cell proliferation, differentiation or survival.

[0379] The function of immunomodulatory proteins can be examined using a variety of methods to assess the ability of the protein to bind to a cognate binding partner. For example, the binding of TACI fusion proteins to APRIL or BAFF can be assessed. A variety of assays are known for assessing binding affinity and / or determining whether a binding molecule (e.g., an immunomodulatory protein) specifically binds to a particular binding partner. Technicians can skillfully determine the binding affinity of a binding molecule (e.g., an immunomodulatory protein) to a binding partner (e.g., APRIL or BAFF), such as by using any of a variety of binding assays well known in the art. Various binding assays are known and include, but are not limited to, for example, ELISA. D , KinExA, flow cytometry and / or surface plasmon resonance devices, including those described herein. Such methods include, but are not limited to, Or flow cytometry methods. For example, in some embodiments, The instrument can be used to determine the binding kinetics and constants of the complex between two proteins using surface plasmon resonance (SPR) analysis (see, for example, Scatchard et al., Ann. NY Acad. Sci. 51: 660, 1949; Wilson, Science 295: 2103, 2002; Wolff et al., Cancer Res. 53: 2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614 or equivalents). When the molecule is bound to the surface or dissociated from the surface, SPR measures the change in the concentration of the molecule at the sensor surface. The change in the SPR signal is proportional to the change in mass concentration close to the surface, thereby allowing the measurement of the binding kinetics between the two molecules. The dissociation constant of the complex can be determined by monitoring the change in the refractive index relative to time when the buffer passes through the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) or by monitoring changes in the spectral or optical properties of the proteins by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR) to determine binding. Other exemplary assays include, but are not limited to, Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting expressed polynucleotides or protein binding.

[0380] The immunomodulatory protein provided can also be assessed in any of a variety of assessments to assess the regulation of B cell activity. One such assay is a cell proliferation assay. In the presence or absence of a test compound (such as an immunomodulatory protein), cells are cultured, and cell proliferation is detected by colorimetric assay (Mosman, J. Immunol. Meth. 65: 55-63, 1983) by, for example, measuring the incorporation of tritiated thymidine or based on the metabolic decomposition of 3- (4,5- dimethylthiazol-2-yl) -2,5- diphenyl tetrazolium bromide (MTT). Alternative assay forms use further engineered cells to express reporter genes. The reporter gene is connected to a promoter element that reacts to a receptor-linked pathway, and the assay detects activation of the reporter gene transcription. A variety of reporter genes that are easy to measure in a cell extract are known in the art, for example, Escherichia coli lacZ, chloramphenicol acetyltransferase (CAT) and serum response element (SRE) (see, for example, Shaw et al., Cell 56: 563-72, 1989). An exemplary reporter gene is the luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:29094-101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase activity assay kits are commercially available, for example, from Promega Corp. (Madison, Wisconsin).

[0381] The immunomodulatory protein provided can be characterized by the ability to inhibit the stimulation of human B cells by soluble APRIL or BAFF, as described in International Publication No. WO 00 / 40716 by Gross et al. In brief, human B cells are isolated from peripheral blood mononuclear cells, such as using CD19 magnetic beads (e.g., Miltenyi Biotec Auburn, California). The purified B cells can be incubated under stimulating conditions (e.g., in the presence of soluble APRIL) and further incubated in the presence of immunomodulatory proteins at gradually adjusted concentrations. The B cells can be labeled with a proliferation dye, or can be labeled with 1 μCi 3 The number of B cells can be determined over time by labeling with H-thymidine to measure proliferation.

[0382] A reporter cell line expressing a reporter gene under the operable control of transcription factors such as NF-kB, NFAT-1 and AP-1 can be prepared, which expresses TACI or BCMA. For example, reporter cells can include Jurkat and other B lymphoma cell lines. These cells are incubated with soluble BAFF or APRIL ligands to signal via the reporter genes in these constructs. The effect of the provided immunomodulatory protein on regulating this signal transduction can be evaluated.

[0383] Perfect animal models are available to test the in vivo efficacy of the provided immunomodulatory proteins in certain disease states, including those involving autoimmune or inflammatory conditions. For example, animal models of autoimmune diseases include, for example, MRL-lpr / lpr or NZB×NZW F1 congenic mouse strains, which are used as models of SLE (systemic lupus erythematosus). Such animal models are known in the art, see, for example, Autoimmune Disease Models A Guidebook, Cohen and Miller edited Academic Press. Offspring of hybrids between New Zealand black (NZB) and New Zealand white (NZW) mice develop spontaneous forms of SLE that are very similar to human SLE. Offspring mice called NZBW begin to produce IgM autoantibodies to T cells at 1 month of age, and by 5-7 months of age, Ig anti-DNA autoantibodies are dominant immunoglobulins. Polyclonal B cell overactivity leads to excessive production of autoantibodies. The deposition of these autoantibodies, particularly autoantibodies to single-stranded DNA, is associated with the development of glomerulonephritis, which clinically manifests as proteinuria, azotemia, and death due to renal failure. Renal failure is the leading cause of death in mice affected by spontaneous SLE, and in the NZBW strain, this process is chronic and occlusive. The disease is faster and more severe in females than in males, with an average survival time of only 245 days, compared to 406 days in males. Although many female mice will have symptoms (proteinuria) by 7-9 months of age, some female mice may be significantly younger or older when symptoms occur. The fatal immune nephritis seen in NZBW mice is very similar to the glomerulonephritis seen in human SLE, making this spontaneous mouse model very attractive for testing potential SLE therapeutics (Putterman and Naparstek, Murine Models of Spontaneous Systemic Lupus Erythematosus, Autoimmune Disease Models: A Guidebook, Chapter 14, pages 217-34, 1994; Mohan et al., J. Immunol. 154: 1470-80, 1995; and Daikh et al., J. Immunol. 159: 3104-08, 1997). The administration of provided immunomodulatory proteins to these mice can be evaluated to evaluate the efficacy of improving symptoms and changes in the course of the disease.

[0384] Another mouse model of inflammation and lupus-like disease is the bm12-induced mouse SLE model (Klarquist and Janssen, 2015. J. Vis. Exp. (105), e53319). bm12B6(C)-H2-Ab1 bm12 Splenocyte suspensions from H2-Ab1 / KhEgJ ("bm12") mice were adoptively transferred into female C57BL / 6NJ recipient mice. bm12 With H2-Ab1 b The difference is 3 nucleotides, resulting in a change of 3 amino acids in the beta chain of the MHC class II IA molecule. Allogeneic activation of donor bm12 CD4+ T cells by recipient antigen-presenting cells leads to chronic GVHD, whose symptoms are very similar to SLE, including autoantibody production, changes in immune cell subsets, and mild renal disease. Glomerulonephritis with immune complex deposition occurs later in the model and is mainly composed of autoantigens bound to IgG1, IgG2b, IgG2c, and IgG3 antibodies. Endpoints of the model can include the concentration of anti-dsDNA antibodies, select IgG isotypes, blood urea nitrogen (BUN), and creatinine in serum, immune cell subset composition in spleen and cervical LN, and renal histology.

[0385] In some embodiments, a mouse model of Sjögren's syndrome (SjS) can be used. Based on a modified form of the scheme disclosed by Zhou et al., 2016 Sci. Rep. 6, 39105, repeated administration of anti-mouse (m) PD-L1 antibodies can be used to induce SjS disease and accelerated onset of diabetes in female non-obese diabetic (NOD) mice susceptible to diabetes. Starting at 6 weeks of age, 100 μg of anti-PD-L1 antibodies were injected intraperitoneally (IP) into mice on days 0, 2, 4, and 6 of the study, and the mice were treated with the provided immunomodulatory proteins on different days. Untreated mice were included as controls for endpoint analysis. All mice were usually killed on day 10 of the study, and submandibular glands (SMGs) and pancreases were collected from each mouse for histopathological evaluation to assess the signs and severity of sialadenitis and insulitis. Blood glucose levels can be measured on different days.

[0386] In some embodiments, a mouse model of experimental allergic encephalomyelitis (EAE) can be used. The model is similar to human multiple sclerosis, and demyelination occurs due to activation of T cells of neural proteins such as myelin basic protein (MBP) or proteolipid protein (PLP). Inoculation with antigen leads to induction of CD4+ class II MHC restricted T cells (Th1). Changes in the scheme for EAE can produce acute, chronic recurrent or passive transmission variants of the model (Weinberg et al., J.Immunol.162:1818-26,1999; Mijaba et al., Cell.Immunol.186:94-102,1999; and Glabinski, Meth.Enzym.288:182-90,1997). It can be assessed to apply the provided immunomodulatory protein to improve symptoms and changes to the course of the disease.

[0387] In some embodiments, a collagen-induced arthritis (CIA) model can be used, in which mice develop a chronic inflammatory arthritis that is very similar to human rheumatoid arthritis (RA). Since CIA shares similar immune and pathological features with RA, this makes it an ideal model for screening potential human anti-inflammatory compounds. Another advantage of using the CIA model is that the pathogenesis is known. T cell and B cell epitopes on type II collagen have been identified, and various immunological (delayed type hypersensitivity and anti-collagen antibodies) and inflammatory (cytokines, chemokines and matrix degrading enzymes) parameters associated with immune-mediated arthritis have been determined and can be used to evaluate the efficacy of test compounds in the model (Wooley, Curr. Opin. Rheum. 3: 407-20, 1999; Williams et al., Immunol. 89: 9784-788, 1992; Myers et al., Life Sci. 61: 1861-78, 1997; and Wang et al., Immunol. 92: 8955-959, 1995). Administration of the provided immunomodulatory proteins can be evaluated to improve symptoms and alter the course of the disease.

[0388] In some embodiments, a model of bronchial infection such as asthma can be created when mice are injected with ovalbumin and restimulated nasally with an antigen, which produces an asthmatic response in the bronchi similar to asthma. Administration of provided immunomodulatory proteins can be evaluated for improvement of symptoms and changes in the course of the disease.

[0389] In some embodiments, myasthenia gravis (MG) is another autoimmune disease for which a mouse model can be obtained. MG is a neuromuscular transmission disorder, which involves the generation of autoantibodies to nicotinic acetylcholine receptors (AChR). MG is acquired or hereditary, and its clinical features include abnormal weakness and fatigue during physical activity. MG mouse model has been established. (Christadoss et al., Establishment of a Mouse Model of Myasthenia Gravis Which Mimics Human Myasthenia Gravis Pathogenesis for Immune Intervention, Immunobiology of Proteins and Peptides VIII, Atassi and Bixler edited, 1995, pp. 195-99.) Experimental autoimmune myasthenia gravis (EAMG) is an antibody-mediated disease characterized by the presence of antibodies to AChR. These antibodies destroy the receptor, resulting in neuromuscular electrical impulse defects, thereby causing muscle weakness. In the EAMG model, mice are immunized with nicotinic acetylcholine receptors. Clinical signs of MG become apparent several weeks after the second immunization. EAMG is evaluated by several methods, including measurement of serum levels of AChR antibodies by radioimmunoassay (Christadoss and Dauphinee, J. Immunol. 136:2437-40, 1986; and Lindstrom et al., Methods Enzymol. 74:432-60, 1981), measurement of muscle AChRs or electromyography (Wu et al. Protocols in Immunology. Vol. 3, ed. Coligen, Kruisbeak, Margulies, Shevach and Strober. John Wiley and Sons, New York, p. 15.8.1, 1997).

[0390] Another use of the in vivo model includes delivering antigenic challenge to the animal, then administering the immunomodulatory protein and measuring T cell and B cell responses. T cell-dependent and non-T cell-dependent immune responses can be measured as described in Perez-Melgosa et al., J.Immunol.163:1123-7, 1999. The immune response in an animal to which a provided immunomodulatory protein is administered after conventional antigenic challenge (e.g., keyhole limpet hemocyanin (KLH), sheep red blood cells (SRBC), ovalbumin or collagen) can be performed to measure the effect on B cell responses.

[0391] Pharmacokinetic studies in conjunction with radiolabeled immunomodulatory proteins can be used to determine the distribution and half-life of such polypeptides in vivo.

[0392] In some embodiments, the pharmacokinetic (PK) and pharmacodynamic (PD) profiles observed in control animals and animal models of disease (e.g., cancer models) can be used to predict or determine patient administration. For example, PK data from non-human primates (e.g., cynomolgus monkeys) can be used to estimate human PK. Similarly, mouse PK and PD data can be used to predict human administration. Observed animal data can be used to provide information for computational models, which can be used to simulate human dose responses. VI. Therapeutic Applications

[0393] The pharmaceutical compositions described herein (including pharmaceutical compositions comprising immunomodulatory proteins described herein (e.g., TACI-Fc)) can be used in a variety of therapeutic applications (e.g., methods of treating diseases). Therapeutic applications of pharmaceutical compositions include methods and uses of any of the provided formulations. For example, in some embodiments, pharmaceutical compositions (e.g., any of the provided formulations) are used to treat inflammatory or autoimmune disorders, cancer, organ transplantation, viral infections, and / or bacterial infections in mammals. Pharmaceutical compositions (e.g., any of the provided formulations) can modulate (e.g., reduce) immune responses to treat diseases.

[0394] Such methods and uses include therapeutic methods and uses, for example, it is designed to administer molecules or compositions containing the molecules to subjects suffering from diseases, disorders or disorders. In some cases, as described, the disease, disorder or disorder is an autoimmune or inflammatory disease or disorder. In some embodiments, the molecules or engineered cells are administered in an effective amount to achieve the treatment of the disease or disorder. Uses include the use of molecules containing immunomodulatory proteins, and the use in the preparation of drugs to perform such therapeutic methods. In some embodiments, the method is performed by administering the provided immunomodulatory protein or a composition containing it to a subject suffering from or suspected of having a disease or disorder. In some embodiments, the method thus treats the disease, disorder or disorder or disorder of the subject.

[0395] Illustrative subjects include mammalian subjects, such as farm animals, livestock, and human patients. In certain embodiments, the subject is a human subject.

[0396] The pharmaceutical compositions described herein can be used in a variety of therapeutic applications, such as the treatment of a disease. For example, in some embodiments, the pharmaceutical compositions are used to treat inflammatory or autoimmune disorders, organ transplantation, viral infections, and / or bacterial infections in mammals. The pharmaceutical compositions can modulate immune responses to treat diseases. In some embodiments, the pharmaceutical compositions suppress immune responses, which can be used in the treatment of inflammatory or autoimmune disorders or in organ transplantation.

[0397] It is believed that the provided methods can be used in a variety of applications, including but not limited to, for example, in preventive or therapeutic methods for treating a variety of immune system diseases or conditions in mammals, in which the regulation or control of the immune system and immune system responses is beneficial. For example, suppressing immune responses can be beneficial in preventive and / or therapeutic methods for inhibiting the rejection of tissues, cells or organ transplants from donors by the recipient. In therapeutic situations, the mammalian subject is typically a subject suffering from an immune system disease or condition, and administration is performed to prevent further progression of the disease or condition.

[0398] The immunomodulatory proteins provided (including TACI fusion proteins) can be used for the treatment of the following diseases: autoimmune diseases, B cell cancers, immunomodulation, EBD and any antibody-mediated pathology (e.g., ITCP, myasthenia gravis, etc.), nephropathy, indirect T cell immune response, graft rejection and graft-versus-host disease. The administration of immunomodulatory proteins (e.g., TACI-Fc) can specifically regulate B cell responses during immune responses. In addition, the administration of the immunomodulatory proteins provided can be used to regulate B cell development, the development of other cells, antibody production and cytokine production. The administration or use of the immunomodulatory proteins provided can also regulate B cell communication, such as by neutralizing the proliferation effect of BAFF or APRIL.

[0399] In some embodiments, the pharmaceutical composition suppresses the immune response, which can be used in the treatment of inflammatory or autoimmune disorders or in organ transplantation. In some embodiments, the pharmaceutical composition contains an immunomodulatory protein that exhibits antagonist activity to B cell stimulatory receptors, thereby reducing or decreasing the immune response. In some embodiments, the composition can be used to treat B cell-mediated diseases.

[0400] In some embodiments, compositions can be used to treat autoimmune diseases. In some embodiments, the therapeutic composition containing the immunomodulatory protein provided herein is applied to a subject suffering from an immune system disease (e.g., an autoimmune disease) and can result in the suppression or inhibition of such an immune system attack or a biological response associated therewith. By suppressing this immune system attack on healthy body tissues, the resulting physical symptoms (e.g., pain, joint inflammation, joint swelling or tenderness) caused by or associated with the attack on healthy tissues can be reduced or alleviated, and the biological and physical damage caused by or associated with the attack on the immune system can be reduced, slowed down or stopped. In preventive situations, the subject can be a subject suffering from, susceptible to or considered to present an immune system disease, disorder or illness, and is generally administered to prevent disease, disorder or illness progression, suppress or alleviate symptoms, signs or biological responses associated therewith, prevent the physical damage that may be caused by it, and / or maintain or improve the body function of the subject.

[0401] In some embodiments, the disease or condition that can be treated by the pharmaceutical compositions described herein is any disease mediated by: immune complex deposition (e.g., lupus nephritis, vasculitis); direct interference with pathways (e.g., catastrophic antiphospholipid antibody syndrome, myasthenic crisis; anti-Jo-1 disease); opsonization or direct injury to cells (e.g., idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia); antibody-mediated rejection of allografts (e.g., highly sensitized renal transplant patients); or anti-drug antibodies to biological replacement factors, carriers (e.g., anti-factor 8).

[0402] In some embodiments, the inflammatory or autoimmune disorder, condition or disease that can be treated by the pharmaceutical compositions described herein is systemic lupus erythematosus (SLE), including flare prevention without the use of glucocorticoids; Sjögren's syndrome; primary biliary cirrhosis (PBC); systemic sclerosis; polymyositis; diabetes prevention; IgA nephropathy; IgA vasculitis; B-cell cancers, such as myeloma; multiple sclerosis, optic neuritis.

[0403] In some embodiments, the inflammatory or autoimmune disorder is inflammatory arthritis.Examples of inflammatory arthritis treated according to the provided methods include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, lupus, Lyme disease, gout, or ankylosing spondylitis.

[0404] In some embodiments, the immunomodulatory proteins provided can be used to treat pre-B cell leukemia or B cell leukemia (such as plasma cell leukemia, chronic or acute lymphocytic leukemia), myeloma (such as multiple myeloma, plasma cell myeloma, endothelial myeloma and giant cell myeloma) and lymphoma (such as non-Hodgkin's lymphoma). In some any embodiments, the type of myeloma includes multiple myeloma, plasma cell tumor, multiple solitary plasma cell tumor and / or extramedullary myeloma. In some any embodiments, the type of myeloma includes light chain myeloma, non-secretory myeloma and / or IgD or IgE myeloma.

[0405] In some embodiments, the immunomodulatory protein provided can be used as an immunosuppressant to selectively block the effect of B lymphocytes for the treatment of diseases. For example, some autoimmune diseases are characterized by the production of autoantibodies, which promote tissue destruction and disease aggravation. Autoantibodies may also cause the occurrence of immune complex deposition complications and cause many symptoms of systemic lupus erythematosus (including renal failure, neuralgia symptoms and death). Regulating antibodies produced unrelated to cell response may also be useful in many disease states. It has also been shown that B cells play a role in the secretion of arthritis-causing immunoglobulins in rheumatoid arthritis. The immunomodulatory protein provided is used to suppress, block or neutralize the effect of B cells, so that the method and use of suppressing antibody production may be useful in the treatment of the following diseases: autoimmune diseases, such as myasthenia gravis, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis and psoriatic arthritis.

[0406] In some embodiments, the immunomodulatory protein provided can be used to block or neutralize the B cell effect associated with end-stage renal disease, which may be associated with autoimmune diseases or may be irrelevant. Such a method will also be used to treat immune nephropathy. Such a method will also be used to treat glomerulonephritis associated with diseases such as: membranous nephropathy, IgA nephropathy or Berger disease, IgM nephropathy, IgA vasculitis, Goodpasture's disease, post-infectious glomerulonephritis, mesangial proliferative disease, chronic lymphocytic leukemia, minimal change nephrotic syndrome. Such a method will also be used as a therapeutic application for treating secondary glomer...

Claims

1. A method of treating an inflammatory or autoimmune disease or disorder in a subject in need thereof, the method comprising administering to the subject a TACI-Fc fusion protein, which is a homodimer of two polypeptides of the formula TACI-Linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13, wherein the TACI-Fc fusion protein is administered once a week up to once every three months at a dose of from at or about 2.4 mg to at or about 960 mg.

2. The method of claim 1, wherein the dose of the TACI-Fc fusion protein is administered once every three months.

3. The method of claim 1, wherein the dose of the TACI-Fc fusion protein is administered once a month (Q4W).

4. The method of claim 1, wherein the dose of the TACI-Fc fusion protein is administered once every other week (Q2W).

5. The method of claim 1, wherein the dose of the TACI-Fc fusion protein is administered once a week (Q1W).

6. The method of any one of claims 1-5, wherein the dose of the TACI-Fc fusion protein is from or about 8 mg to 960 mg.

7. The method of any one of claims 1-6, wherein the dose of the TACI-Fc fusion protein is from or about 80 mg to 960 mg.

8. The method of any one of claims 1-7, wherein the dose of the TACI-Fc fusion protein is from at or about 80 mg to at or about 720 mg, from at or about 160 mg to at or about 560 mg, or from at or about 240 mg to at or about 480 mg.

9. The method of any one of claims 1-7, wherein the dose of the TACI-Fc fusion protein is from at or about 24 mg to at or about 480 mg, optionally from at or about 40 mg to at or about 480 mg, from at or about 80 mg to at or about 320 mg, or from at or about 80 mg to at or about 120 mg.

10. A formulation comprising a TACI-Fc fusion protein, an acetate buffer having a pH of from about 4.0 to about 6.0, a proline concentration of from about 1% to about 10%, and a surfactant concentration of from about 0.005% to about 0.05% (w / v), wherein the TACI-Fc fusion protein is a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising amino acid substitutions K77E, F78Y and Y102D in the amino acid sequence shown in SEQ ID NO:13.

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