Use of mutated TACI-FC fusion proteins for treatment of autoantibody mediated diseases

Abstract

Provided herein are methods of treatment and uses relating to immunomodulatory TACI-Fc fusion proteins that exhibit BAFF and APRIL (or BAFF / APRIL heterotrimer) neutralizing activity. The TACI-Fc proteins provided may comprise variant domains of a transmembrane activator and 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

[0001] Cross - reference to related applications

[0002] This application claims priority to the following applications: U.S. Provisional Application No. 63 / 378,361 (filed Oct. 4, 2022), U.S. Provisional Application No. 63 / 382,094 (filed Nov. 2, 2022), U.S. Provisional Application No. 63 / 383,243 (filed Nov. 10, 2022), U.S. Provisional Application No. 63 / 385,948 (filed Dec. 2, 2022), U.S. Provisional Application No. 63 / 483,936 (filed Feb. 8, 2023), U.S. Provisional Application No. 63 / 486,946 (filed Feb. 24, 2023), U.S. Provisional Application No. 63 / 491,526 (filed Mar. 21, 2023), U.S. Provisional Application No. 63 / 497,691 (filed Apr. 21, 2023), U.S. Provisional Application No. 63 / 502,611 (filed May 16, 2023), U.S. Provisional Application No. 63 / 505,053 (filed May 30, 2023), and U.S. Provisional Application No. 63 / 581,609 (filed Sep. 8, 2023), all titled "Methods and Uses of TACI - FC Fusion Immunomodulatory Proteins", the contents of which are hereby incorporated by reference in their entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence listing (761612004340SEQLIST.xml; size: 345,811 bytes; creation date: Sep. 27, 2023) are hereby incorporated by reference in their entirety. Technical field

[0005] The present disclosure provides therapeutic methods and uses related to immunomodulatory TACI - Fc fusion proteins that exhibit neutralizing activity against BAFF and APRIL (or BAFF / APRIL heterotrimers). The provided TACI - Fc fusion proteins may include variant domains of the transmembrane activator and CAML interactor (TACI). The methods and uses provide therapeutic utility for a variety of immune diseases, disorders, or afflictions, such as B - cell mediated diseases, disorders, or afflictions. Background art

[0006] The regulation of immune responses by intervening in the interaction between soluble ligands and their receptors has received increasing medical attention. Currently, biologics for enhancing or suppressing immune responses are generally limited to antibodies (e.g., anti - PD - 1 antibodies) or soluble receptors against single cell - surface molecules (e.g., CTLA - 4 - Fc). Improved therapeutic agents are needed to regulate immune responses, particularly B - cell immune responses. Embodiments meeting such needs are provided. SUMMARY OF THE INVENTION

[0007] In some aspects, provided herein is a method of treating an autoimmune-related disease or disorder in a subject, the method comprising administering to the subject 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 that comprises one or more amino acid substitutions selected from K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO:13, and wherein the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks (Q4W). In some aspects, provided herein is a method of treating an autoimmune-related disease or disorder in a subject, the method comprising administering to the subject 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 that comprises one or more amino acid substitutions selected from K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO:13, and wherein the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 24 mg to from or about 480 mg once every four weeks (Q4W), once every eight weeks (Q8W), or once every twelve weeks (Q12W).

[0008] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E, F78Y, and Y102D.

[0009] In some embodiments, the dose is from or about 80 mg to from or about 240 mg Q4W. In some embodiments, the dose is from or about 80 mg Q4W. In some embodiments, the dose is from or about 240 mg Q4W. In some embodiments, the dose is from or about 24 mg to from or about 240 mg once every four weeks (Q4W), once every eight weeks (Q8W), or once every twelve weeks (Q12W). In some embodiments, the dose is from or about 24 mg Q4W. In some embodiments, the dose is from or about 24 mg Q8W. In some embodiments, the dose is from or about 24 mg Q12W. In some embodiments, the dose is from or about 80 mg Q8W. In some embodiments, the dose is from or about 80 mg Q12W. In some embodiments, the dose is from or about 240 mg Q8W. In some embodiments, the dose is from or about 240 mg Q12W.

[0010] In some embodiments, the autoantibody-related disease or disorder is selected from rheumatic diseases or disorders, kidney (renal) diseases or disorders, blood diseases or disorders, skin diseases or disorders, or neurological diseases or disorders. In some embodiments, the autoantibody-related disease or disorder is a rheumatic disease or disorder. In some embodiments, the autoantibody-related disease or disorder is Sjögren's syndrome. In some embodiments, the autoantibody-related disease or disorder is systemic lupus erythematosus (SLE).

[0011] In some embodiments, the TACI-Fc fusion protein reduces the risk of a subject developing hypogammaglobulinemia or severe hypogammaglobulinemia. In some embodiments, hypogammaglobulinemia is characterized by a circulating IgG ≤ 7 g / L. In some embodiments, severe hypogammaglobulinemia is characterized by a circulating IgG < 3 g / L. In some embodiments, severe hypogammaglobulinemia is characterized by a circulating IgG < 1.5 g / L. In some embodiments, severe hypogammaglobulinemia is characterized by a circulating IgG < 1.0 g / L. In some embodiments, the TACI-Fc fusion protein reduces the amount of circulating immunoglobulin G (IgG). In some embodiments, the circulating IgG is reduced by about 35% compared to the subject's baseline.

[0012] In some aspects, provided herein is a method of treating systemic lupus erythematosus (SLE), the method comprising: a) selecting a subject diagnosed with SLE for administration of a TACI-Fc fusion protein; and b) administering the TACI-Fc fusion protein to the selected subject, 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; and the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks.

[0013] In some embodiments, the dose is from or about 80 mg to from or about 240 mg Q4W. In some embodiments, the dose is from or about 80 mg Q4W. In some embodiments, the dose is from or about 240 mg Q4W.

[0014] In some embodiments, the systemic lupus erythematosus is mild to moderate systemic lupus erythematosus or moderate to severe systemic lupus erythematosus. In some embodiments, the systemic lupus erythematosus is mild systemic lupus erythematosus. In some embodiments, the systemic lupus erythematosus is moderate systemic lupus erythematosus. In some embodiments, the systemic lupus erythematosus is severe systemic lupus erythematosus.

[0015] In some embodiments, the subject is selected for treatment if the subject has had active SLE for ≥6 months at the time of screening.

[0016] In some embodiments, the subject is selected for treatment if SLE is characterized by one or more of the following at the time of screening: (i) a combined SELENA-SLEDAI score ≥8 or combined SELENA-SLEDAI ≥6 if there are high anti-dsDNA or low complement (C) levels; (ii) ≤6 g / g total urinary protein to creatinine ratio (proteinuria); (iii) a BILAG score of grade A in ≥1 organ; (iv) a BILAG score of grade B in ≥2 organs; and (v) a Physician's Global Assessment (PGA) score ≥1.0.

[0017] In some embodiments, the subject is receiving standard therapy for treating SLE.

[0018] In some embodiments, the subject is selected for treatment if, at the time of screening or administration of the TACI-Fc fusion protein, the subject is receiving a stable standard treatment regimen, which is characterized by the stable use of standard therapy for treating SLE. In some embodiments, stable use is the stable use of standard therapy for at least 30 days.

[0019] In some embodiments, the TACI-Fc fusion protein is administered to the subject in combination with standard therapy for treating SLE.

[0020] In some embodiments, the standard therapy includes one or more of corticosteroids, antimalarials (such as hydroxychloroquine), non-steroidal anti-inflammatory drugs (NSAIDs), or immunosuppressants or immunomodulators, or any combination thereof.

[0021] In some embodiments, the immunosuppressant or immunomodulator is selected from azathioprine, mycophenolate (such as mycophenolate mofetil or sodium mycophenolate), cyclophosphamide, methotrexate, leflunomide, tacrolimus, cyclosporine, and any combination of the foregoing.

[0022] In some embodiments, the standard therapy includes corticosteroids, and the administration of corticosteroids is tapered after administration of the TACI-Fc fusion protein.

[0023] In some embodiments, a subject is selected for treatment if the subject is characterized at screening by one or more of the following: (i) severe lupus nephritis, such as defined as urine protein >6 g / 24 hours or serum creatinine >2.5 mg / dL or 221 μmol / L; (ii) requiring hemodialysis; (iii) having received high-dose corticosteroids for ≥14 days within the past 2 months, such as where the high-dose corticosteroids are treatment with prednisone >100 mg / day or equivalent; and (iv) a central nervous system disease, whether caused by SLE or not, within the past 2 months. In some aspects, the central nervous system disease is epilepsy, psychosis, organic brain syndrome, cerebrovascular accident, encephalitis, or central nervous system vasculitis.

[0024] In some embodiments, the autoantibody-related disease or disorder is a kidney (renal) disease or disorder. In some embodiments, the autoantibody-related disease or disorder is glomerulonephritis. In some aspects, provided herein is a method of treating glomerulonephritis, the method comprising: a) selecting a subject diagnosed with glomerulonephritis for administration of a TACI-Fc fusion protein; and b) administering the TACI-Fc fusion protein to the selected subject, 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; and the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks.

[0025] In some embodiments, the dose is from or about 80 mg to from or about 240 mg Q4W. In some embodiments, the dose is from or about 80 mg Q4W. In some embodiments, the dose is from or about 240 mg Q4W.

[0026] In some embodiments, a subject is selected for treatment if the subject has active glomerulonephritis at screening. In some embodiments, the glomerulonephritis is selected from IgA nephropathy, lupus nephritis, and primary membranous nephropathy.

[0027] In some embodiments, the glomerulonephritis is IgA nephropathy, and the subject is selected for treatment if the subject is characterized by one or both of the following at screening: (i) the subject was diagnosed with IgA nephropathy ≤5 years prior to screening; and (ii) urine total protein to creatinine ratio ≥0.75 g / g (proteinuria). In some embodiments, the glomerulonephritis is IgA nephropathy, and the subject is selected for treatment if the subject is characterized by one or more of the following at screening: (i) the subject was diagnosed with IgA nephropathy ≤5 years prior to screening; (ii) urine total protein to creatinine ≥0.75 g / g (proteinuria); and (iii) elevated galactose-deficient IgA1 (Gd-IgA1). In some embodiments, the TACI-Fc fusion protein reduces Gd-IgA1. In some embodiments, the Gd-IgA1 is reduced by more than 50%.

[0028] In some embodiments, the glomerulonephritis is lupus nephritis, and the lupus nephritis is class III (active focal), class IV (diffuse), or class V (lupus membranous nephropathy).

[0029] In some embodiments, the glomerulonephritis is lupus nephritis, and the subject is selected for treatment if the subject is characterized by one or more of the following at screening: (i) the subject was diagnosed with lupus nephritis class II-V ≤3 years prior to screening; (ii) urine total protein to creatinine ratio ≥1 g / g (proteinuria); (iii) active urinary sediment; (iv) positive for anti-dsDNA and antinuclear antibody (ANA), such as where positive anti-dsDNA is a titer ≥30 IU / mL and positive ANA is a titer ≥1:80; and (v) a stable standard treatment regimen, where the standard treatment regimen is characterized by the stable use of standard therapy for SLE, such as where stable use is the stable use of standard therapy for at least 30 days; and (v) has received stable background immunosuppression, such as where stable background immunosuppression is receiving a stable dose of MMF ≥1 g / day for at least 8 weeks prior to screening or at the time of administration of the TACI-Fc fusion protein, with or without corticosteroids.

[0030] In some embodiments, the glomerulonephritis is primary membranous nephropathy.

[0031] In some embodiments, the glomerulonephritis is primary membranous nephropathy (pMN), and the subject is selected for treatment if the subject is characterized by one or more of the following at screening: (i) the subject was diagnosed with pMN ≤5 years prior to screening; (ii) urine total protein to creatinine ratio ≥3.5 g / g (proteinuria); and (iii) positive for anti-PLA2R1 antibody or anti-THSD7A antibody.

[0032] In some embodiments, a subject is selected for treatment if the subject has been receiving angiotensin converting enzyme (ACE) inhibitor and / or angiotensin II receptor blocker (ARB) treatment at the time of screening or at the time of administration of the TACI-Fc fusion protein, such as where the subject has been receiving the maximum recommended dose of an ACE inhibitor or ARB treatment.

[0033] In some embodiments, a subject is selected for treatment if the subject's blood pressure is stable at the time of screening or at the time of administration of the TAC-Fc fusion protein.

[0034] In some embodiments, the autoantibody-related disease or disorder is a blood disease or disorder. In some embodiments, the autoantibody-related disease or disorder is autoimmune cytopenia.

[0035] In some aspects, provided herein is a method of treating autoimmune cytopenia, the method comprising: a) selecting a subject diagnosed with autoimmune cytopenia for administration of a TACI-Fc fusion protein; and b) administering the TACI-Fc fusion protein to the selected subject, 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; and the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks.

[0036] In some embodiments, the dose is from or about 80 mg to from or about 240 mg Q4W. In some embodiments, the dose is from or about 80 mg Q4W. In some embodiments, the dose is from or about 240 mg Q4W.

[0037] In some embodiments, a subject is selected for treatment if the subject has active cytopenia at the time of screening.

[0038] In some embodiments, the autoimmune cytopenia is selected from immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA).

[0039] In some embodiments, the autoimmune cytopenia is ITP, and a subject is selected for treatment if the subject is characterized by one or more of the following at the time of screening: (i) the subject has been diagnosed with ITP for ≥ 3 months prior to screening; (ii) the platelet count has been consistently < 30,000 / μL; and (iii) the subject has received ≥ 4 prior treatments for ITP.

[0040] In some embodiments, the autoimmune cytopenia is AIHA, and the AIHA is warm-type AIHA (wAIHA) or cold-type AIHA (cold agglutinin disease, CAD).

[0041] In some embodiments, the autoimmune cytopenia is wAIHA or CAD, and the subject is selected for treatment if the subject is characterized by one or more of the following at screening: (i) the subject has been diagnosed with wAIHA or CAD for ≥ 3 months prior to screening; (ii) persistent hemoglobin (Hb) < 9 g / dL; and (iii) has received ≥ 2 prior treatments for AIHA.

[0042] In some embodiments, the autoimmune cytopenia is wAIHA. In some embodiments, the autoimmune cytopenia is CAD.

[0043] In some embodiments, the subject is selected for treatment if the subject is receiving stable immunosuppression at the time of screening or at the time of administration of the TACI-Fc fusion protein.

[0044] In some embodiments, the TACI-Fc fusion protein is co-administered to the subject in combination with a stable immunosuppressant.

[0045] In some embodiments, the stable immunosuppression comprises a stable dose of a steroid, such as a corticosteroid, for at least two weeks prior to screening or at the time of administration of the TACI-Fc fusion protein; and / or the stable immunosuppression comprises a stable dose of azathioprine, MMF, or cyclosporine for at least four weeks prior to screening or at the time of administration of the TACI-Fc fusion protein.

[0046] In some embodiments, the subject is not characterized as having secondary cytopenia (e.g., a systemic autoimmune disease or malignancy) or Evans syndrome.

[0047] In some embodiments, the autoantibody-related disease or disorder is a skin disease or disorder. In some embodiments, the autoantibody-related disease or disorder is an autoimmune bullous skin disease.

[0048] In some aspects, the present disclosure provides a method of treating an autoimmune bullous (vesicular) skin disease, the method comprising: a) selecting a subject diagnosed with an autoimmune bullous (vesicular) skin disease for administration of a TACI-Fc fusion protein; and b) administering a TACI-Fc fusion protein to the selected subject, 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 set forth in SEQ ID NO:13; and the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks.

[0049] In some embodiments, the dose is from or about 80 mg to from or about 240 mg Q4W. In some embodiments, the dose is from or about 80 mg Q4W. In some embodiments, the dose is from or about 240 mg Q4W.

[0050] In some embodiments, the subject is selected for treatment if the subject has an active vesicular disease at the time of screening. In some embodiments, the autoimmune bullous (vesicular) skin disease is selected from pemphigus vulgaris, pemphigus foliaceus, or bullous pemphigoid.

[0051] In some embodiments, the autoimmune bullous (vesicular) skin disease is pemphigus vulgaris or pemphigus foliaceus, and the subject is selected for treatment if the subject is characterized by one or both of the following at the time of screening: (i) a pemphigus disease area index (PDAI) ≥ 15; and (ii) positive anti-Dsg1 antibodies or positive anti-Dsg3 antibodies.

[0052] In some embodiments, the autoimmune bullous (vesicular) skin disease is pemphigus vulgaris. In some embodiments, the autoimmune bullous (vesicular) skin disease is pemphigus foliaceus.

[0053] In some embodiments, the autoimmune bullous (vesicular) skin disease is pemphigoid, and the subject is selected for treatment if the subject is characterized by one or both of the following at the time of screening: (i) IgA antibodies; and (ii) positive anti-Bp180 antibodies or positive anti-Bp230 antibodies.

[0054] In some embodiments, the subject is selected for treatment if the subject is receiving stable immunosuppression at the time of screening or at the time of administration of the TACI-Fc fusion protein.

[0055] In some embodiments, the TACI-Fc fusion protein is co-administered to a subject in combination with a stable immunosuppressant.

[0056] In some embodiments, stable immunosuppression includes a stable dose of steroids, such as corticosteroids, for at least two weeks prior to screening or administration of the TACI-Fc fusion protein; and / or stable immunosuppression includes a stable dose of azathioprine, MMF, or cyclosporine for at least four weeks prior to screening or administration of the TACI-Fc fusion protein.

[0057] In some embodiments, the subject is not characterized as having a secondary disease (e.g., paraneoplastic).

[0058] In some embodiments, the autoantibody-related disease or disorder is a neurological disease or disorder. In some embodiments, the autoantibody-related disease or disorder is encephalitis.

[0059] In some aspects, provided herein is a method of treating encephalitis, the method comprising: a) selecting a subject diagnosed with encephalitis for administration of a TACI-Fc fusion protein; and b) administering the TACI-Fc fusion protein to the selected subject, 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 the amino acid substitutions K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13; and the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks.

[0060] In some embodiments, the dose is from or about 80 mg to from or about 240 mg Q4W. In some embodiments, the dose is from or about 80 mg Q4W. In some embodiments, the dose is from or about 240 mg Q4W.

[0061] In some embodiments, the encephalitis is autoimmune encephalitis. In some embodiments, the encephalitis is limbic encephalitis.

[0062] In some embodiments, the TACI-Fc fusion protein is administered to a subject Q4W for 12 to 72 weeks. In some embodiments, the TACI-Fc fusion protein is administered to a subject Q4W for 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks or longer. In some embodiments, the TACI-Fc fusion protein is administered to a subject Q4W for 12 weeks. In some embodiments, the TACI-Fc fusion protein is administered to a subject Q4W for 16 weeks. In some embodiments, the TACI-Fc fusion protein is administered to a subject Q4W for 24 weeks. In some embodiments, the TACI-Fc fusion protein is administered to a subject Q4W for 48 weeks.

[0063] In some embodiments of any of the provided methods, alternative embodiments contemplate administering the TACI-Fc fusion protein to a subject Q8W or Q12W instead of Q4W. In some of any such embodiments of any of the provided methods, the dose administered to the subject is from or about 240 mg to from or about 480 mg Q8W, such as the TACI-Fc fusion protein at from or about 240 mg Q8W, from or about 320 mg Q8W, or from or about 480 mg Q8W. In some of any such embodiments of any of the provided methods, the dose administered to the subject is from or about 240 mg to from or about 480 mg Q12W, such as the TACI-Fc fusion protein at from or about 240 mg Q12W, from or about 320 mg Q12W, or from or about 480 mg Q12W.

[0064] In some embodiments, the variant TACI polypeptide is as shown in SEQ ID NO:26.

[0065] In some embodiments, the linker is a GS linker having a length between 5 and 20 amino acids. In some embodiments, the linker 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), GGGGS SA (SEQ ID NO:80) or GSGGGGSGGGGS (SEQ ID NO:194) or a combination thereof. In some embodiments, the linker is as shown in SEQ ID NO:74.

[0066] In some embodiments, the Fc is an IgG1 Fc domain. In some embodiments, the Fc is a variant IgG1 Fc that exhibits a reduced binding affinity for Fc receptors and / or reduced effector function compared to the wild-type IgG1 Fc domain.

[0067] In some embodiments, the variant IgG1 Fc domain comprises one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to the EU numbering. In some embodiments, the variant IgG1 Fc comprises the amino acid substitutions L234A, L235E, and G237A according to the EU numbering.

[0068] In some embodiments, the Fc comprises the amino acid substitution C220S, where the residue is numbered according to the EU index of Kabat.

[0069] In some embodiments, the Fc lacks the hinge sequence EPKSS or EPKSC.

[0070] In some embodiments, the Fc region comprises K447del, where the residue is numbered according to the EU index of Kabat. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO:73.

[0071] In some embodiments, the TACI-Fc fusion protein is as shown in SEQ ID NO:167.

[0072] In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO:81.

[0073] In some embodiments, the TACI-Fc fusion protein is as shown in SEQ ID NO:168.

[0074] In some embodiments, the TACI-Fc fusion protein is provided in a formulation comprising an acetate buffer having a pH of from about 4.0 to about 6.0, proline at a concentration of from or about 1% to about 10%, and a surfactant at a concentration of from about 0.005 to about 0.05% (w / v).

[0075] In some embodiments, the pH of the formulation is about 5.2.

[0076] In some embodiments, the acetate buffer comprises acetate at a concentration of from or about 5 mM to or about 15 mM. In some embodiments, the acetate buffer comprises acetate at a concentration of or about 10 mM.

[0077] In some embodiments, the concentration of proline is from about 2% to about 5%. In some embodiments, the concentration of proline is or about 3%.

[0078] In some embodiments, the concentration of the surfactant is from about 0.01 to about 0.025% (w / v), such as or about 0.015% (w / v). In some embodiments, the surfactant is polysorbate 80.

[0079] In some embodiments, the amount of the TACI-Fc fusion protein in the formulation is from about 50 mg to about 100 mg. In some embodiments, the amount of the TACI-Fc fusion protein in the formulation is or about 80 mg.

[0080] In some embodiments, the concentration of the TACI-Fc fusion protein is between about 50 mg / mL and about 200 mg / mL. In some embodiments, the concentration of the TACI-Fc fusion protein is or about 100 mg / mL.

[0081] In some embodiments, the B cell immune response or activity of the subject is reduced. In some embodiments, the number of mature and total circulating B cells in the subject is decreased.

[0082] In some embodiments, the circulating serum immunoglobulins of the subject are reduced.

[0083] In some embodiments, one or more of B cell maturation, differentiation, and / or proliferation are reduced or inhibited.

[0084] In some embodiments, the circulating levels of APRIL or BAFF protein in a subject are reduced. In some embodiments, the APRIL or BAFF protein is an APRIL homotrimer, a BAFF homotrimer, an APRIL / BAFF heterotrimer, or a BAFF 60-mer.

[0085] In some embodiments, the subject is a human.

[0086] In some embodiments, the subject is an adult subject. In some embodiments, the subject is 18 years of age or older, such as 18 - 65 years of age. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Shows a schematic diagram of a functional inhibition assay involving the recombination of APRIL and BAFF via TACI. In this assay, Jurkat cells were transduced with a luciferase-based NF-κB reporter gene and stably expressed murine or human TACI on the cell surface. After activation by recombinant APRIL or BAFF, the endogenous NF-κB transcription factor binds to the DNA response element that controls the transcription of the firefly luciferase gene. Luciferase expression can be monitored, such as by detecting with Bio-Glo TM reagent or measuring using a Cytation 3 reader.

[0088] Figure 2 Shows exemplary human TACI TD Fc fusion molecules for blocking human APRIL (upper panel) and BAFF (lower panel)-mediated signal transduction. The TACI TD Fc fusion was incubated with APRIL or BAFF for 20 min (room temperature, shaking), then added to wells containing 150,000 Jurkat / TACI / NFκB-luciferase cells for 5 hours.

[0089] Figure 3A Shows the function of an exemplary TACI TD Fc fusion molecule for blocking APRIL (upper panel of this figure) or BAFF (lower panel of this figure).

[0090] Figure 3B Shows human TACI TD Fc fusion molecules for blocking murine APRIL (left panel) and BAFF (right panel)-mediated signal transduction.

[0091] Figure 4A Shows human TACI TD Fc fusion molecules for blocking human APRIL (upper panel) and BAFF (lower panel)-mediated signal transduction relative to TACI 13 - 118-Fc, TACI 30 - 110-Fc, and belimumab.

[0092] Figure 4B-4CDescribes the evaluation of APRIL and BAFF inhibitory activities and of the TACI variant 26TACI CRD2-Fc optimized for binding and affinity by various TACI Fc fusion proteins. Figure 4B Shows APRIL and BAFF inhibition by the indicated TACI variants and 26TACI CRD2-Fc evaluated in the TACI / Jurkat / NF-κB reporter assay. Decreased luciferase production indicates increased inhibitory activity. Figure 4C Shows the SPR sensorgrams (shown in black) of 26TACI CRD2-Fc and midostaurin and the results of nonlinear least-squares regression analysis of the data (shown in orange). Midostaurin is from Clinigen.

[0093] Figure 5A -C shows the exemplary human TACI TD Fc fusion molecule 26TACICRD2-Fc for blocking BAFF-( Figure 5A )、APRIL-( Figure 5B ) and BAFF+APRIL combination-mediated ( Figure 5C ) signaling relative to belimumab, BION-1301 and WT TACI-Fc molecules, including WT TACI30-110 (abatacept) and WT TACI 13-118-Fc (midostaurin).

[0094] Figure 5D-5F Describes that 26TACI CRD2-Fc inhibits APRIL and BAFF more potently than comparator molecules. Figure 5D-5F Shows APRIL, BAFF or APRIL plus BAFF inhibition by 26TACI CRD2-Fc and the indicated comparator molecules evaluated in the TACI / Jurkat / NF-κB reporter assay.

[0095] Figure 5G-5J Describes inhibition of BAFF multimers and BAFF / APRIL heterotrimers by 26TACI CRD2-Fc relative to comparator molecules in the TACI / Jurkat / NF-κB assay. Figure 5G Shows BAFF 60-mer, while Figure 5H-5J Shows inhibition of the heterotrimer BAFF / APRIL and homotrimer BAFF by 26TACI CRD2-Fc, TACI 30-110-Fc, midostaurin, belimumab and an anti-APRIL mAb based on the VIS649 mAb sequence. Curve fitting was performed using GraphPad Prism log(agonist) vs. response. Constraints: Hillslope = -1, F = 50.

[0096] Figure 5K Shows the affinity of exemplary human 26TACI CRD2-Fc fusion molecules for human BAFF (left panel) and APRIL (right panel) as determined by surface plasmon resonance (SPR) relative to WT TACI-Fc (Taiticept).

[0097] Figure 6A-6L Shows the parametric analysis evaluated in the NZB / NZW mouse model of human SLE. Evaluation was started from 20 weeks of age for proteinuria score ( Figure 6A ), percentage change in mean body weight ( Figure 6B ), and percentage survival ( Figure 6C ). Anti-double-stranded DNA IgG titers ( Figure 6D ) and blood urea nitrogen (BUN) ( Figure 6E and Figure 6L ) in serum were analyzed (by Student's t-test, **** p < 0.0001 for anti-dsDNA IgG compared to Fc; by Student's t-test, *** p = 0.0008 for BUN compared to Fc). Kidneys were processed and histological analysis was performed on sections by repeated periodic acid Schiff (PAS) staining, where individual components and total histological scores are as shown in Figure 6F . Frozen kidney sections were also stained for immunohistochemical analysis of murine IgG and complement C3 glomerular depositions, as shown in Figure 6G and Figure 6H , respectively. Figure 6I Shows histological scores ± SEM. Figure 6J Shows sialadenitis as measured by histological scores of submandibular glands. Figure 6K Shows the kidney IgG deposition score (mean ± SD) evaluated by IHC from the right kidney at termination ( Figure 6K , left panel) and representative IHC (10X) of kidney IgG depositions from mice treated with Fc control or 26TACI CRD2-Fc ( Figure 6K , left panel).

[0098] Figure 7 Shows the ability of the TACI mutant (K77E / F78Y / Y102D) to inhibit APRIL (left panel) and BAFF (right panel)-mediated signaling, which was quantified by luciferase production in Jurkat / NF-κB / TACI cells.

[0099] Figure 8A and Figure 8B Describe the schematic diagrams of exemplary TACI-Fc fusion proteins. Figure 8A Describes an exemplary TACI-Fc fusion protein containing two cysteine-rich pseudorepeat sequences (CRD). Figure 8BDescribes an exemplary TACI-Fc fusion protein containing a cysteine-rich pseudo-repeat sequence (CRD, such as CRD2).

[0100] Figure 9 Describes 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 the same. The symbol "-" indicates a gap in the alignment. Exemplary, non-limiting positions of amino acid substitutions described herein are in bold text. Based on the alignment of two similar sequences with common identical residues, one of ordinary skill in the art can identify the "corresponding" positions in a sequence by using conserved and identical amino acid residues as a guide and comparing to the reference sequence. Figure 9 Provides an exemplary alignment of the reference TACI extracellular domain sequence shown in SEQ ID NO:122 (containing the full extracellular domain with CRD1 and CRD2 and the starting methionine residue) with the TACI extracellular domain sequence shown in SEQ ID NO:13 (containing only a single CRD, CRD2); alignment of identical residues indicates that, for example, the amino acid residue E7 in SEQ ID NO:13 corresponds to the residue E74 in SEQ ID NO:122, the amino acid residue K10 in SEQ ID NO:13 corresponds to the residue K77 in SEQ ID NO:122, the amino acid residue Y12 in SEQ ID NO:13 corresponds to the Y79 in SEQ ID NO:122, the amino acid residue L15 in SEQ ID NO:13 corresponds to the L82 in SEQ ID NO:122, the amino acid residue R17 in SEQ ID NO:13 corresponds to the R84 in SEQ ID NO:122; and the amino acid residue D16 in SEQ ID NO:13 corresponds to the D85 in SEQ ID NO:122. One of ordinary skill in the art is able to perform similar alignments of two similar protein sequences to identify corresponding residues, including based on the examples and descriptions herein.

[0101] Figure 10A-10D Shows a parametric analysis evaluating the mouse keyhole limpet hemocyanin (KLH) model. Serum-KLH IgM OD levels were evaluated as the primary response ( Figure 10A ) and the secondary response ( Figure 10B ). Similarly, serum anti-KLH IgG1 OD levels were evaluated as the primary response ( Figure 10C ) and the secondary response ( Figure 10D ).

[0102] Figure 11A-11B Shows an analysis of harvested spleens evaluated from the mouse keyhole limpet hemocyanin (KLH) immunization model. The spleens were processed and by weight (Figure 11A ) and total cell number ( Figure 11B ) were analyzed.

[0103] Figure 11C It shows that in KLH-immunized mice, 26TACI CRD2-Fc affects splenocytes more potently than WT TACI-Fc. The total number of splenocytes was counted by flow cytometry.

[0104] Figure 12A It describes the spleen analysis evaluated for a population of cell subtypes. Figure 12A It shows the spleen composition of the murine keyhole limpet hemocyanin (KLH) model and shows the results of the number of B cell subsets relative to the group mean. Figure 12B-12C It describes the analysis evaluating the cellular subtype phenotypic composition of the spleen from the murine keyhole limpet hemocyanin (KLH) model and shows the results of the number of germinal center B cells and plasma cells ( Figure 12B ). Figure 12C It shows spleen plasma cells, plotted for individual mice.

[0105] Figure 12D-1 2J describes that 26TACI CRD2-Fc has a more potent effect on spleen B cell and T cell subsets in KLH-immunized mice than WT TACI-Fc. Figure 12D-1 2J shows the total number of the indicated spleen B cell subsets (i.e., T1 B cells, B cells, T2 B cells, GC cells, FOL B cells, MZ B cells, and plasma cells) counted by flow cytometry on day 20.

[0106] Figure 13A It describes the gating protocol for quantifying B cell subsets and plasma cells in the murine spleen. In the FSC-A / SSC-A dot plot, cells were gated out of debris. This gating was analyzed by FSC-H / FSC-A and then by SSC-H / SSC-W dot plots to gate cells along an established diagonal excluding doublet cell populations. CD45+ / LiveDeadAqua viable negative cells were gated by SSC-H / SSC-W singlet gate to identify live CD45+ cells. Then the live CD45± cell gate was analyzed by B220 / Gr1 dot plot. Then B220+ / Gr1- cells were analyzed by GL7 / CD95 dot plot to identify GL 7+ / CD95+ GC B cells. B220+ / Gr1- cells were also analyzed by CD138 / CD19 dot plot to identify CD19+ cells which were subsequently analyzed by CD23 / CD19 dot plot. CD23+ / CD19+ cells were further analyzed by CD21 / IgM expression to identify CD21+ / Ig M+ follicular (FOL) B cells and CD21br / IgM br T2 B cells. CD23- / CD19+ cells were also analyzed by CD21 / IgM dot plots to identify CD21- / IgM br transitional type 1 (T1) B cells and CD21 br / IgM br MZ B cells. The live CD45+ cell gate was also analyzed by B220 / CD19 dot plots to identify B220 + / lo / CD19+ B cells. B220 lo / CD138+ plasma cells were gated by B220 + / lo / CD19+ gate. FSC-A = forward scatter area; SSC-A = side scatter area; H = height; W = width.

[0107] Figure 13B A gating protocol for quantifying CD4+ T FH cells in mouse spleen is described. In the FSC-A / SSC-A dot plot, cells were gated out of debris. This gating was analyzed by FSC-H / FSC-A and then by SSC-H / SSC-W dot plots to gate cells along an established diagonal excluding doublet cell populations. CD45 + / LiveDead Aqua viable negative cells were gated by SSC-H / SSC-W single threshold to identify live CD45 + cells. Then the live CD45 + cell gate was analyzed by B220 / CD3 dot plots. Then CD3 + T cells were analyzed by CD4 / CD8 dot plots. CD4 + T cells were analyzed by PD1 / CXCR5 dot plots to identify PD1+CXCR5+T FH cells. T FH = T follicular helper; FSC-A = forward scatter area; SSC-A = side scatter area; H = height; W = width.

[0108] Figure 14A -D describes the number of T cells in the mouse keyhole limpet hemocyanin (KLH) model. Splenic CD3+, CD8+, CD4+ and follicular helper T cells are depicted in Figure 14A 、 Figure 14B 、 Figure 14C and Figure 14D respectively.

[0109] Figure 14E-14FSpleen T cells were also counted by flow cytometry. Plots were made for individual mice, and the mean ± SD are shown as horizontal lines and error bars, respectively. GC = germinal center; T1 = transitional-1 B cells; T2 = transitional-2 B cells; FOL = follicle; MZ = marginal zone.

[0110] Figure 14G Total T cells counted by flow cytometry in the spleens of mice challenged with KLH or naive mice on day 20 are shown, and plots were made for individual mice. FH cells were plotted for individual mice.

[0111] Figure 15 Tcm and Tem cell populations in the murine keyhole limpet hemocyanin (KLH) model are described.

[0112] Figure 16A-16B and Figure 17A-17B describes the overall incidence and severity of sialadenitis ( Figure 16A-16B ) and insulitis ( Figure 17A-17B ) in diabetic-prone mice after treatment with a test molecule.

[0113] Figure 18 and Figure 19 describes serum immunoglobulin (IgM, IgA, and IgG) concentrations of an exemplary test molecule in a pharmacokinetics / pharmacodynamics study after a single intravenous infusion in male Sprague Dawley rats.

[0114] Figure 20A and Figure 20B describes the individual animal serum concentration versus time curves of an exemplary test molecule administered to cynomolgus monkeys in a PK / PD model. Figure 20B The results for atacicept shown in

[0115] Figure 21A-21B are based on published data (Carbonatto et al. (2008) Toxicol Sci 105:200 - 210). Figure 21A IgM, IgA, and IgG PK / PD in the cynomolgus monkey PK / PD model are shown. Figure 21B Levels of serum IgM, IgA, and IgG (mean + range) measured by ELISA at each time point in each treatment group are shown and plotted as a percentage of the baseline serum concentration obtained from serum collected on day - 8.

[0116] Figure 22 Absolute cell counts of animals receiving an exemplary test molecule in the cynomolgus monkey PK / PD model are described.

[0117] Figure 23 Describes the percent cells from baseline in animals receiving an exemplary test molecule in a cynomolgus monkey PK / PD model.

[0118] Figure 24 Describes the absolute count or relative percentage of proliferating T cells in animals receiving an exemplary test molecule in a cynomolgus monkey PK / PD model.

[0119] Figure 25A-25B Describes predicted human PK curves after repeated intravenous dosing every four weeks ( Figure 25A ) or every two weeks ( Figure 25B ) in a two-compartment PK model.

[0120] Figure 26A-26E Describes the inhibition of class-switched memory B cells ( Figure 26A ), plasma cells ( Figure 26B ), and immunoglobulin secretion ( Figure 26C-26E ).

[0121] Figure 26F and 26G Shows CD19+ B cells activated with rhCD40L and recultured with exogenous APRIL, BAFF, and 26TACICRD2-Fc or the indicated control drug molecule. Cells are stained and analyzed by flow cytometry to identify class-switched memory B cells (IgD, IgM, CD27+) or plasma cells (IgM, IgD, CD38+, CD319+). After 7 days, supernatants are collected and IgM ( Figure 26H ), IgA ( Figure 26I ), IgG1 ( Figure 26J ), IgG2 ( Figure 26K ), IgG3 ( Figure 26L ), and IgG4 ( Figure 26M ) secretion is quantified by multiplex analysis. The Kruskal-Wallis test and uncorrected Dunn's test are used to determine statistical significant differences between group medians; p values < 0.05 are considered statistically significant.

[0122] Figure 27A-27C Describes the plasma cell levels in the bone marrow ( Figure 27A ), spleen ( Figure 27B ), and lymph nodes ( Figure 27C ) in a CIA mouse model receiving a test molecule.

[0123] Figure 28 Describes the number of plasma cells in bone marrow smears from cynomolgus monkeys receiving an exemplary TACI-Fc fusion protein.

[0124] Figure 29A-29BDescribes the dose-dependent serum concentration versus time curves ([ Figure 29A ]) and percent cells from baseline ([ Figure 29B ]) in animals receiving an exemplary TACI-Fc fusion protein in a 1-month GLP toxicology study in cynomolgus monkeys. Figure 29A ) and percent cells from baseline ([ Figure 29B ]) Figure 29B ).

[0125] Figure 30A-30B Describes serum IgA, IgG, IgM, and IgE levels in animals receiving an exemplary TACI-Fc fusion in a 1-month GLP toxicology study ([ Figure 30A ]) and a 6-month GLP toxicology study ([ Figure 30B ]). Figure 30A ) and a 6-month GLP toxicology study ([ Figure 30B ]) Figure 30B ).

[0126] Figure 31 Is an analysis of harvested spleens evaluated from a murine chronic graft-versus-host disease (cGVHD) model. The spleens were processed and analyzed by weight and total cell count.

[0127] Figure 32 Describes an analysis of the spleen cell population composition from a murine chronic graft-versus-host disease model and shows the results of CD45 + cell and B220 + B cell numbers.

[0128] Figure 33 Describes an analysis of the spleen cell subtype population composition and shows the results of CD4 + and CD8 + T cell subset numbers.

[0129] Figure 34 Describes the CD4 + T cell subset numbers in a cGVHD model.

[0130] Figure 35A Describes the B220 + B cells and CD1d hi CD5 + B-1 cell numbers in a cGVHD model. Figure 35B Describes the transitional-1 (T1) and transitional-2 (T2) B cell numbers in a cGVHD model.

[0131] Figure 36A Describes follicular and marginal zone (MZ) B cells, while Figure 36B Describes germinal center (GC) B cells and plasma cell numbers in a cGVHD model.

[0132] Figure 37 Describes early plasma cells, plasmablasts, and long-lived plasma cell (LL-PC) numbers in a cGVHD model.

[0133] Figure 38A Describes renal IgM immune complex deposits in the kidney measured by immunohistochemical staining with a specific fluorescently labeled antibody against mouse IgG.

[0134] Figure 38B Shows representative IHC (20X) of renal IgG deposits from Fc control, TACI CRD2-Fc (DAPI overlap in the lower right corner), or naïve mice.

[0135] Figure 39 Shows the analysis of anti-dsDNA autoantibody serum titers at week 8 and week 13.

[0136] Figure 40A-40B Shows at 56 days, i.e., week 8 ( bm12 ) and week 8 ( Figure 40A ) in a mouse model of autoantibody-associated glomerulonephritis, the analysis of anti-dsDNA autoantibody serum titers. Figure 40B ) of anti-dsDNA autoantibody serum titers.

[0137] Figure 41 Describes renal IgM immune complex deposits in the kidney measured by immunohistochemical staining with a specific fluorescently labeled antibody against mouse IgG.

[0138] Figure 42 Describes the levels of serum IgA, IgM, and IgG (IgG1, IgG2b, and IgG3) in animals receiving an exemplary TACI-Fc fusion in a mouse model of autoantibody-associated glomerulonephritis.

[0139] Figure 43A-43B Describes the levels of anti-SRBC IgG1 ( Figure 43A ) and plasma cells ( Figure 43B ) in animals receiving 26TACI CRD2-Fc compared to BAFF- and APRIL-specific biologics.

[0140] Figure 43C-43L Describes that 26TACI CRD2-Fc exhibits stronger immunosuppressive activity than atacicept and BAFF or APRIL inhibitors in a mouse SRBC immunization model. Figure 43C-43F Shows the anti-SRBC Ig concentration in serum measured on day 15. Germinal center (GC) B cells / spleen ( Figure 43G ), CD4+T FH cells / spleen ( Figure 43H ), plasma cells (PC) / spleen ( Figure 43I ), and plasmablasts (PB) / spleen ( Figure 43J) and plotted as values per individual mouse. The percentages of long-lived plasma cells (LL-PC) ( Figure 43K ) and total plasma cells (PC) ( Figure 43L ) in the bone marrow were also determined by flow cytometry and plotted as data per individual mouse. Data are represented as median ± interquartile range ( Figure 43G-43H ) or mean ± SD ( Figure 43C-43F , 43I-43L).

[0141] Figure 44A-44D Describes the individual serum concentrations of the 26TACI CRD2-Fc fusion molecule over time in a human cohort administered 26TACI CRD2-Fc via the IV or SC route. Figure 44A-44B Describes the 26TACI CRD2-Fc serum concentrations over 56 days. Figure 44C-44D Describes the 26TACI CRD2-Fc serum concentrations over 112 days.

[0142] Figure 45A-45B Describes the serum IgA, IgG, IgM levels and their corresponding changes from baseline in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 45A ) or SC route ( Figure 45B ).

[0143] Figure 45C-45F Describes the serum galactose-deficient IgA1 (Gd-IgA1) levels and their corresponding changes from baseline in a human cohort administered 26TACI CRD2-Fc via the IV or SC route. Figure 45C-45D Describes the serum Gd-IgA1 levels over 28 days. Figure 45E-45F Describes the serum Gd-IgA1 levels over 112 days.

[0144] Figure 46 Describes the serum IgA, IgG or IgM levels and their corresponding changes from baseline in a human cohort administered 80 mg 26TACI CRD2-Fc SC compared to the levels of the control drugs atacicept (first column on the left), taci-cept (second column), BION 1301 (third column) or sibelizumab (sibelizumab) (fourth column), as determined from publicly available data.

[0145] Figure 47A-47B Describes the dose-dependent, targeted reduction in the frequency of circulating CD19+CD38+CD27+IgD antibody-secreting cells (including plasmablasts and plasma cells) in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 47A ) or SC route ( Figure 47B ).

[0146] Figure 47C-47D Describes the frequency of circulating CD27-IgD+ antibody-secreting cells (including naive B cells) in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 47C ) or the SC route ( Figure 47D ).

[0147] Figure 47E-47F Describes the frequency of circulating CD27+IgD- antibody-secreting cells (including memory B cells) in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 47E ) or the SC route ( Figure 47F ).

[0148] Figure 48A-48D Shows the dose-dependent reduction and its duration (pg / mL or % change from baseline) in free APRIL in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 48A , Figure 48C ) or the SC route ( Figure 48B , Figure 48D ), as observed on day 28 or day 56 after dosing.

[0149] Figure 48E-48F Shows the dose-dependent reduction and its duration (% change from baseline) in free BAFF in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 48E ) or the SC route ( Figure 48F ), as observed on day 28 after dosing.

[0150] Figure 48G-48J Shows the dose-dependent reduction and its duration in free APRIL (pg / mL) or free BAFF (pg / mL) in a human cohort administered 26TACI CRD2-Fc via the IV route ( Figure 48G , Figure 48I ) or the SC route ( Figure 48H , Figure 48J ), as observed on day 112 after dosing.

[0151] Figure 49A-49G Describes that 26TACI CRD2-Fc exhibits enhanced immunosuppressive activity compared to atacicept and WTTACI CRD2-Fc in a murine SRBC immunization model. Figure 49A-49D Shows the anti-SRBC Ig serum concentration measured on day 15. Figure 49E Shows the percentage of plasma cells in the bone marrow counted by flow cytometry and plotted for individual mice. Figure 49E-49GShows the total number of germinal center (GC) B cells / spleen or CD4+ TFH cells / spleen counted by flow cytometry and plotted per individual mouse. Data are represented as median ± interquartile range or mean ± SD.

[0152] Figure 50A-50G Describes that 26TACI CRD2-Fc exhibits enhanced immunosuppressive activity compared to tacirolimus and anti-CD20 antibody in a murine SRBC immunization model. Figure 50A-50D Shows the anti-SRBC Ig serum levels measured on day 15. Figure 50E Shows the percentage of plasma cells in the bone marrow counted by flow cytometry and plotted per individual mouse. Bone marrow was not collected from the naïve group. Figure 50F-50G Shows the total number of germinal center (GC) B cells / spleen or CD4+ T FH cells / spleen counted by flow cytometry and plotted per individual mouse. Data are represented as mean ± SD.

[0153] Figure 51A-51C Describes the results after administration of 26TACI CRD2-Fc to subjects with IgA nephropathy (IgAN) or primary membranous nephropathy (pMN). Figure 51A Shows the urinary protein:creatinine ratio and anti-SRBC Ig serum concentration measured on days 3, 8, and 15 after TACI CRD2-Fc administration. Figure 51B Shows the urinary protein:creatinine ratio and anti-SRBC Ig serum concentration measured on days 3, 8, 15, 22, 29, 43, and 57 after TACI CRD2-Fc administration. Figure 51C Shows the urinary protein:creatinine ratio measured 12 weeks after TACI CRD2-Fc administration in patients with IgAN and pMN. Figure 51D Shows serum IgA, IgG, and IgM in patients with IgAN.

[0154] Figure 51E-51J Describes the results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). Figure 51E Describes the percentage change of UPCR from baseline (mean ± SD) in a single (spot) UPCR test. Figure 51F Describes the percentage change of UPCR from baseline (mean ± SD) in a 24-hour UPCR test. Figure 51G Describes the percentage change from baseline over time (mean ± SD) of IgA, IgG, and IgM (mg / dL) in IgAN patients receiving 80 mg Q4W or 240 mg Q4W. Figure 51H Describes the percentage change of Gd-IgA1 from baseline over time (mean ± SD). Figure 51IDescribes the change in eGFR from baseline (mean ± SD). Figure 51J Describes the percentage change in eGFR from baseline (mean ± SD).

[0155] Figure 51K-51L Describes the results after administration of 26TACI CRD2-Fc to subjects with primary membranous nephropathy (pMN). Figure 51K Describes the percentage change from baseline in the 24-hour UPCR test (mean ± SD). Figure 51L Describes the change from baseline in the circulating levels of the disease-specific biomarker anti-phospholipase A2 receptor (RU / mL) (mean ± SD).

[0156] Figure 52A-52G Describes that 26TACI CRD2-Fc provides benefits in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). Figure 52A Shows an RBC-restricted triple fusion protein that can be bound by a T cell receptor to initiate the pathogenesis of AIHA. Figure 52B Shows HOD autoantibodies in HOD mice: no CTLA-4, IL-10R, LAG-3, or PD-1 antibodies were administered (first 4 Aby); 4 days before administration of 26TACI CRD2-Fc, Fc control, or PBS (day -4); and on days 9, 15, 23, and 28 after administration of TACI CRD2-Fc, Fc control, or PBS. Figure 52C Shows the change in HOD autoantibodies on day 28 in HOD mice receiving 26TACI CRD2-Fc, Fc control, or PBS. Figure 52D Shows the number of plasma cells per spleen in HOD mice receiving 26TACICRD2-Fc, Fc control, or PBS. Figure 52E Shows the number of plasma cells per spleen and bone marrow in HOD mice receiving 26TACICRD2-Fc, Fc control, anti-CD20, or PBS. Figure 52F Shows that before 4 Aby of receiving 26TACI CRD2-Fc, Fc control, or PBS, as Figure 52B shown, the hematocrit levels in HOD mice on day 3 before administration of 26TACI CRD2-Fc, Fc control, or PBS (day -3) and on days 15 and 28 after administration of 26TACI CRD2-Fc, Fc control, or PBS. Figure 52GShows autoantibodies (antiglobulins) bound to red blood cells (RBCs) in HOD mice treated on day 4 before administration of 26TACI CRD2-Fc, Fc control, or PBS, and on days 9, 15, 23, and 28 after administration of 26TACI CRD2-Fc, Fc control, or PBS.

[0157] Figure 53A-53F Describes the benefits provided by 26TACI CRD2-Fc in an experimental autoimmune myasthenia gravis (EAMG) mouse model. Figure 53A Describes the timeline of the EAMG mouse model. Figure 53B Describes the mean EAMG clinical score over time. Figure 53C Describes the serum anti-AChR IgG concentration on day 91 (end of study). Figure 53D and Figure 53E Describes the serum Ig isotype concentration on day 91 (end of study). Figure 53F Describes the muscle AChR content on day 91 (end of study).

[0158] Figure 54A-54E Describes the diagnostic plot for population pharmacokinetic analysis. Figure 54A Describes the PK model structure, where Vc refers to the blood compartment and Vp refers to the tissue compartment. Figure 54B Describes the individual predicted values (μg / mL) compared to the observed concentrations (μg / mL). Figure 54C Describes the population predicted values (μg / mL) compared to the observed concentrations (μg / mL). Figure 54D Describes time (days) compared to the conditional weighted residuals. Figure 54E Describes the population predictions (μg / mL) compared to the conditional weighted residuals.

[0159] Figure 55 Describes the concentration of 26TACI CRD2-Fc within 8 weeks after intravenous (IV) administration at doses of 2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, or 960 mg or subcutaneous (SC) administration at doses of 80 mg, 240 mg, 480 mg, or 960 mg.

[0160] Figure 56 The described simulations show the concentration of 26TACI CRD2-Fc within 40 weeks after subcutaneous (SC) administration at doses of 24 mg, 80 mg, and 240 mg. Dosing is repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W) for a duration of 24 weeks of dosing and 16 weeks after dosing.

[0161] Figures 57A - 57FDescribes the PK and PD modeling of circulating APRIL and immunoglobulins (IgA, IgG, IgM). Figure 57A Describes the PK / PD model structure of circulating free APRIL. Figure 57B Describes the PK / PD model structures of circulating IgA, IgM, and IgG. Figure 57C Describes free APRIL (pg / mL) within 12 weeks in response to IV (2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, and 960 mg) or SC (80 mg, 240 mg, 480 mg, and 960 mg) administration of 26TACI CRD2-Fc. Figure 57D Describes IgA (g / L) within 17 weeks in response to IV (2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, and 960 mg) or SC (80 mg, 240 mg, 480 mg, and 960 mg) administration of 26TACI CRD2-Fc. Figure 57E Describes IgG (g / L) within 17 weeks in response to IV (2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, and 960 mg) or SC (80 mg, 240 mg, 480 mg, and 960 mg) administration of 26TACI CRD2-Fc. Figure 57F Describes IgM (g / L) within 17 weeks in response to IV (2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, and 960 mg) or SC (80 mg, 240 mg, 480 mg, and 960 mg) administration of 26TACI CRD2-Fc.

[0162] Figures 58A - 58G Describes the PK / PD simulation of 26TACI CRD2-Fc. Figure 58A Describes the percent change from baseline of free APRIL and APRIL within 40 weeks after subcutaneous (SC) administration (80 mg or 240 mg) repeated every 4 weeks (Q4W). Figure 58B Describes the percent change from baseline of APRIL within 40 weeks after 24 weeks of subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). Figure 58C Describes the percent change from baseline of IgA within 40 weeks after 24 weeks of subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). Figure 58DDescribes the percent change from baseline in IgG over 40 weeks after 24 weeks of subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). Figure 58E Describes the percent change from baseline in IgM over 40 weeks after 24 weeks of subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). Figure 58F Describes the percent change from baseline in free APRIL over 40 weeks after 24 weeks of subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W); and describes the percent change from baseline in IgA, IgG, and IgM over 72 weeks after 24 weeks of subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). Figure 58G Describes the percent change from baseline in free IgG and IgG over 72 weeks after subcutaneous (SC) administration (80 mg or 240 mg) repeated every 4 weeks (Q4W).

[0163] Figures 59A - 59D Describes PK and PD modeling of circulating BAFF and Gd-IgA1. Figure 59A Describes the PK / PD model structure of circulating free BAFF. Figure 59B Describes the PK / PD model structure of circulating Gd-IgA1. Figure 59C Describes free BAFF (pg / mL) within 16 weeks in response to IV (2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, and 960 mg) or SC (80 mg, 240 mg, 480 mg, and 960 mg) administration of 26TACI CRD2-Fc. BLQ = below limit of quantification. Figure 59D Describes Gd-IgA1 (pg / mL) within 16 weeks in response to IV (2.4 mg, 8 mg, 24 mg, 80 mg, 240 mg, 480 mg, and 960 mg) or SC (80 mg, 240 mg, 480 mg, and 960 mg) administration of 26TACI CRD2-Fc.

[0164] Figures 60A - 60B Describes the PK / PD simulation of 26TACI CRD2-Fc. Figure 60A Describes the percent change from baseline in free APRIL and APRIL over 40 weeks after subcutaneous (SC) administration (80 mg or 240 mg) repeated every 4 weeks (Q4W). Figure 60BDescribes the percent change from baseline of free Gd-IgA1 and Gd-IgA1 over 72 weeks following subcutaneous (SC) administration (80 mg or 240 mg) repeated every 4 weeks (Q4W).

[0165] Figures 61A - 61B Describes a summary of the target coverage in the simulation. Figure 61A Shows patients with >95% APRIL coverage. Figure 61B Shows the percentage of patients with IgG below 1.5 g / L.

[0166] Figures 62A - 62B Describes a summary of the target coverage in the APRIL / BAFF and IgG / Gd-IgA1 simulations. Figure 62A Shows patients with 95% APRIL / BAFF coverage. Figure 62B Predicts that the reduction in Gd-IgA1 in patients will be greater than 50%.

[0167] Figures 63A - 63E Shows the proteinuria and urinary creatinine scores over time and at day 48 in the IFNα-accelerated NZB / W lupus model. Figure 63A Shows the mean proteinuria score (+SD) over time for each treatment group, and the last observation carried forward (LOCF) for any mice terminated before day 50. Figure 63B Shows the mean creatinine score (+SD) over time, and the LOCF for any mice terminated before day 50. Figure 63C Shows the proteinuria scores for the treatment groups at day 48 (last measurement before termination). Figure 63D Shows the creatinine scores for the treatment groups at day 48. Figure 63E Shows the ratio of proteinuria to urinary creatinine scores at day 48. For Figures 63C - 63E , data are presented as median ± IQR. Study details and methods are provided in Example 32. Statistical differences between groups were determined by the Kruskal-Wallis test and multiple comparisons were made using the uncorrected Dunn test, as described in Example 32; only significant differences (p<0.05) are listed.

[0168] Figures 64A - 64F Shows the histological analysis of the kidneys, spleen, submandibular gland, and lacrimal gland at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Figure 64A Shows the total glomerular lesion score in the kidneys. Figure 64B Shows the total tubulointerstitial lesion score in the kidneys. Figure 64C Shows the mean follicle diameter in the spleen. Figure 64D Shows the spleen follicle score. Figure 64E Shows the number of inflammatory foci in the submandibular gland.Figure 64F Shows the number of inflammatory foci in the lacrimal gland. Data are presented as median ± IQR. Study details and methods are provided in Example 32. Statistical differences between groups were determined by the Kruskal-Wallis test and multiple comparisons were made using the uncorrected Dunn's test as described in Example 32; only significant differences p < 0.05 are listed.

[0169] Figures 65A - 65E Shows hemoglobin (HGB), hematocrit (HCT), RBC, and direct antiglobulin test (DAT) levels in whole blood at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Figure 65A Shows hemoglobin concentration. Figure 65B Shows hematocrit (%). Figure 65C Shows RBC concentration. Figure 65D Shows pan-Ig DAT level (MFI). As Figure 65E Shows IgA DAT level (MFI). MFI: Mean fluorescence intensity recorded using flow cytometry. Data are presented as median ± IQR. Study details and methods are provided in Example 32. Statistical differences between groups were determined by the Kruskal-Wallis test and multiple comparisons were made using the uncorrected Dunn's test as described in Example 32; only significant differences p < 0.05 are listed.

[0170] Figures 66A - 66B Shows blood urea nitrogen (BUN) and anti-double-stranded (ds)DNA IgM levels in serum at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Figure 66A Shows BUN concentration. Figure 66B Shows anti-dsDNA IgM level. Data are presented as median ± IQR. Study details and methods are provided in Example 32. Statistical differences between groups were determined by the Kruskal-Wallis test and multiple comparisons were made using the uncorrected Dunn's test as described in Example 32; only significant differences p < 0.05 are listed.

[0171] Figures 67A - 67C Shows H&E score, serum Ig, and B cell frequency in the experimental model of epidermolysis bullosa acquisita (EBA). Figure 67A Shows that mice in the EBA model treated with 26TACI CRD2-Fc starting when the disease covered at least 2% of the surface area had significantly lower dermal H&E scores compared to Fc control-treated mice at termination. P values are shown by the Mann-Whitney test. Figure 67BShows serum immunoglobulins, including total and collagen VII-specific isotypes. P values are shown by Mann-Whitney test. Figure 67C Shows the frequency of COL7 vWFA2 -(antigen)-specific B cells in the lymph nodes of mice treated with 26TACI CRD2-Fc. Statistical significance differences between the Fc control and 26TACI CRD2-Fc treatment groups were determined by Mann-Whitney test. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

[0172] Figures 68A - 68B Shows the serum levels of anti-GluN1 peptide IgG antibodies at week 4 (before treatment initiation) and at termination (week 10). P values show the statistical differences between treatment groups determined using Mann-Whitney test at each serum dilution.

[0173] Detailed description

[0174] The present disclosure provides immunomodulatory proteins that bind to one or more ligands, such as produced as soluble factors to inhibit or reduce B cell responses or activities. The provided immunomodulatory proteins include proteins that bind to BAFF or APRIL ligands to neutralize their activities and block or antagonize the activities of B cell-stimulating receptors such as TACI or BCMA. The provided immunomodulatory proteins can be fusion proteins of the extracellular domain of TACI or a binding portion thereof (TACI ECD hereinafter) with a multimerization domain such as immunoglobulin Fc. For example, the present disclosure provides TACI-Fc fusion proteins. In some embodiments, the immunomodulatory proteins provided herein can be used to treat diseases, disorders, or conditions associated with dysregulated immune responses, such as diseases, disorders, or conditions associated with inflammatory or autoimmune symptoms, including inflammatory diseases or autoimmune diseases.

[0175] The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and protect tissues from excessive damage during immune responses, such as during resistance to pathogen infections. However, in some cases, the immune system may be dysregulated, resulting in abnormal immune responses against normal body parts or tissues, leading to autoimmune diseases or disorders or autoimmune symptoms. In other cases, unwanted immune responses may occur against foreign tissues such as grafts, resulting in transplant rejection.

[0176] For a long time, B cells have been associated with autoimmune diseases such as systemic lupus erythematosus (SLE) because B cells are capable of presenting antigens to autoreactive T cells, secreting inflammatory cytokines (Lund, Curr Opin Immunol 2008, 20(3):332-338), and differentiating into antibody-secreting cells (ASC), namely plasmablasts and plasma cells (PC) responsible for the production of pathogenic autoantibodies (Banchereau et al., Cell, 2016, 165(3):551-565). Therefore, depletion or inhibition of B cells and ASC is an effective approach for treating many rheumatic and other autoimmune diseases.

[0177] Similarly, since B cells are key mediators of the systemic immune response, B cells also play a role in kidney, hematological, dermatological, and neurological autoimmune diseases.

[0178] In the immunopathogenesis of a form of kidney disease, namely IgA nephropathy (IgAN), B cells produce a small amount of antibodies (e.g., Gd-IgA1), while plasma cells produce a large amount of autoantibodies (e.g., anti-Gd-IgA1). This leads to the formation of antibody: autoantibody complexes, which deposit and accumulate in mesangial cells, thereby activating the alternative and lectin pathways of the complement system, resulting in chronic inflammation, loss of renal function, hematuria, proteinuria, and reduced glomerular filtration rate (Maixnerova et al., (2022) J Clin Med, 11(10):2810). Therefore, targeting BAFF and APRIL has become a promising approach to reducing the levels of pathogenic autoantibodies (e.g., Gd-IgA1).

[0179] In autoimmune cytopenias, targeting BAFF and APRIL can reduce pathogenic autoantibodies that cause platelet destruction in immune thrombocytopenia (ITP) and red blood cell destruction in warm autoimmune hemolytic anemia (wAIHA) and cold autoimmune hemolytic anemia (cAIHA or CAD).

[0180] It is known that B cells play a substantial role in the pathogenesis of autoimmune diseases with cutaneous manifestations. These autoimmune diseases include autoimmune bullous diseases, lupus erythematosus, dermatomyositis, systemic sclerosis, psoriasis, pemphigus, and pemphigoid, the latter two being particularly antibody-driven (Fetter et al., Cells (2020) 9(12):2627). Traditionally, the skin has been considered devoid of B cells. However, recent data indicate that B cells are localized in the skin of humans and other mammalian species (Debes and McGettigan, J Immunol (2019) 202(6):1659-1666). Once localized to the skin, autoreactive skin-associated B cells can locally promote autoantibody production, cytokine expression, and crosstalk with autoreactive T cells (Fetter et al., Cells (2020) 9(12):2627).

[0181] Autoimmune bullous diseases (ABD) are characterized by autoantibodies targeting structural skin proteins. Treatment is limited: rituximab is the only biologic approved for pemphigus vulgaris (Uzawa et al. (2021) Clin Exp Immunol 203:366; Ma et al. (2023) Front Immunol 13:1064007), but may be associated with frequent relapses and is often accompanied by elevated cytokine BAFF3. BAFF and its related cytokine APRIL play key roles in a broader spectrum of B cell activation and are elevated in ABD, correlating with disease activity. BAFF / APRIL inhibition may lead to more sustained reduction of autoantibodies, thus improving clinical outcomes.

[0182] For autoimmune-mediated neurological diseases, there is a rapidly expanding group of central nervous system (CNS) diseases caused by pathogenic autoantibodies that are clinically distinct. Some of these autoantibodies target glial surface proteins. Autoantigen-specific B cells have consistently been identified in the circulation of patients with diseases mediated by glial surface autoantibodies (NSAb) (Sun et al., Nat Rev Neurol, (2020) 16(9):481-492). The efficacy of certain anti-B cell therapies has been described in detail for the treatment of patients with multiple sclerosis, neuromyelitis optica spectrum disorder, autoimmune encephalitis and excitatory CNS diseases, autoimmune neuropathy, myasthenia gravis, and inflammatory myopathies (Stathopoulos and Dalakas, Neurotherapeutics, (2022) 19(3):691-710). More specifically, myasthenia gravis (MG) is a classic B cell-mediated autoimmune disease because the presence of autoantibodies specifically targeting components of the acetylcholine receptor (AChR) impairs neuromuscular transmission in the postsynaptic membrane (Yi et al., Muscle Nerve (2018) 57(2):172-184).

[0183] In addition, BAFF and APRIL play key roles in B cell biology. It has been reported that one or both cytokines are upregulated and are associated with clinical parameters in MG, autoimmune encephalitis, NMOSD, MS, and other autoantibody-related neurological diseases (Uzawa et al. (2021) Clin Exp Immunol 203:366; Ma et al. (2023) Front Immunol 13:1064007; Ashida et al. (2022) Front Neurol 13:1012857). Therapeutic agents targeting the B cell pathway, including BAFF and APRIL, have shown promising clinical potential in the treatment of myasthenia gravis (MG) and other autoantibody-related neurological diseases; however, safer and more effective therapies are still needed. Targeting BAFF and APRIL can reduce the levels of pathogenic autoantibodies (such as anti-NMDAR, anti-AChR, anti-MOG) and autoantibodies against proteins at the neuromuscular junction or other sites of neuron-neuron or neuron-tissue interaction.

[0184] Therefore, depleting or inhibiting B cells and ASCs is an effective approach for treating many kidney, blood, skin, and nervous system autoimmune diseases.

[0185] Key regulators of B cell development, differentiation, and survival include tumor necrosis factor (TNF) family cytokines, B cell-activating factor (BAFF / TNFSF13B), and a proliferation-inducing ligand (APRIL / TNFSF13), which are mainly expressed by myeloid cells and signal through multiple receptors. BAFF binds with different affinities to BAFF-R (TNFRSF13C), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI; TNFRSF13B), and B cell maturation antigen (BCMA; TNFRSF17) expressed on B cells, while APRIL binds to TACI and BCMA (Samy, et al., Int Rev Immunol, 2017, 36(1):3-19), heparan sulfate proteoglycan (HSPG) (Ingold, et al., J Exp Med, 2005, 201(9):1375-1383), and polysaccharides such as CD138 (Moreaux et al., Eur J Haematol, 2009, 83(2):119-129; Ingold, et al., J Exp Med, 2005, 201(9):1375-1383). BAFF can exist in three functional forms: membrane-bound, soluble trimer, and soluble BAFF 60-mer (Eslami and Schneider, Curr Opin Immunol, 2021, 71:75-80), with the soluble trimer formed via proteolytic cleavage of membrane BAFF (Samy et al., Int Rev Immunol, 2017, 36(1):3-19). APRIL and BAFF can also form functionally active heterotrimers; all forms of these cytokines have been shown to be elevated in various antibody-related diseases, including SLE (Roschke et al., J Immunol, 2002, 169(8):4314-4321; Dillon et al., Arthritis Res Ther, 2010, 12(2):R48).

[0186] Thus, BAFF and APRIL are TNF superfamily members that can bind to the TACI and BCMA receptors on B cells; BAFF can also bind to a third receptor, the BAFF receptor (BAFF-R). Both BAFF and APRIL can bind to and activate BCMA and TACI; BAFF can also bind to and activate BAFF-R (Xu et al., 2020 Cancers (Basel) 12(4):1045). BAFF and APRIL together support B cell development, differentiation, and survival, particularly plasmablasts and plasma cells, and play a role in the pathogenesis of B cell-related autoimmune diseases. BAFF and APRIL were initially identified as transmembrane proteins, mainly expressed in stromal cells and myeloid-derived cells (Smulski et al. Front. Immunol. 2018 9:2285) and can be cleaved to release soluble cytokines. BAFF circulates as homotrimers, hexamers, or heterotrimers containing two APRIL and one BAFF or two BAFF and one APRIL protomers. APRIL circulates as homotrimers or heterotrimers and can be localized to the extracellular matrix or cell surface by interacting with heparan sulfate proteoglycans.

[0187] Despite structural similarities and involvement in common signaling pathways, APRIL and BAFF play non-redundant roles in B cell regulation, in part due to differential receptor expression during partially overlapping stages of B cell development. When BAFF-R is expressed, BAFF plays a key role in early B cell development, while APRIL plays a key role in the function of differentiated ASCs that express TACI, BCMA, and HSPG (e.g., syndecan-1 / CD138).

[0188] The expression of BAFF and APRIL is increased under pro-inflammatory conditions (Smulski et al. 2018), and 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). Binding of BAFF / APRIL to their receptors triggers events in B cell and plasma cell development, differentiation, and activation. For example, activation of BAFF-R contributes to the survival and maturation of transitional and naive B cells, while TACI is involved in T cell-independent B cell responses to certain antigens, B cell regulation, and immunoglobulin (Ig) class switch recombination. BCMA, which is upregulated in activated B cells, is essential for the long-term survival of plasma cells.

[0189] In some aspects, immunotherapies that alter immune cell activity, such as B cell activity, can treat certain diseases, disorders, and conditions in which the immune response is dysregulated. Specifically, inhibition or attenuation of the immune response, such as the B cell response, may help reduce or prevent unwanted inflammation, autoimmune symptoms, and / or transplant rejection. However, therapeutic approaches that seek to modulate the interaction between ligands and their receptors that mediate the immune response are not entirely satisfactory. In some cases, therapies that intervene and alter the immunomodulatory effects of immune cell (e.g., B cell) activation are limited by spatial orientation requirements and size constraints imposed by the extent of the immune synapse. In some aspects, existing therapeutic agents, including antibody drugs, may not be able to interact with multiple target proteins involved in regulating these interactions simultaneously. For example, soluble receptors and antibodies typically bind competitively (e.g., binding to no more than one target species at a time), thus lacking the ability to bind multiple targets simultaneously. In addition, pharmacokinetic differences between drugs that target only one of these receptors may make it difficult to appropriately maintain the blood concentrations required for a drug combination targeting two different targets throughout the course of treatment.

[0190] The use of BAFF and / or APRIL inhibitors for the treatment of various autoimmune diseases or other B cell-related diseases has been investigated in clinical trials. The BAFF inhibitor belimumab (Benlysta (registered trademark)) has been approved for the treatment of SLE (Benlysta product information, 2020), and APRIL single-pathway inhibitors (e.g., BION1301 and VIS649) are currently being evaluated in phase 2 studies [NCT04684745; NCT04287985].

[0191] Among several B cell-targeting strategies, blockade of BAFF or APRIL has shown clinical promise. Belimumab, an anti-BAFF antibody, is approved for the treatment of SLE (Hahn, N Engl J Med, 2013, 368(16):1528-1535) and SLE-related lupus nephritis (LN) (Asif et al., Curr Opin Nephrol Hypertens, 2022), but clinical remission measured by low lupus disease activity state (LLDAS) or complete renal response (CRR) occurs in only a minority of patients (12 - 4% or 30%, respectively) (Oon et al., Ann Rheum Dis, 2019, 78(5):629-633; Furie et al., N Engl Med, 2020, 383(12):1117-1128). Therefore, more active agents are still needed. Other antibodies targeting BAFF / APRIL include ananalumab, a blocking and cell-depleting anti-BAFF-R antibody (McWilliams et al., Blood Adv, 2019, 3(3):447-460) and the anti-APRIL antibodies BION-1301 (Dulos, America Society of Hematology, 2016) and sibelimumab (VIS649) (Myette et al., Kidney Int, 2019, 96(1):104-116). These antibodies have shown promising pharmacodynamic activity in phase I clinical trials (Barratt, American Society of Nephrology, 2021; Mathur et al., Kidney Int Rep, 2022, 7(5):993-1003), but are limited to inhibiting BAFF or APRIL (Ramanujam et al., J Clin Invest, 2006, 116(3):724-734; Benson et al., J Immunol, 2008, 180(6):3655-3659; Liu et al., Exp Cell Res, 2011, 317(9):1270-1277; Huard et al., PloS One, 2012, 7(2):e31837, Haselmayer et al., Eur J Immunol, 2017, 47(6):1075-1085; Samy et al., Int Rev Immunol, 2017, 36(1):3-19; Stohl et al., Arthritis Rheumatol, 2020, 72(2):292-302).BAFF-Trap is a WT TACI and WT BAFF-R hybrid Fc fusion protein (Zhou et al., Signal Transduct Target Ther, 2019, 4:19), and also has limitations due to only inhibiting BAFF.

[0192] The co-neutralization of BAFF and APRIL significantly reduces B cell functions, including antibody production, while the inhibition of BAFF or APRIL alone has relatively mild effects. The Fc fusion of the wild-type (WT) extracellular domain of TACI with the Fc domain of IgG1 (such as atacicept and tabalumab) is in clinical development and targets BAFF and APRIL. Atacicept (Samy et al., Int Rev Immunol, 2017, 36(1): 3-19) and tabalumab (Shi et al., Immunopharmacol Immunotoxicol, 2021, 1-8) are soluble WT TACI extracellular domain (ECD) Fc fusion proteins that strongly inhibit BAFF and weakly inhibit APRIL signaling.

[0193] These dual BAFF / APRIL antagonists have been shown to inhibit the survival of immature and mature B cells and plasma cells while preserving 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 have reduced serum IgG, IgM, and IgA levels as well as the numbers 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). In preclinical studies, when directly compared to inhibiting BAFF or APRIL alone, the dual inhibitors exhibited more pronounced pharmacodynamic (PD) effects and greater disease model modification (Ramanujam et al. 2006 J Clin Invest. 116:724-34; Benson et al. 2008 J Immunol. 180:3655-3659; Haselmeyer et al. 2017 Eur J Immunol. 47:1075-1085; Samy et al. 2017). Abatacept and tacirolimus have shown promising clinical potential in certain autoimmune diseases such as systemic lupus erythematosus (SLE) and IgA nephropathy, but have not clearly demonstrated long-term and / or complete disease remission. For example, both abatacept and tacirolimus have shown clinical activity against SLE (Merrill et al., Arthritis Rheumatol, 2018, 70(2):266-276; Dhillon, Drugs, 2021; Shi et al., Immunopharmacol Immunotoxicol, 2021, 1-8). However, abatacept failed to reach its primary endpoint in pivotal trials (Merrill et al., Arthritis Rheumatol, 2018, 70(2):266-276) and no longer appears to be actively developed for SLE (Vera, Therapeutics Provides Business Update and Reports Second Quarter 2022 Financial Results, 2022). In contrast, tacirolimus has been conditionally approved in China for the treatment of SLE based on a Phase 2b study and recently reported positive confirmatory Phase 3 results; however, most subjects still appear to experience disease flares within the first 6 months of treatment (Wu et al., American College of Rheumatology, 2019).

[0194] Although B cell-targeted therapies have shown promising therapeutic potential, they are not entirely satisfactory. So far, co-targeting of BAFF and APRIL has only been attempted in the context of developing the WT TACI-Fc molecules atacicept and tabalumab, although the affinity of WT TACI-Fc for APRIL may not be optimal and is far lower than that achieved by anti-APRIL mAbs, which have an affinity range of KD = 0.95 to 400 pM depending on the method used (Dulos, American Society of Hematology, 2016; Myette et al., Kidney Int, 2019, 96(1):104-116). For example, soluble recombinant TACI (e.g., atacicept or tabalumab) has shown considerable promise as a therapeutic agent, but its utility appears to be hampered by low to moderate affinity for APRIL. Thus, although these molecules demonstrably neutralize BAFF adequately, their efficiency in blocking APRIL activity is low, and there is clearly room for improvement. These findings provide clinical validation of the BAFF / APRIL pathway in SLE and also suggest that further improvement of the drug design of atacicept and tabalumab, perhaps by improving APRIL inhibition, could specifically provide opportunities for more effective and safer treatment options.

[0195] The provided embodiments include those that provide improved neutralizing activity and inhibit or reduce B cell responses. In some embodiments, the improved activity is mediated by increased or improved binding or interaction of the provided immunomodulatory protein (such as a TACI-Fc fusion protein) with BAFF and / or APRIL. The provided immunomodulatory protein blocks or antagonizes the interaction of BAFF or APRIL (such as homotrimers of BAFF or APRIL, heterotrimers of BAFF / APRIL, or BAFF 60-mers) with cognate B cell stimulatory receptors, thereby neutralizing the activity of the 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., a 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 provided aspects, this activity is improved or better than that achieved by a WT TACI-Fc fusion protein (such as atacicept or belimumab). In some embodiments, the provided immunomodulatory protein (TACI-Fc fusion protein) is a candidate therapeutic agent for treating a variety of autoimmune and inflammatory diseases, particularly B cell-related diseases (such as SLE, SjS, and other connective tissue diseases).

[0196] The provided embodiments include methods and uses of specific Fc fusion proteins of a TACI variant TNF receptor domain (TD, i.e., CRD2) that simultaneously inhibit BAFF and APRIL cytokines. The provided embodiments relate to the identification of variant TACI polypeptides following random mutagenesis and directed evolution of the second cysteine-rich domain (CRD2) of TACI (spanning residues 68 - 110), which variant TACI polypeptides are engineered to have improved affinity for APRIL and / or BAFF. As shown herein, affinity maturation involves five rounds of selection alternating between APRIL and BAFF while reducing the concentration of the selection reagent to maintain selection pressure. The results show that the variant TACI polypeptides exhibit substantially enhanced affinity for BAFF and APRIL compared to wild-type TACI. For example, provided herein are variant TACI polypeptides containing one or more amino acid substitutions (replacements or mutations) that confer improved binding affinity of the protein for BAFF and / or APRIL. Specifically, the provided embodiments include those that provide improved, combined BAFF and APRIL inhibition. Thus, the provided immunomodulatory proteins provide effective and durable disease inhibition in the treatment of autoimmune or inflammatory diseases, including severe B cell-related autoimmune diseases such as SLE.

[0197] For example, the provided embodiments are based on the discovery that directed evolution of the TNFR domain (TD) of the TACI extracellular domain to modify affinity has facilitated the development of molecules with improved affinity for APRIL and / or BAFF. Thus, affinity modification has generated variant TACI comprising a variant TNFR domain (vTD). Fusion of these molecules with immunoglobulin Fc produces immunomodulatory proteins that inhibit B cell activity and responses. For example, reformatted as soluble Fc fusion proteins, the affinity matured TACI variants exhibit inhibition of APRIL and BAFF, as shown in the TACI-dependent reporter gene assay herein, and their IC 50 values are lower than those of wild-type TACI-Fc and the control drug belimumab. In addition, the results of animal model evaluations also showed a rapid and significant reduction in key lymphocyte subsets, including plasma cells, germinal center B cells, and follicular T helper cells. Furthermore, the variant molecules tested exhibited improved activity in mouse models, including significantly reduced autoantibodies and sialadenitis in the spontaneous SjS model, inhibited glomerular IgG deposition in the bm12-induced lupus model, and effectively inhibited anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, sialadenitis, kidney lesions, and kidney immune complex deposition in the NZB / W lupus model. In addition, compared to wild-type TACI-Fc, the TACI-Fc fusions tested exhibited significantly and persistently reduced serum IgM, IgG, and IgA antibody titers in mice. The findings herein indicate that these immunomodulatory proteins continuously exhibit potent immunosuppressive activity and efficacy in vitro and in vivo, outperforming existing and / or approved immunomodulators such as belimumab, abatacept, atacicept, or taci-cept. Thus, such biologics may 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.

[0198] In addition, the observations herein demonstrate that the TACI-Fc fusion proteins exhibit high serum exposure when administered to mice and cynomolgus monkeys. The provided TACI-Fc fusion proteins achieve favorable and higher serum exposure as well as more potent immunosuppressive activity, supporting their use at lower clinical doses and / or lower dosing frequencies (or longer dosing intervals) than existing WT TACI-Fc therapies. For example, existing WT TACI-Fc therapeutic agents such as atacicept and abatacept must be administered at least once per week. Reducing the dosing frequency can provide better symptom control for the treated subject, improve compliance with the dosing regimen, improve patient quality of life or patient satisfaction, and / or generally reduce the cost of treatment. In addition, reducing the dose, even at a more regular frequency such as once per week, may mitigate certain adverse reactions.

[0199] In certain embodiments, the provided TACI-Fc fusion proteins are used to treat SLE and other autoantibody-related rheumatic diseases, which are indications with still far unmet needs. In SLE, the complex pathogenesis and heterogeneity of the disease hinder treatment options, suggesting that multiple pathways or aspects of B cell development and differentiation may need to be inhibited simultaneously to achieve a durable response. Although B cell depleting agents such as rituximab / ocrelizumab / obinutuzumab (anti-CD20) and obexelimab (anti-CD19) have shown favorable clinical impacts in certain autoimmune disease settings, this has not translated to SLE, where rituximab has failed to show benefit in SLE and LN trials (Merrill et al., Arthritis Rheum, 2010, 62(1):222-233; Rovin et al., Arthritis Rheum, 2012, 64(4):1215-1226). A possible limitation of these therapies is that CD20 and CD19 are not expressed on all ASCs or LL-PCs, and only early B cells (including pro / pre-, immature, mature, and memory B cells) are depleted, thus sparing most pathogenic plasmablasts and PCs (Lee et al., Nat Rev Drug Discov, 2021, 20(3):179-199; Arbitman et al., JAutoimmun, 2022, 102873).

[0200] Targeting or co-targeting BAFF and / or APRIL is an alternative approach to ADCC-mediated B cell depletion. Preclinical studies have shown that the lack of these two key B cell survival and differentiation factors in B cells can significantly reduce all B cell subsets after the immature T1 developmental stage, including LL-PC, without affecting CD19+CD20+ pro / pre-B cell precursors (Gross et al., Immunity, 2001, 15(2):289-302). Inhibiting ASC can significantly affect pathogenic antibody production, thus potentially reducing disease activity. Although early efforts to target the BAFF / APRIL pathway mainly focused on drugs such as belimumab that only neutralize BAFF, it may be necessary to inhibit both APRIL and BAFF to affect the survival of more differentiated and pathogenic TACI+ / BCMA+ ASCs (Samy et al., Int Rev Immunol, 2017, 36(1):3-19).

[0201] APRIL plays a particularly important role in IgA class switch, production, and glycosylation, as first shown by studies in APRIL knockout mice (Castigli et al., Proc Natl Acad Sci USA, 2004, 101(11):3903-3908). In addition, elevated plasma APRIL levels in patients with IgA nephropathy (IgAN) are associated with more severe clinical manifestations such as high proteinuria and levels of Gd (galactose-deficient) IgA1, which are important pathogenic factors and contribute to the disease pathogenesis. In fact, early trials of BION-1301 and sibelizumab have shown that APRIL inhibition alone can mediate a significant decrease in Ig (especially IgA) in healthy subjects, and BION-1301 has affected proteinuria in IgAN patients in ongoing trials (Barratt et al., J Immunol, 2022, 180(6):3655-3659). However, targeting APRIL alone has its own limitations and is not expected to affect less mature BAFF-dependent B cells, which may also contribute to disease pathogenesis (Lee et al., Nat Rev Drug Discov, 2021, 20(3):179-199). BAFF neutralization leads to downregulation of B cell function, reduced autoantibody production, and inhibition of tertiary lymphoid structure formation in the kidney (Samy et al., Int Rev Immunol, 2017, 36(1):3-19).

[0202] Belimumab was the first approved therapeutic agent for SLE and LN (Lee et al., Nat Rev Drug Discov, 2021, 20(3):179-199), highlighting the need for new therapies. Another advance in SLE therapy is the recent approval of the anti-type I interferon receptor antibody anifrolumab (Morand et al., N Engl J Med, 2020, 382(3):211-221; Deeks, Drugs, 2021). Anifrolumab targets myeloid dendritic cells rather than B cells, thus targeting the pathophysiology of SLE that is different from B cell modulators, although type I interferon is known to indirectly promote B cell differentiation and loss of tolerance. In SLE, interferon-regulated gene expression is significantly increased; however, interferon gene signature expression does not predict response, highlighting the pleiotropic effects of the interferon system (Morand et al., N Engl J Med, 2020, 382(3):211-221). In contrast, high levels of BAFF and APRIL in the sera of SLE patients have been well established and described in many studies (Samy et al., Int Rev Immunol, 2017, 36(1):3-19). High serum BAFF levels are also associated with elevated autoantibody levels, particularly anti-dsDNA antibodies (Samy et al., Int Rev Immunol, 2017, 36(1):3-19).

[0203] The provided TACI-Fc fusion protein is a potential best-in-class BAFF / APRIL inhibitor for SLE and other autoantibody-related diseases. The provided TACI-Fc fusion protein: has significantly improved ligand affinity; is superior to WT TACI-Ig, BAFF, and / or APRIL-specific inhibitors; and is well tolerated in healthy adults following IV or SC administration, with dose-dependent PK / PD. For example, the present disclosure Figure 46 demonstrates the superiority of the provided TACI-Fc fusion protein in reducing circulating immunoglobulins (i.e., IgA, IgG, and IgM) compared to current biologics. This indicates that the provided TACI-Fc fusion protein is suitable for a variety of autoantibody-related inflammatory diseases.

[0204] In addition, the TACI-Fc fusion protein is well tolerated at low doses (e.g., 80 mg) to high doses (e.g., 960 mg) without adverse reactions. When administered once every four weeks (Q4W), the TACI-Fc fusion protein is also well tolerated. In addition, the TACI-Fc fusion protein is effective at low doses (e.g., 80 mg) whether administered SC or IV.

[0205] All publications mentioned in this application (including patent documents, scientific articles, and databases) are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference separately. If the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall control over the definitions incorporated by reference herein.

[0206] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0207] I. Definitions

[0208] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terminology 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 pertains. In some instances, for purposes of clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the meaning commonly understood in the art.

[0209] As used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms "a", "an", and "the" include plural referents.

[0210] As used herein, the term "about" refers to the ordinary error range of the corresponding value that would be readily known to one of ordinary skill in the art. The description of "about" a value or parameter herein includes (and describes) embodiments that are about the value or parameter itself. For example, the description of "about X" includes the description of "X".

[0211] The term "affinity modification" as used in the context of protein domains refers to a mammalian protein having an altered amino acid sequence in its extracellular domain or a specific binding portion thereof (relative to the corresponding wild-type parent or unmodified domain) such that, compared to the parental wild-type or unmodified (i.e., non-affinity-modified domain) protein, it has increased or decreased binding activity, such as binding affinity, for at least one of its binding partners (or "counterstructures"). 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 the wild-type or unmodified domain. The increase or decrease in binding activity (e.g., binding affinity) can be determined using well-known binding assays, including flow cytometry. Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005). See also Linsley et al., Immunity, 1: 7930801 (1994). The increased value of the binding activity (e.g., affinity) of a protein for its binding partner is at least 10% higher than the wild-type control value, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000% or 10000% higher than the wild-type control value. The decreased value of the binding activity (e.g., affinity) of a protein for at least one of its binding partners 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% of the wild-type control value, but no less than 10% of the wild-type control value. An affinity-modified protein alters the primary amino acid sequence of the extracellular domain or its specific binding portion by substitution, addition or deletion of amino acid residues. The term "affinity modification" should not be construed as imposing any conditions on any particular starting composition or method for producing an affinity-modified protein. Thus, an affinity-modified protein is not limited to a wild-type protein domain that is then converted to an affinity-modified domain by any particular affinity-modification process. For example, an affinity-modified domain polypeptide can be generated starting from wild-type mammalian domain sequence information, then docked to its binding partner by computer modeling, and finally produced as an affinity-modified domain material composition by recombinant or chemical synthesis. But in an alternative instance, an affinity-modified domain can be produced by site-directed mutagenesis of a wild-type domain. Thus, an affinity-modified TD domain represents a product, and not necessarily a product produced by any given process. A variety of techniques can be employed, including recombinant methods, chemical synthesis or combinations thereof.

[0212] The term "affinity-modified TD domain" refers to an affinity-modified domain of a member of the tumor necrosis factor receptor superfamily (TNFRSF) protein or its TNF ligand, which has 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 the TNFR domain, which is composed of at least one cysteine-rich domain (CRD) within the extracellular domain of the TNFRSF protein or its specific binding portion (relative to the corresponding wild-type parent or unmodified domain), such that it has increased or decreased binding activity, such as binding affinity, for at least one of its binding partners (or "counterstructure") compared to the parental wild-type or unmodified protein containing a non-affinity-modified or unmodified TD domain.

[0213] "Affinity-modified TACI" (also referred to as variant TACI) refers to a TACI protein molecule that antagonizes or blocks B cell stimulatory receptor activity. For example, TACI binds to APRIL and / or BAFF, which are ligands of the B cell stimulatory receptors B cell maturation antigen (BCMA), B cell activation factor receptor (BAFF-R), and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). In certain embodiments, BIM includes the extracellular domain of TACI, or a portion of the extracellular domain of TACI, which portion contains a TNF receptor family domain (e.g., TD, e.g., CRD) that binds to the cognate ligands APRIL and / or BAFF and the heterotrimer of APRIL and BAFF. Affinity-modified variants of the extracellular domain of TACI or portions thereof may include one or more amino acid modifications (e.g., amino acid substitutions) in the TD, thereby increasing the binding affinity for the cognate ligands (e.g., APRIL and / or BAFF, and the heterotrimer of APRIL and BAFF).

[0214] As used herein, "B cell stimulating receptor" refers to one or more of B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which are related tumor necrosis factor (TNFR) superfamily receptors expressed on B cells. Binding or ligation of these related receptors by their cognate ligands BAFF and / or APRIL or heterotrimers of APRIL and BAFF can regulate B cell homeostasis, including B cell survival, B cell maturation and differentiation, and immunoglobulin class switching. B cell stimulating receptors typically 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. Various TRAF molecules are recruited to the cytoplasmic domain and 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.

[0215] As used herein, "bind", "bound", or grammatical variations thereof refer to any attractive interaction in which a molecule participates 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.

[0216] As used herein, binding activity refers to a property of a molecule (e.g., a polypeptide) relating to whether and how it binds one or more binding partners. Binding activity can include any measurement of the binding of a molecule to a binding partner. Binding activity includes the ability to bind a binding partner, the affinity for binding a binding partner (e.g., high affinity), the avidity for binding a binding partner, the bond strength with a binding partner, and / or the specificity or selectivity for binding a binding partner.

[0217] As used herein, the term "binding affinity" refers to the specific binding affinity of a protein for its binding partner (i.e., its counterstructure) under specific binding conditions. Binding affinity refers to the strength of the interaction between two or more molecules, such as binding partners, and is typically the strength of non-covalent interactions between two binding partners. The increase or decrease in the binding affinity of an affinity-modifying domain or an immunomodulatory protein containing an affinity-modifying domain for a binding partner is determined relative to the binding affinity of an unmodified domain (e.g., a native or wild-type TD domain). Methods for determining binding affinity or relative binding affinity are known in the art, including 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 the mean fluorescence intensity (MFI) in a flow binding assay.

[0218] As used herein, the term "binding avidity" refers to the specific binding avidity of a protein for its binding partner (i.e., its counterstructure) under specific binding conditions. In biochemical kinetics, avidity refers to the cumulative strength of multiple non-covalent binding interactions, such as the multiple affinities between a protein and its binding partner (i.e., its counterstructure). Thus, avidity is different from affinity, which describes the strength of a single interaction.

[0219] The term "biological half-life" refers to the time required for a substance, such as an immunomodulatory protein, to lose half of its pharmacological or physiological activity or concentration. The biological half-life can be affected by the elimination, excretion, degradation (e.g., enzymatic degradation / digestion), or absorption and concentration in certain organs or tissues of the body. In some embodiments, the biological half-life can be evaluated by determining the time required for the plasma concentration of a substance to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used for derivatization and increasing the biological half-life of proteins are known in the art and include, but are not limited to, polymerization domains (e.g., Fc immunoglobulin domains), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0220] As used herein, the term "cell surface counterstructure" (or "cell surface binding partner") is a counterstructure (or a binding partner) expressed on a mammalian cell. Typically, a cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor.

[0221] For a protein such as a receptor, a soluble ligand, or an extracellular domain or a portion thereof or an affinity-modified variant thereof, the term "binding partner" or "counterstructure" refers to at least one molecule (usually a native mammalian protein) to which the protein in question 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, a 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 and by a cell such as a mammalian cell, thereby forming an immunological synapse, such as an immune cell.

[0222] The term "cis" with respect to binding to cell surface molecules refers to binding to two or more different cell surface molecules, each present on the surface of the same cell. In some embodiments, cis refers to two or more cell surface molecules located only on one of the two mammalian cells forming an IS or only on the other (but not both).

[0223] As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (such as charge or hydrophobicity). Examples of groups of amino acids with side chains of similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl 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, glutamic acid - aspartic acid, and asparagine - glutamine.

[0224] The term "corresponding to" with respect to the position of a protein, such as when reciting that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (such as that shown in a 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 sequences, one of ordinary skill in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Figure 9 Identifying corresponding residues by aligning two sequences is illustrated.

[0225] As used herein, a "domain" (which is typically a sequence of three or more amino acids, generally a sequence of 5 or 7 or more amino acids, such as 10 to 200 amino acid residues) refers to a portion of a molecule, such as a protein or a coding nucleic acid, that is distinct and identifiable in structure and / or function from other portions of the molecule. For example, domains include those portions of a polypeptide chain that can form independently folded structures within a protein consisting of one or more structural motifs and / or can be identified by functional activity (such as binding activity). A protein can have one or more different domains. For example, domains can be identified, defined, or distinguished by homology to related family members based on primary sequence or structure (such as homology to a motif). In another example, domains can be distinguished by their function, such as the ability to interact with a biomolecule (such as a cognate binding partner). A domain can exhibit a biological function or activity independently such that the domain can function independently or when fused to another molecule, such as binding, for example. A domain can be a linear amino acid sequence or a non-linear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by one of ordinary skill in the art. For purposes of illustration herein, definitions are provided, but it should be understood that one of ordinary skill in the art is fully capable of identifying a particular domain by name. If desired, appropriate software can be employed to identify domains. It should be understood that the reference to amino acids, including reference to the specific sequences shown in SEQ ID NOs used to describe domain organization (e.g., the TD domain), is for illustrative purposes and does not mean to limit the scope of the provided embodiments. It should be understood that the description of polypeptides and their domains is theoretically based on homology analysis and alignment with similar molecules. Moreover, in some cases, the adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., the TD domain) can also be included in the sequence, such as to ensure proper folding of the domain upon expression. Thus, the exact locus can vary and may not be the same for each protein. For example, a particular TD domain, such as a particular CRD domain, can be several amino acids (1 - 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) longer or shorter.

[0226] The terms "extracellular domain", "extracellular region", or "ECD" are used interchangeably herein and refer to the region of a membrane protein, such as a transmembrane protein, that is outside the vesicle membrane (e.g., the space outside the cell) when the full-length form of the membrane protein is expressed from the cell. For the purposes of this disclosure, it should be understood that reference to an ECD refers to the sequences and domains that make up the region and does not require that the protein containing the ECD be a membrane protein or that the domain be present extracellularly. For example, a soluble immunomodulatory protein can contain the ECD sequence of a membrane protein fused to another moiety, such as a multimerization domain, e.g., an Fc region. The extracellular domain typically interacts with a specific ligand or a specific cell surface receptor, such as via a binding domain that specifically binds the ligand or cell surface receptor. Examples of binding domains include cysteine-rich domains (CRDs). The extracellular domain of TNFR superfamily members contains a TD domain (e.g., a CRD domain). Thus, the ECD as referred to herein includes the full-length sequence of the ECD of a membrane protein and its specific binding fragments containing a CRD that binds to a ligand or a cognate binding partner.

[0227] As used herein, an "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition, such as one containing an immunomodulatory protein or an Fc fusion protein, that, when administered in vitro (by contacting a patient's cells) or in vivo (by administering to a patient), alone (i.e., as a single therapy) or in combination with other therapeutic agents, produces a statistically significant inhibition of disease progression, e.g., by ameliorating or eliminating the symptoms and / or causes of the disease. An effective amount for treating a disease, disorder, or condition, such as an immune system disease, disorder, or condition, can be an amount that alleviates, reduces, or mitigates at least one symptom or biological response or effect associated with the disease, disorder, or condition, prevents the progression of the disease, disorder, or condition, or improves the physiological function of the patient. In the case of cell therapy, the effective amount is the effective dose or number of cells administered to the patient. In some embodiments, the patient is a human patient.

[0228] As used herein, a fusion protein refers to a polypeptide encoded by a nucleic acid sequence containing the coding sequences of two or more proteins (in some cases 2, 3, 4, 5, or more proteins), wherein the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated in a host cell, a protein containing 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 containing 1, 2, 3, or more but usually fewer than 20, 15, 10, 9, 8, 7, or 6 amino acids. The protein product encoded by the fusion construct is called a fusion polypeptide. An example of a fusion protein according to the provided embodiments is an Fc fusion protein containing an affinity-modifying domain (e.g., a variant of the extracellular domain of TACI or a portion thereof containing a CRD) linked to an immunoglobulin Fc domain.

[0229] The term "half-life extension moiety" refers to a moiety of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum as compared to the half-life of the protein without conjugation of that moiety. In some embodiments, the half-life extension is greater than or about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold or about 6.0-fold. In some embodiments, following in vivo administration, the half-life extension is greater than 6 hours, greater than 12 hours, greater than 24 hours, greater than 48 hours, greater than 72 hours, greater than 96 hours or greater than 1 week as compared to a protein without a half-life extension moiety. Half-life refers to the time required for a protein to lose half of its concentration, amount or activity. For example, the half-life can be determined by using an ELISA assay or an activity assay. Exemplary half-life extension moieties include Fc domains, multimerization domains, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS) and polyglutamic acid (glutamylated).

[0230] The Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also referred to as an Fc polypeptide) largely corresponds to the constant region of the immunoglobulin heavy chain and in some cases is responsible for various functions, including effector functions of antibodies. The Fc domain contains part or all of the hinge domain of the immunoglobulin molecule plus the CH2 and CH3 domains. In some cases, all or part of the Fc hinge sequence may be deleted for inclusion in the provided fusion protein. The Fc domain can form a dimer of two polypeptide chains joined by one or more disulfide bonds. In some embodiments, the 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 functions. In some embodiments, unless otherwise described with reference to a specific SEQ ID NO, references to amino acid substitutions in the Fc region are in the EU numbering system. The EU numbering is known and conforms to the most recently updated IMGT Scientific Chart (IMGT (registered trademark), International ImMun oGeneTics information http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created on: May 17, 2001, last updated on: January 10, 2013) and the EU index reported by Kabat, E.A. et al. in Sequences of Proteins of Immunol ogical interest. 5th Edition U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0231] Immunoglobulin Fc fusions (“Fc-fusions”), such as immunomodulatory Fc fusion proteins, are molecules that contain one or more polypeptides operably linked to an immunoglobulin Fc region. The Fc fusion can contain, for example, an Fc region operably linked to the extracellular domain of TACI or a CRD-containing portion thereof, including any provided affinity-modified variants. The immunoglobulin Fc region can be indirectly or directly linked to one or more polypeptides. A variety of linkers are known in the art and can optionally be used to link the Fc to a fusion partner to produce an Fc fusion. The same type of Fc fusion can dimerize to form an Fc fusion homodimer. Non-identical types of Fc fusions (e.g., knobs-into-holes engineering) can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as murine or human Fc.

[0232] 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 prokaryote, such as Escherichia coli, or it can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a hybridoma cell. 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 media or the CHO cell line DX-B11 lacking DHFR.

[0233] As used herein, the term "immunological synapse" or "immune synapse" (abbreviated "IS") refers to the interface between a mammalian cell expressing MHC I (major histocompatibility complex) or MHC II, such as an antigen-presenting cell or a tumor cell, and a mammalian lymphocyte, such as an effector T cell or a natural killer (NK) cell.

[0234] As used herein, the term "immunoglobulin" (abbreviated "Ig") is synonymous with the term "antibody" (abbreviated "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 immunoglobulins that are less than full length, whether synthesized in whole or in part (e.g., recombinantly or chemically synthesized) or naturally occurring, including any fragment thereof that contains at least a portion of the variable heavy chain (VH) and / or variable light chain (VL) region of the immunoglobulin molecule sufficient to form an antigen-binding site and that can specifically bind an antigen upon assembly. Antibodies can also include all or part of the 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 linked together on one chain, and other antibody V-region fragments, such as Fab′, F(ab)2, F(ab′)2, dsFv diabodies, Fc, and Fd polypeptide fragments. Thus, it is understood that antibodies referred to herein include full-length antibodies and antigen-binding fragments. The term antibody also includes antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules. The meaning of the term includes bispecific antibodies, homobispecific antibodies, and heterobispecific antibodies. Definitions include polyclonal or monoclonal antibodies. Antibodies also include synthetic or recombinantly produced antibodies. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, pages 71 and chapter 6.

[0235] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably and refer to an antibody in a substantially intact form as compared 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 an antibody produced by an antibody-secreting B cell of a mammalian species (e.g., human, mouse, rat, rabbit, non-human primate, etc.) and a synthetically produced antibody having the same domains. Specifically, the whole antibody includes an antibody having a heavy chain and a light chain (including the Fc region). The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody can have one or more effector functions.

[0236] "Antibody fragment" includes a part of a whole antibody, the antigen-binding region and / or variable region of a whole antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (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 Fvs (scFv) or single-chain Fabs (scFab); any of the above antigen-binding fragments and multispecific antibodies derived from antibody fragments.

[0237] "Fv" consists of a variable domain of a heavy chain and a variable domain of a light chain that are linked by non-covalent association. The folding of these two domains gives rise to six complementarity-determining regions (CDRs) (3 from each of the heavy and light chains), which provide the amino acid residues for antigen binding and confer antigen-binding specificity on the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for the antigen) has the ability to recognize and bind an antigen, although in some cases its affinity is lower than that of the complete binding site.

[0238] "dsFv" refers to an Fv with engineered intermolecular disulfide bonds, which can stabilize the V H -V L Yes.

[0239] "Fd fragment" is an antibody fragment containing the variable region of the antibody heavy chain (V H ) and a constant domain (C H 1).

[0240] "Fab fragment" is an antibody fragment produced by pepsin digestion of a full-length immunoglobulin or a fragment having the same structure synthesized, for example, by recombinant methods. The Fab fragment contains a light chain (containing V L and C L ) and another chain containing the variable domain of the heavy chain (V H ) and a constant domain of the heavy chain (C H 1).

[0241] "F(ab')2 fragment" is an antibody fragment produced by pepsin digestion of an immunoglobulin at pH 4.0 - 4.5 or a fragment having the same structure synthesized, for example, by recombinant methods. The F(ab')2 fragment essentially contains two Fab fragments, where each heavy chain portion contains several additional amino acids, including cysteine residues that form the disulfide bonds joining the two fragments.

[0242] "Fab' fragment" is a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain).

[0243] "Fd' fragment" is an antibody fragment containing a heavy chain portion of an F(ab')2 fragment.

[0244] "Fv' fragment" is a fragment containing only the V H and V L domains of an antibody molecule.

[0245] "scFv fragment" refers to an antibody fragment containing a variable light chain (V L ) and a variable heavy chain (V H ) covalently linked in any order by a polypeptide linker. The length of the linker is such that the two variable domains can be bridged without substantial interference. An exemplary linker is (Gly-Ser) n residues, with some Glu or Lys residues dispersed therein to increase solubility.

[0246] "Diabody" is a dimer of scFv; diabodies typically have a shorter peptide linker than scFv and preferentially dimerize.

[0247] As used herein, the term "immunoreactivity" 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 (e.g., interferon-γ), cytotoxic activity, or the ability to activate the NF-κB pathway or other signaling cascades, resulting in the activation of transcription factors in immune cells. Assays for evaluating the immunoreactivity of immunomodulatory proteins can be compared to control proteins with known activity.

[0248] An "immunomodulatory protein" or "immunomodulatory polypeptide" is a protein that modulates immunoreactivity. "Modulation" or "modulating" an immune response refers to enhancing or suppressing immunoreactivity. Such modulation includes any induction, degree or extent of change, or inhibition of the immunoreactivity of immune cells such as B cells or T cells. For example, a soluble Fc fusion protein herein can inhibit the immunoreactivity of B cells. An immunomodulatory protein can be a single polypeptide chain or a multimer (dimer or higher multimer) in which at least two polypeptide chains are covalently bonded, e.g., by interchain disulfide bonds. Thus, monomeric, dimeric, and higher order multimeric proteins are all within the scope of this defined term. Multimeric proteins can be homomultimers (composed of identical polypeptide chains) or heteromultimers (composed of different polypeptide chains).

[0249] As used herein, a modification refers to a modification of the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule, including altering the amino acids or nucleotides of the sequence, respectively. Amino acid modifications or alterations can be, respectively, deletions, insertions, or substitutions (replacements) of amino acids or nucleotides. Methods for modifying polypeptides are conventional methods well known to those skilled in the art, such as by using recombinant DNA methods.

[0250] The term "oligomerization domain" refers to an amino acid sequence that promotes the formation of a multimer of two or more polypeptides. An oligomerization domain includes sequences that promote stable interactions of a polypeptide molecule with one or more additional polypeptide molecules, each of the additional polypeptide molecules containing a complementary oligomerization domain (e.g., a first oligomerization domain and a second oligomerization domain), and these oligomerization domains can be the same or different oligomerization domains. The interaction between complementary oligomerization domains (e.g., the interaction between a first oligomerization domain and a second oligomerization domain) forms a stable protein-protein interaction, thereby producing a multimer of a polypeptide molecule and an additional polypeptide molecule. In some cases, the oligomerization domains are the same and interact with themselves to form a stable protein-protein interaction between two polypeptide chains. Generally, a polypeptide is directly or indirectly conjugated to an oligomerization domain. Exemplary oligomerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. An oligomerization domain can be, for example, an immunoglobulin constant region or domain, such as the Fc domain or a portion thereof from IgG (including IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM and modified forms thereof).

[0251] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form. Unless otherwise restricted, these terms encompass nucleic acids containing known analogs of natural nucleotides, and these nucleic acids have binding properties similar to those of natural nucleotides and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences and the explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixture of bases and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0252] As used herein, the terms "operable combination", "in operable order", and "operably linked" refer to nucleic acid sequences linked in such a way or orientation that the arrangement of the fragments enables them to function in concert for a desired purpose. In some embodiments, the term refers to nucleic acid linkage to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or producing a desired functional protein molecule. For example, fragments of a DNA sequence, such as a coding sequence and regulatory sequences, are linked in such a way that gene expression is permitted when an appropriate molecule (e.g., a transcriptional activator protein) binds to the regulatory sequences.

[0253] The term "pharmaceutical composition" refers to a composition suitable for use as a medicament in a mammalian subject, typically 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 typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0254] 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. These terms do not refer to a specific length of the product. Thus, both "peptide" and "oligopeptide" are included within the definition of polypeptide. These terms include post-translational modifications of polypeptides, such as, glycosylation, acetylation, phosphorylation, etc. These terms also include molecules that contain one or more amino acid analogs or non-canonical or non-natural amino acids that can be synthesized or recombinantly expressed using known protein engineering techniques. In addition, a protein can be derivatized by well-known organic chemistry techniques as described herein.

[0255] When the term "purified" is applied to a nucleic acid (such as one encoding an immunomodulatory protein) or a protein (e.g., an immunomodulatory protein), it generally means a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band 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 substantially one band in an electrophoretic gel is "purified". The purity of a purified nucleic acid or protein is at least about 50%, and typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or higher (e.g., by weight or mole percentage).

[0256] The term "recombinant" refers to a material (e.g., nucleic acid or polypeptide) that has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be made to the material within its natural environment or state, or to the material removed from its natural environment or state. For example, "recombinant nucleic acid" is a nucleic acid prepared by recombinant nucleic acids, such as during cloning, affinity modification, DNA shuffling, or other well-known molecular biology procedures. "Recombinant DNA molecules" are composed of DNA fragments joined together by these molecular biology techniques. As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule (e.g., an immunomodulatory protein) expressed using recombinant DNA molecules. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid or a cell that has been otherwise altered by genetic engineering, such as by introducing a nucleic acid molecule encoding a recombinant protein (such as the immunomodulatory proteins provided herein) into the cell. Transcription control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically 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, as well as viruses (similar control elements, i.e., promoters, also exist in prokaryotes). The choice of a particular promoter and enhancer depends on the cell type used for expressing the protein of interest.

[0257] 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 the appropriate nucleic acid sequences necessary for the expression of the coding sequence operably linked therein in a particular cell. The nucleic acid sequences required for expression in prokaryotes include a promoter, an optional operator sequence, a ribosome binding site, and other possible sequences. It is known that eukaryotic cells utilize promoters, enhancers, as well as termination signals and polyadenylation signals. A secretion signal peptide sequence may also optionally be encoded by the recombinant expression vector and operably linked to the coding sequence such that the expressed protein can be secreted by the recombinant host cell, such as for its expression as a secreted protein or for easier isolation or purification of the immunomodulatory protein from the cell, if desired. The term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell into which the vector has been introduced. Vectors include viral vectors, such as lentiviral vectors.

[0258] As used herein, the term "sequence identity" refers to the sequence identity of a gene or protein at the nucleotide or amino acid level, respectively. "Sequence identity" measures the identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequences at a given position in each sequence when aligning the sequences. Similarly, when aligning sequences, nucleic acid sequence identity can be determined by comparing the nucleotide sequences at a given position in each sequence. Sequence alignment methods for comparison 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 the percentage of sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI). In some cases, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity, the percentage 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. Reference to sequence identity includes sequence identity throughout the full length of each sequence being compared. Those skilled in the art can determine the parameters suitable for aligning sequences, including any algorithms required to achieve the maximum alignment within the full length of the sequences being compared.

[0259] As used herein, the term "soluble" with respect to a protein means that the protein is not a membrane protein or is not anchored to the cell membrane. A protein can be constructed as a soluble protein by including only an extracellular domain or a portion thereof without including a transmembrane domain. In some cases, the solubility of a protein can be improved by directly or indirectly linking or attaching to an Fc domain or other molecule that prolongs the half-life, which can also improve the stability and / or half-life of the protein in some cases. In some aspects, the soluble protein is an Fc fusion protein.

[0260] As used herein, the term "specifically binds" refers to the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or avidity is at least 10-fold, but optionally 50, 100, 250, or 500-fold, or even at least 1000-fold, the average affinity or avidity of the same protein for a statistically significant sized random peptide or polypeptide collection. A specifically binding protein need not bind exclusively to a single target molecule, but may specifically bind to more than one target molecule. In some instances, a specifically binding protein may bind to a protein having a structure conformation similar to the target protein (e.g., paralog or ortholog). One of skill in the art will recognize that specific binding to a molecule having the same function in different animal species (i.e., ortholog) or to a molecule having an epitope substantially similar to the target molecule (e.g., paralog) is possible and does not compromise binding specificity as determined with respect to a statistically valid unique non-target collection (e.g., random polypeptides). Thus, the immunomodulatory proteins of the present invention may specifically bind more than one different class of target molecules due to cross-reactivity. Solid-phase ELISA immunoassays, ForteBio Octet, or Biacore measurements can be used to determine specific binding between two proteins. Generally, the interaction between two binding proteins has a dissociation constant (Kd) of less than about 1x10 -5 M, and is typically as low as about 1x10 -12 M. In certain aspects of the present disclosure, the interaction between two binding proteins has a dissociation constant of less than about 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, or 1x10 -11 M or less.

[0261] As used herein, the term "specific binding fragment" or "fragment" with respect to a protein refers to a polypeptide that is shorter than the full-length protein or its specific domain or region and specifically binds to the binding partner of the full-length protein or the specific domain or region, both in vitro and / or in vivo. A specific binding fragment refers to the full-length extracellular domain of a polypeptide or the polypeptide binding domain, but still a fragment that binds to the binding partner of the binding domain. For example, a specific binding fragment refers to a fragment of the extracellular domain of a full-length TNFR family member or its full-length TNFR domain (TD) (such as CRD), but still binds to the binding partner of the TNFR family member or the CRD of the TNFR family member. In some embodiments, the specific binding fragment is at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the 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 can 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 the CRD1 and / or CRD2 domain. In some embodiments, the specific binding fragment includes the CRD2 domain.

[0262] 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 infant, juvenile, adolescent, adult, and elderly subjects.

[0263] As used herein, "synthetic" with respect 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 produced by recombinant methods and / or chemical synthesis methods.

[0264] As used herein, the term "TNF receptor superfamily" or "TNFRSF" refers to a group of cell surface cytokine receptors that are all type I (N-terminal extracellular) transmembrane glycoproteins and contain one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on common structural features, including the presence of one or more cysteine-rich domains (CRDs) in their N-terminal extracellular region, which domains typically function in the protein binding of their cognate binding partner or ligand. TNFRSF proteins may have only one or a few CRDs (such as CRD1, CRD2, etc.). Typically, the ECD or extracellular domain of TNFRSF members contains 1 to 6 CRD pseudorepeats. For example, BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are typically trimeric or multimeric complexes stabilized by their intramolecular disulfide bonds. The binding of TNFRSF proteins to their ligands promotes various biological activities in cells, such as inducing apoptotic cell death or cell survival and proliferation.

[0265] The term "TD" refers to a structural domain or domain of a TNFRSF protein or a TNF family ligand. For example, the TD of a TNFRSF protein is a cysteine-rich domain (CRD) module having approximately 40 amino acids containing six (6) conserved cysteines. Thus, with respect to the TD of a TNFRSF protein, reference to CRD can also be used interchangeably with the term TD. The six cysteines are involved in the formation of intrastrand disulfide bonds. The extracellular domain (ECD) of TNFRSF members contains one or more CRD domains; thus, the term TD is also used to refer to the ECD of such protein molecules. Reference to variant TD (vTD) refers to a variant or modified sequence of TD.

[0266] The term "trans" with respect to binding to cell surface molecules refers to binding to two different cell surface molecules, where each is present on the surface of a different cell. In some embodiments, trans refers to two different cell surface molecules, the first being present only on one of two mammalian cells forming an IS and the second being present only on the second of two mammalian cells forming an IS.

[0267] As used herein, the term "transmembrane protein" refers to 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). A transmembrane protein contains transmembrane domains ("transmembrane domains") through which the transmembrane protein is integrated into the lipid bilayer and through which the integration is thermodynamically stable under physiological conditions. Transmembrane domains can generally be predicted from their amino acid sequences via any number of commercially available bioinformatics software applications based on their elevated hydrophobicity relative to the regions of the protein that interact with the aqueous environment (e.g., cytosol, extracellular fluid). Transmembrane domains are generally hydrophobic α-helices that span the membrane. Transmembrane proteins can cross both layers of the lipid bilayer one or more times.

[0268] As used herein, the terms "treating", "treatment", or "therapy" of a disease, disorder, or condition refer to administering the immunomodulatory protein or engineered cell of the invention, alone or in combination with another compound described herein, to slow, stop, or reverse the progression of the disease or condition, as evidenced by a decrease, cessation, or elimination of clinical or diagnostic symptoms. "Treating", "treatment", or "therapy" also refers to reducing the severity of symptoms in an acute or chronic disease, disorder, or condition, or reducing the recurrence rate (e.g., in the case of a relapsing or remitting autoimmune disease process or an inflammatory disorder), or reducing inflammation in the case of the inflammatory aspects of an autoimmune disease or inflammatory disorder. "Preventing", "prophylaxis", or "prevention" of a disease, disorder, or condition as used in the context of the present invention refers to administering the immunomodulatory protein of the invention, alone or in combination with another compound, to prevent the occurrence or onset of the disease, disorder, or condition, or some or all of the symptoms of the disease, disorder, or condition, or to reduce the likelihood of the onset of the disease, disorder, or condition.

[0269] When used to refer to variant proteins or polypeptides, the term "variant" (which may also be referred to as "modified" or "mutant" and used interchangeably) refers to a protein produced by artificial intervention, such as a mammalian (e.g., human or murine) protein. A variant is a polypeptide having an altered or modified amino acid sequence relative to an unmodified or wild-type protein or its domain, such as by one or more amino acid substitutions, deletions, additions, or combinations thereof. Variant polypeptides 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. 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 the signal sequence) or the portion thereof containing the extracellular domain or binding domain. Both non-naturally occurring amino acids and naturally occurring amino acids are included within the scope of allowable substitutions or additions. Variant proteins are not limited to any particular method of manufacture and include, for example, chemical synthesis, recombinant DNA technology, or combinations thereof. The 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 an alteration (i.e., increase or decrease) in the binding activity (such as binding affinity or avidity) for one or more binding partners. Thus, the variant proteins can be "affinity-modified" proteins as described herein.

[0270] As used herein, the terms "wild-type" or "native" or "natural" are used interchangeably and refer to biomaterials found in nature and not modified by artificial intervention, such as nucleic acid molecules, proteins, host cells, etc.

[0271] II. TACI Immunomodulatory Proteins and Variant TACI Polypeptides

[0272] The present disclosure provides TACI immunomodulatory proteins that comprise a portion of the extracellular domain (ECD) of the TACI receptor or variants thereof and that can bind to at least one TACI cognate binding partner. The present disclosure also provides TACI polypeptide variants that exhibit altered (e.g., increased) binding activity or affinity for one or more TACI cognate binding partners. In some embodiments, the TACI cognate binding partner is one or more of BAFF or APRIL, or a BAFF / APRIL heterotrimer. The provided TACI immunomodulatory proteins and polypeptides include soluble fusion proteins thereof, wherein the TACI portion of the extracellular domain or variant thereof is linked to another moiety, such as an immunoglobulin Fc or other multimerization domain or half-life extension moiety. Thus, in some embodiments, the immunomodulatory protein is a TACI-Fc fusion protein. In some embodiments, provided is a TACI-Fc fusion protein comprising: (1) a TACI polypeptide consisting of the extracellular domain of the TACI receptor or a portion thereof, or a variant TACI polypeptide that can bind to at least one TACI cognate binding partner, and (2) an Fc domain. The TACI polypeptide or variant TACI polypeptide can be linked directly or indirectly (e.g., via a peptide linker) to the Fc domain.

[0273] TACI is a member of the tumor necrosis factor receptor family and is characterized by having an extracellular domain (ECD) containing cysteine-rich pseudorepeat domains (CRDs). TACI is a membrane-bound receptor having an extracellular domain containing two cysteine-rich pseudorepeats (CRD1 and CRD2), a transmembrane domain, and a cytoplasmic domain that interacts with CAML (calcium modulator and cyclophilin ligand), an integral membrane protein located in intracellular vesicles and a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with B cells as well as subsets of T cells. The TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family. One ligand is designated BAFF (B cell activating factor of the TNF family), also variously designated ZTNF4, "neutrophil factor-α", "BLyS", "TALL-1", and "THANK" (Yu et al., International Publication No. WO98 / 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)). The other ligand is designated APRIL, also variously designated "ZTNF2" and "TNRF death ligand-1" (Hahne et al., J. Exp. Med. 188:1185 (1998); Kelly et al., Cancer Res. 60:1021 (2000)). These two ligands also bind to the B cell maturation antigen (BCMA) (Gross et al., Nature 404:995 (2000)). Binding of the TACI receptor to its ligand BAFF or APRIL can stimulate B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.

[0274] 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 (CRD, also referred to hereinafter as the tumor necrosis family receptor domain or TD), each of which exhibits an 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 stalk 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.

[0275] In some embodiments, the variant TACI polypeptides provided herein contain one or more amino acid modifications, such as one or more substitutions (or, "mutations" or "replacements"), deletions or additions in the extracellular domain of a reference TACI polypeptide, such as a wild-type or unmodified TACI polypeptide containing a CRD (also referred to hereinafter as TD). Thus, the variant TACI polypeptides provided are or comprise variant TDs ("vTDs") in which one or more amino acid modifications (e.g., substitutions) are located in the CRD. In some embodiments, one or more amino acid modifications, such as one or more substitutions (or, "mutations" or "replacements"), deletions or additions, are located in the CRD1 region. In some embodiments, one or more amino acid modifications, such as one or more substitutions (or, "mutations" or "replacements"), deletions or additions, are located in the CRD2 region. In some embodiments, one or more amino acid modifications, such as one or more substitutions (or, "mutations" or "replacements"), deletions or additions, are located in the amino acids of the CRD1 and CRD2 regions.

[0276] In some embodiments, a reference (e.g., unmodified) TACI sequence is a wild-type TACI sequence or a portion thereof containing one or two CRDs. In some embodiments, a 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 a reference (e.g., unmodified) TACI polypeptide comprises CRD1 and CRD2. However, a variant TACI polypeptide need not contain both CRD1 and CRD2. In some embodiments, a variant TACI polypeptide comprises CRD1 or a specific binding fragment thereof or consists essentially of the same. In some embodiments, a variant TACI polypeptide comprises CRD2 or a specific binding fragment thereof or consists essentially of the same. In some embodiments, a variant TACI is a soluble polypeptide and lacks a transmembrane domain. In some embodiments, a variant TACI polypeptide further comprises a transmembrane domain and, in some instances, a cytoplasmic domain.

[0277] In some embodiments, a reference (e.g., unmodified) TACI sequence is a mammalian TACI sequence. In some embodiments, a reference (e.g., unmodified) TACI sequence can be mammalian TACI, including but not limited to human, mouse, cynomolgus monkey, or rat. In some embodiments, a reference (e.g., unmodified) TACI sequence is a human sequence. The extracellular domain of an exemplary human TACI sequence is shown in SEQ ID NO:122.

[0278] In some embodiments, a 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 thereof, (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 higher sequence identity to SEQ ID NO:122 and binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer, or (iii) is a fragment or portion of (i) or (ii) containing CRD1 and / or CRD2, wherein the portion binds to an APRIL, BAFF, or APRIL / BAFF heterotrimer. In some embodiments, a reference (e.g., unmodified) TACI sequence lacks the N-terminal methionine shown in SEQ ID NO:122.

[0279] TACI extracellular domain (ECD): SEQ ID NO:122

[0280] MSGLGRSRRGGRSRVDQEERFPQGLWTGVAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELRRQRSGEVENNSDNSGRYQGLEHRGSEASPALPGLKLSADQVALVYST

[0281] In some embodiments, the reference (e.g., unmodified) TACI sequence is the extracellular domain sequence of TACI, which is a part of the ECD with an N-terminal deletion relative to the amino acid sequence shown in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of the N-terminal amino acid residues 1-28 corresponding to the residues shown in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of the N-terminal amino acid residues 1-29 corresponding to the residues shown in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of the N-terminal amino acid residues 1-30 corresponding to the residues shown in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of the N-terminal amino acid residues 1-31 corresponding to the residues shown in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of the N-terminal amino acid residues 1-32 corresponding to the residues shown in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of the N-terminal amino acid residues 1-33 corresponding to the residues shown in SEQ ID NO:122.

[0282] In embodiments of any of the provided embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion that contains a deletion of one or more residues of the stalk portion of the TACI extracellular domain. In some embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion that lacks one or more consecutive C-terminal amino acid residues starting at residue 105 and up to and including amino acid residue 166, corresponding 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 are deleted.

[0283] In some embodiments, a reference (e.g., unmodified) TACI sequence contains an ECD portion having a contiguous amino acid sequence including CRD1 and / or CRD2 (e.g., CRD1 and CRD2 or only CRD2) and a fragment or portion of only the stalk sequence. Suitable stalk fragments include one or more amino acids of amino acid residues 105 to 154 of SEQ ID NO: 122. For example, the stalk fragment can consist of: amino acid residue 105 of SEQ ID NO: 122, 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.

[0284] In some embodiments, the reference (e.g., unmodified) TACI sequence lacks or is mutated at one or more potential furin cleavage sites. In some cases, the reference (e.g., unmodified) TACI sequence is the ECD or a portion thereof in which the arginine residue at position 119 is mutated, such as R119G. In some cases, the reference (e.g., unmodified) TACI sequence is the ECD or a portion thereof in which the glutamine residue at position 121 is mutated, such as Q121P. In some cases, the reference (e.g., unmodified) TACI sequence is the ECD or a portion thereof in which the arginine residue at position 122 is mutated, such as R122Q.

[0285] In some embodiments, the reference TACI sequence is the TACI ECD sequence as shown in International PCT Publication Nos. WO2000 / 067034, WO2002 / 094852, or WO2008 / 154814.

[0286] In some embodiments, the reference TACI sequence is the TACI ECD sequence having or consisting of the sequence shown in SEQ ID NO:131.

[0287] TACI ECD(CRD1 / CRD2):SEQ ID NO:131

[0288] SRVDQEER FPQGLWTGVA MRSCPEEQYW DPLLGTCMSCKTICNHQSQR TCAAFCRSLSCRKEQGKFYD HLLRDCISCA SICGQHPKQCAYFCENKLRS PVNLPPEL

[0289] In some embodiments, the reference TACI sequence is the TACI ECD sequence having or consisting of the sequence shown in SEQ ID NO:130.

[0290] TACI ECD(CRD1 / CRD2):SEQ ID NO:130

[0291] AMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLS CRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

[0292] In some embodiments, the reference TACI sequence is the 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).

[0293] TACI ECD(CRD1 / CRD2):SEQ ID NO:1

[0294] VAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSL SCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

[0295] In some embodiments, the reference TACI sequence is the extracellular domain region of TACI that consists essentially of only the CRD2 sequence and lacks or is missing the entire sequence of CRD1 and substantially all of the stalk region. Although previous studies have suggested that residues in the stalk region may contain protease cleavage sites, it is believed that at least CRD1 and CRD2 are required for the full expression and / or binding activity of TACI to its cognate ligands. For example, International PCT Publication No. WO2002 / 094852 demonstrates that a TACI molecule containing CRD1 and CRD2 but lacking the entire amino-terminal region and a partial sequence of the stalk region exhibits reduced protein degradation upon expression. 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. WO2008 / 154814, which identifies residues 13-118 or 13-108 of the extracellular region of TACI as being required for biological activity while minimizing degradation of TACI during expression. Surprisingly, it has been found herein (e.g., in Example 3) that the extracellular region of TACI consisting essentially of only CRD2 and a small portion of the stalk region exhibits significantly improved cognate binding activity compared to longer TACI molecules that contain both CRD1 and CRD2.

[0296] The present disclosure provides an immunomodulatory protein (e.g., a TACI-Fc fusion protein) comprising a TACI polypeptide, which is part of the extracellular domain (ECD) region of TACI containing CRD2, wherein the N-terminal region and CRD1 are deleted, and one or more residues of the TACI extracellular domain stalk portion are deleted, e.g., relative to the amino acid sequence shown in SEQ ID NO:122. In some embodiments, the portion of the TACI extracellular domain containing CRD2 comprises amino acid residues 71-104 corresponding to the residues shown in SEQ ID NO:122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-66 corresponding to the residues shown in SEQ ID NO:122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-67 corresponding to the residues shown in SEQ ID NO:122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-68 corresponding to the residues shown in SEQ ID NO:122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-69 corresponding to the residues shown in SEQ ID NO:122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-70 corresponding to the residues shown in SEQ ID NO:122. In embodiments of any such embodiment, the TACI polypeptide of the immunomodulatory protein lacks one or more consecutive C-terminal amino acid residues starting at residue 105 and up to and including amino acid residue 166, corresponding 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 are deleted.

[0297] In some embodiments, the immunomodulatory proteins provided herein (e.g., TACI-Fc fusion proteins) have an ECD portion in the TACI polypeptide having a contiguous amino acid sequence with TACI ECD, wherein the TACI ECD includes CRD2 (e.g., residues 71-104 of SEQ ID NO:122), but lacks the N-terminal region and CRD1, and lacks one or more residues of the TACI extracellular domain stem portion, e.g., relative to the amino acid sequence shown in SEQ ID NO:122. For example, the TACI ECD portion can consist of the following amino acid residues with respect to those shown 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 instances, the TACI ECD portion can consist of the following residues with respect to those shown in SEQID 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 instances, the TACI ECD portion can consist of the following residues with respect to those shown 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 instances, the TACI ECD portion can consist of the residues shown below with respect to 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 instances, the TACI ECD portion can consist of the residues shown below with respect to 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 that is consistent with the immunomodulatory protein provided herein, wherein such immunomodulatory protein contains a variant TACI polypeptide that is modified by one or more amino acid modifications (e.g., substitutions) described herein as compared to such TACI reference sequence.

[0298] Specifically, the TACI polypeptide provided herein is 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 provided variant TACI polypeptide is modified by one or more amino acid modifications (e.g., substitutions) described herein as compared to this reference TACI sequence.

[0299] TACI ECD sequence (CRD2): SEQ ID NO:13

[0300] SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLR S

[0301] In some embodiments, the reference TACI sequence comprises the amino acid sequence shown in SEQ ID NO:204. In some embodiments, the reference TACI sequence consists of the amino acid sequence shown in SEQ ID NO:204. In some embodiments, the reference TACI sequence comprises the amino acid sequence shown in SEQ ID NO:206. In some embodiments, the reference TACI sequence consists of the amino acid sequence shown in SEQ ID NO:206. In some embodiments, the reference TACI sequence comprises the amino acid sequence shown in SEQ ID NO:215. In some embodiments, the reference TACI sequence consists of the amino acid sequence shown in SEQ ID NO:215. In some embodiments, the reference TACI sequence comprises the amino acid sequence shown in SEQ ID NO:217. In some embodiments, the reference TACI sequence consists of the amino acid sequence shown in SEQ ID NO:217. In some embodiments, the reference TACI sequence comprises the amino acid sequence shown in SEQ ID NO:240. In some embodiments, the reference TACI sequence consists of the amino acid sequence shown in SEQ ID NO:240. In some embodiments, the reference TACI sequence comprises the amino acid sequence shown in SEQ ID NO:241. In some embodiments, the reference TACI sequence consists of the amino acid sequence shown in SEQ ID NO:241.

[0302] The provided TACI polypeptides are variant TACI polypeptides. Also provided are immunomodulatory proteins, such as TACI-Fc fusion proteins, which contain the provided variant TACI polypeptides. In embodiments of any of the provided embodiments, the variant TACI sequence has the sequence of a reference (e.g., unmodified) TACI sequence, such as any of the sequences above, but additionally contains one or more amino acid modifications, such as one or more amino acid substitutions. Specifically, provided herein are variant TACI polypeptides or specific binding fragments thereof that contain at least one affinity-modified TD domain (e.g., CRD1 and / or CRD2), the fragment containing 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 altered (e.g., increased) binding activity or affinity for one or both of APRIL or BAFF as compared to the reference (e.g., unmodified or wild-type) TACI polypeptide. In some embodiments, the binding affinity of the variant TACI polypeptide for APRIL and / or BAFF is different from the binding affinity of a reference (e.g., unmodified or wild-type) TACI polypeptide control sequence, as determined by solid-phase ELISA immunoassay, flow cytometry, or Biacore assay. The binding affinity for each cognate binding partner is independent; that is, in some embodiments, the variant TACI polypeptide has an increased binding affinity for one or both of APRIL and BAFF relative to the reference (e.g., unmodified or wild-type) TACI polypeptide, while having a decreased or unchanged binding affinity for the other of APRIL or BAFF.

[0303] In some embodiments, the variant TACI polypeptide has an enhanced binding affinity for BAFF relative to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has an enhanced binding affinity for APRIL relative to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has an enhanced binding affinity for both 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 or murine proteins. In certain embodiments, the cognate ligands BAFF and / or APRIL are human. In some embodiments, a 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%, such as 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 or wild-type) TACI polypeptide. In any embodiment, the reference (e.g., unmodified or wild-type) TACI polypeptide has the same sequence as the variant TACI polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0304] In some embodiments, for any of the foregoing embodiments, the equilibrium dissociation constant (K d ) for BAFF can be less than 1 x 10 -5 M, 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 M or 1 x 10 -12 M. In some embodiments, for any of the foregoing embodiments, the K d for BAFF is less than or about 1 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 M, or 1 x 10 -12 M. In some embodiments, for any of the foregoing embodiments, the K d for BAFF is between 1 x 10 -9 M and less than or about 1 x 10 -12 M. In some embodiments, for any of the foregoing embodiments, the K dis for or about 1x10 -9 M, for or about 2x10 -9 M, for or about 4x10 -9 M, for or about 6x10 -9 M, for or about 8x10 -9 M, for or about 1x10 -10 M, for or about 2x10 -10 M, for or about 4x10 -10 M, for or about 6x10 -10 M, for or about 8x10 -10 M, for or about 1x10 -11 M, for or about 2x10 -11 M, for or about 4x10 -11 M, for or about 6x10 -11 M, for or about 8x10 -11 M, or for or about 1x10 -12 M, or any value between any of the foregoing values. In some embodiments, the provided embodiments include variant TACI polypeptides as described above, and the K d for BAFF is reduced (higher binding affinity) by 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.

[0305] In some embodiments, the equilibrium dissociation constant (K d ) for any of the foregoing embodiments with respect to APRIL can be less than 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or 1x10 -11 M or 1x10 -12 M. In some embodiments, the K d for any of the foregoing embodiments with respect to APRIL is less than for or about 1x10 -9 M, 1x10 -10 M or 1x10 -11 M, or 1x10 -12 M. In some embodiments, the K d for any of the foregoing embodiments with respect to APRIL is between 1x10 -9 M and for or about 1x10 -12 M. In some embodiments, the K d for any of the foregoing embodiments with respect to APRIL is for or about 1x10 -9M is or is about 2x10 -9 M is or is about 4x10 -9 M is or is about 6x10 -9 M is or is about 8x10 -9 M is or is about 1x10 -10 M is or is about 2x10 - 10 M is or is about 4x10 -10 M is or is about 6x10 -10 M is or is about 8x10 -10 M is or is about 1x10 -11 M is or is about 2x10 -11 M is or is about 4x10 -11 M is or is about 6x10 -11 M is or is about 8x10 -11 M, or is or is about 1x10 -12 M, or any value between any of the foregoing values. In some embodiments, the provided embodiments include variant TACI polypeptides as described above, and for APRIL, the K d is reduced (higher binding affinity) by greater than or greater than 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.

[0306] A reference (e.g., unmodified or wild-type) TACI sequence does not necessarily have to be used as the starting composition to generate the variant TACI polypeptides described herein. Thus, the use of terms such as "modified", such as "substituted", does not mean that the present embodiments are limited to a particular method of making variant TACI polypeptides or immunomodulatory proteins containing them. Variant TACI polypeptides can be prepared, for example, by de novo peptide synthesis and thus do not necessarily require modifications such as "substitutions", i.e., changing the codons encoding the modifications (e.g., substitutions). This principle also extends to "additions" and "deletions" of amino acid residues and similarly does not imply a particular method of preparation. The methods of designing or generating variant TACI polypeptides are not limited to any particular method. However, in some embodiments, 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 the 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 from many public databases and then subjected to subsequent screening. The National Center for Biotechnology Information in the United States provides this information, and its website is publicly accessible via the Internet, like the UniProtKB database discussed previously.

[0307] Unless otherwise indicated, as throughout this disclosure, amino acid modifications in variant TACI polypeptides are designated by the amino acid position number corresponding to the position number in the reference ECD sequence shown in SEQ ID NO: 122. One of ordinary skill in the art can identify the corresponding positions of modifications (such as amino acid substitutions) in the TACI polypeptide, including the portion thereof that contains its TD (such as CRD1 and / or CRD2), such as by aligning a reference sequence (such as SEQ ID NO: 1 or 13) with SEQ ID NO: 122. Figure 9 Alignments for identifying the corresponding residues are illustrated in Figure 9 . In the modification lists throughout this disclosure, the amino acid position is indicated in the middle, where the corresponding reference (such as unmodified or 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 at that position, "del" is indicated, and if the modification is an insertion at that position, "ins" is indicated. In some cases, the insertion is listed and the amino acid position is indicated in the middle, with the corresponding reference amino acids listed before and after the number, and the identified variant amino acid insertion listed after the unmodified (such as wild-type) amino acid.

[0308] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions in a reference (such as unmodified or wild-type) TACI sequence, such as any of those described. One or more amino acid modifications, such as substitutions, can be located in the extracellular domain of the reference (such as unmodified or wild-type) TACI sequence. In some embodiments, one or more amino acid modifications (such as substitutions) are located in the CRD1 domain or a specific binding fragment thereof. In some embodiments, one or more amino acid modifications (such as 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, such as substitutions, are located in the CRD1 domain or a specific binding fragment thereof, and some of the one or more amino acid modifications, such as substitutions, are located in the CRD2 domain or a specific binding fragment thereof.

[0309] In some embodiments, the variant TACI polypeptide has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, such as substitutions, in the reference TACI sequence. The modifications (such as substitutions) can be made in the CRD1 domain or the CRD2 domain. In some embodiments, the variant TACI polypeptide has at most 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 or a specific binding fragment thereof of the reference TACI sequence. In some embodiments, the variant TACI polypeptide has at most 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 or a specific binding fragment thereof of the reference TACI sequence.

[0310] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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 as set forth in SEQ ID NO: 122 or a specific binding fragment thereof comprising the CRD1 and / or CRD2 domain. In some embodiments, the specific binding fragment comprises the CRD1 domain, e.g., the specific binding fragment comprises the sequence as set forth in amino acids 34-66 of SEQ ID NO: 122. In some cases, the CRD1 domain is the only intact CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is or comprises the CRD2 domain, e.g., the specific binding fragment comprises the sequence as set forth in amino acids 71-104 of SEQ ID NO: 122. In some cases, the CRD2 domain is the only intact CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is or comprises the CRD1 domain and the CRD2 domain, e.g., the specific binding fragment comprises amino acids 34-104 of SEQ ID NO: 122. In some embodiments, the specific binding fragment comprises a contiguous portion of the stalk domain, e.g., the specific binding fragment comprises a contiguous portion of amino acids 105-165 of SEQ ID NO: 122. In any of the embodiments, the specific binding fragment of SEQ ID NO: 122 is less than the full-length ECD as shown in SEQ ID NO: 122. In some embodiments, the specific binding fragment is as set forth in SEQ ID NO: 1. In some embodiments, the specific binding fragment is as set forth in SEQ ID NO: 13. In some embodiments, the specific binding fragment is as set forth in SEQ ID NO: 130. In some embodiments, the specific binding fragment is as set forth in SEQ ID NO: 131.

[0311] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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, such as the amino acid sequence of SEQ ID NO: 1, 13, or 122.

[0312] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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.

[0313] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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.

[0314] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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.

[0315] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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.

[0316] In some embodiments, a variant TACI polypeptide comprising one or more of the amino acid modifications (e.g., amino acid substitutions) 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.

[0317] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions at numbered 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 corresponding to reference SEQ ID NO:122 in the reference TACI polypeptide or its specific binding fragment. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions selected from 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 their conservative amino acid substitutions. In some embodiments, the reference TACI polypeptide comprises the CRD1 domain or the CRD2 domain, such as the reference TACI polypeptide as shown in SEQ ID NO:1 or SEQ ID NO:122.

[0318] In some embodiments, the amino acid substitutions occur only in the CRD2 domain. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions at numbered positions 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103 corresponding to reference SEQ ID NO:122 in the reference TACI polypeptide or its specific binding fragment. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions selected from 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 their conservative amino acid substitutions. In some embodiments, in the CRD domain, the reference TACI polypeptide comprises only the CRD2 domain but lacks the CRD1 domain, such as the reference TACI polypeptide as shown in SEQ ID NO:13. Thus, in some embodiments, the variant TACI polypeptide comprises a portion of the ECD sequence of a TACI polypeptide that comprises the CRD2 domain but lacks the CRD1 domain.

[0319] Conservative amino acid modifications, such as substitutions, are any amino acids that belong to the same amino acid class as the substituted amino acid, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids include 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).

[0320] In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 75 of the numbered positions of reference SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 75 confers increased binding to BAFF or APRIL as compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain an amino acid substitution. In some embodiments, the substituted amino acid is an acidic amino acid or amide, such as an acidic amino acid or amide that is different as 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).

[0321] In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 77 of the numbered positions of reference SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 77 confers increased binding to BAFF or APRIL as compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain an amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or 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).

[0322] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at numbered position 78 of reference 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 an aromatic amino acid different from 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).

[0323] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at numbered position 84 of reference 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).

[0324] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at numbered position 101 of reference 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).

[0325] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 102 numbered according to reference 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).

[0326] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of E74V. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of Q75E. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of K77E. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of F78Y. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of Y79F. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of L82H. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of L82P. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of R84G. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of R84L. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of R84Q. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of D85V. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of C86Y. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of A101D. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution of Y102D. In some embodiments, the variant TACI polypeptide contains two or more of any two or more of the foregoing amino acid substitutions. 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 provided embodiments, the variant TACI polypeptide comprises at least one amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

[0327] In some embodiments, the variant TACI polypeptide comprises the amino acid substitution E74V. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution K77E. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution L82H. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution L82P. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution R84G. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution R84L. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution D85V. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution A102D. In some embodiments, the variant TACI polypeptide comprises the amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide contains two or more of any two or more of the foregoing amino acid substitutions. 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 provided embodiments, the variant TACI polypeptide comprises the amino acid substitution in any of the reference TACI polypeptide sequences 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 substitution is in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence shown in SEQ ID NO:131.

[0328] 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 provided embodiments, the variant TACI polypeptide comprises the amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

[0329] In embodiments of any embodiment, a variant TACI polypeptide comprises one or more amino acid substitutions of Q75E, K77E, F78Y, R84G, R84Q, A101D, or Y102D, or any combination thereof. In some embodiments, a variant TACI polypeptide comprises any 1, 2, 3, 4, 5, or 6 of the foregoing amino acid substitutions. In some embodiments, a variant TACI polypeptide contains one of the foregoing amino acid substitutions. In some embodiments, a variant TACI polypeptide contains two of the foregoing amino acid substitutions. In some embodiments, a variant TACI polypeptide contains three of the foregoing amino acid substitutions. In some embodiments, a variant TACI polypeptide contains four of the foregoing amino acid substitutions. In some embodiments, a variant TACI polypeptide contains five of the foregoing amino acid substitutions. In some embodiments, a variant TACI polypeptide contains six of the foregoing amino acid substitutions.

[0330] In embodiments of any embodiment, one or more amino acid substitutions comprise Q75E / R84Q. In embodiments of any embodiment, one or more amino acid substitutions comprise Q75E / K77E. In embodiments of any embodiment, one or more amino acid substitutions comprise Q75E / F78Y. In embodiments of any embodiment, one or more amino acid substitutions comprise Q75E / A101D. In embodiments of any embodiment, one or more amino acid substitutions comprise Q75E / Y102D. In embodiments of any embodiment, one or more amino acid substitutions comprise F77E / F78Y. In embodiments of any embodiment, one or more amino acid substitutions comprise K77E / R84Q. In embodiments of any embodiment, one or more amino acid substitutions comprise K77E / A101D. In embodiments of any embodiment, one or more amino acid substitutions comprise K77E / Y102D. In embodiments of any embodiment, one or more amino acid substitutions comprise F78Y / R84Q. In embodiments of any embodiment, one or more amino acid substitutions comprise F78Y / A101D. In embodiments of any embodiment, one or more amino acid substitutions comprise F78Y / Y102D. In embodiments of any embodiment, one or more amino acid substitutions comprise R84Q / A101D. In embodiments of any embodiment, one or more amino acid substitutions comprise R84Q / Y102D. In embodiments of any embodiment, one or more amino acid substitutions comprise A101D / Y102D. In the provided embodiments, a variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences described, such as in the sequences shown in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131.

[0331] In some embodiments, the variant TACI polypeptides include the 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 provided embodiments, the variant TACI polypeptides include the amino acid substitutions in any of the reference TACI polypeptide sequences, such as in the sequences shown in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131.

[0332] In some embodiments, the variant TACI polypeptides include the amino acid substitutions K77E and F78Y (K77E / F78Y). In the provided embodiments, the variant TACI polypeptides include the amino acid substitutions in any of the reference TACI polypeptide sequences. 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.

[0333] In some embodiments, the variant TACI polypeptides include the amino acid substitutions K77E and Y102D (K77E / Y102D). In the provided embodiments, the variant TACI polypeptides include the amino acid substitutions in any of the reference TACI polypeptide sequences. 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.

[0334] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions F78Y and Y102D (F78Y / Y012D). In the provided embodiments, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:131.

[0335] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions K77E, F78Y and Y102D (K77E / F78Y / Y102D). In the provided embodiments, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:131.

[0336] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions Q75E / R84Q. In the provided embodiments, the variant TACI polypeptide comprises the amino acid substitutions in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQID NO:130. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:131.

[0337] In some embodiments, variant TACI polypeptides comprise any of the mutations listed in Table 1. Table 1 also provides exemplary sequences of a reference (e.g., unmodified) TACI polypeptide and of exemplary variant TACI polypeptides, along with their SEQ ID NOs. As indicated, the exact locus or residues corresponding to a given domain may vary, such as depending on the method used to identify or classify the domain. Also, in some cases, adjacent N-terminal 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, such as to ensure proper folding of the domain upon expression. Accordingly, it should be understood that the exemplification of SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain of a variant TACI polypeptide (such as the ECD domain or a portion thereof containing only CRD1 / CRD2 or 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 amino acids longer or shorter, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0338] In some embodiments, variant TACI polypeptides comprise any of the mutations (amino acid substitutions) listed in Table 1. In some instances, the mutation (amino acid substitution) is made in a reference TACI containing the amino acid sequence shown in SEQ ID NO:122. In some instances, the mutation (amino acid substitution) is made in a reference TACI containing the CRD1 and CRD2 domains of TACI, e.g., as shown in SEQ ID NO:1. In some instances, the mutation (amino acid substitution) is made in a reference TACI that has been further truncated by deleting N-terminal and C-terminal amino acid residues to retain CRD2, e.g., as shown in SEQ ID NO:13.

[0339] The use of terms such as "modified" like "substituted" or "mutated" does not mean that the present embodiments are limited to a particular method of making immunomodulatory proteins. Variant TACI polypeptides can be prepared, for example, by de novo peptide synthesis and thus do not necessarily require modification such as "substitution", i.e., changing the codon encoding the modification (e.g., substitution). This principle also extends to "addition" and "deletion" of amino acid residues and similarly does not imply a particular method of preparation. The method of designing or generating vTDs is not limited to any particular method. However, in some embodiments, wild-type or unmodified TD-encoding nucleic acids are mutagenized from wild-type or unmodified TD genetic material and screened for the desired specific binding activity, such as binding affinity, and / or alteration of NF-κB regulation or other functional activity. In some embodiments, vTDs are synthesized de novo using protein or nucleic acid sequences available from many public databases and then subjected to subsequent screening. This information is provided by the National Center for Biotechnology Information, the website of which is publicly accessible via the Internet, as is the case with the UniProtKB database.

[0340] In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence containing CRD1 and CRD2, such as the variant TACI polypeptide shown in any of SEQ ID NOs: 2-12, 21, 22, 101-120. 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 of SEQ ID NOs: 2-12, 21, 22, 101-120, and wherein amino acid modifications (e.g., substitutions) that are 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 of SEQ ID NOs: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds to BAFF, APRIL, or the BAFF / APRIL heterotrimer, and wherein the continuous sequence contains amino acid modifications (e.g., substitutions) that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0341] In some embodiments, the variant TACI polypeptide consists of or consists essentially of the variant TACI extracellular domain (ECD) sequence shown in any 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 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 of SEQ ID NOs: 2-12, 21, 22, 101-120, and wherein amino acid modifications (e.g., substitutions) that are not present in the 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 of SEQ ID NOs: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds to BAFF, APRIL, or the APRIL / BAFF heterotrimer, and wherein the continuous sequence contains amino acid modifications (e.g., substitutions) that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0342] In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence that contains CRD2 of a reference TACI polypeptide but lacks CRD1, such as the variant TACI polypeptides shown in any 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 with any of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and wherein amino acid modifications (e.g., substitutions) that are 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 of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or the BAFF / APRIL heterotrimer, and wherein the continuous sequence contains amino acid modifications (e.g., substitutions) that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0343] In some embodiments, the variant TACI polypeptide consists of or consists essentially of the sequence shown in any 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 with any of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and wherein amino acid modifications (e.g., substitutions) that are not present in the 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 of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or the BAFF / APRIL heterotrimer, and wherein the continuous sequence contains amino acid modifications (e.g., substitutions) that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] In some embodiments, the variant TACI polypeptide comprises the sequence shown in SEQ ID NO:107. In some embodiments, the variant TACI polypeptide consists essentially of the sequence shown in SEQ ID NO:107. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO:107.

[0348] In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence shown in any one of SEQ ID NO: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 with any one of SEQ ID NO:37 - 47, 56 or 57, and wherein amino acid modifications (e.g., substitutions) that are not present in the reference (e.g., unmodified or wild-type) TACI are retained. Also provided herein are nucleic acids comprising the sequence shown in any one of SEQ ID NO: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, such as at least 96% identity, 97% identity, 98% identity or 99% identity with any one of SEQ ID NO:37 - 47, 56 or 57.

[0349] In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence shown in any 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 with any of SEQ ID NOs: 49-55 or 58-70, and wherein amino acid modifications (e.g., substitutions) that are not present in the reference (e.g., unmodified or wild-type) TACI are retained. Also provided herein are nucleic acids containing the sequence shown in any 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, such as at least 96% identity, 97% identity, 98% identity, or 99% identity with any of SEQ ID NOs: 549-55 or 58-70.

[0350]

[0351]

[0352]

[0353] In some embodiments, also provided herein are TACI ECD fusion sequences, wherein any of the above TACI ECD sequences is linked or fused to a multimerization domain, such as any of the domains described herein.

[0354] The interaction of two or more polypeptides of an immunomodulatory protein can be facilitated by their direct or indirect linkage to any moiety or other polypeptide that is itself capable of interacting to form a stable structure. For example, individual polypeptide chains can be joined by multimerization, wherein multimerization of the polypeptides is mediated by a multimerization domain. Generally, the multimerization domain provides for the formation of stable protein-protein interactions between a first polypeptide and a second polypeptide.

[0355] In some embodiments, two or more individual polypeptides of an immunomodulatory protein can be joined by multimerization, such as into a dimer, trimer, tetramer, or pentamer molecule. In some cases, the individual polypeptides are the same. For example, a trimer molecule can be formed from three copies of the same individual polypeptide. In other instances, a tetramer molecule is generated from four copies of the same individual polypeptide. In additional instances, a pentamer molecule is generated from five copies of the same individual polypeptide. A multimerization domain can be a domain that promotes dimerization, trimerization, tetramerization, or pentamerization of polypeptide chains.

[0356] In some embodiments, the immunomodulatory protein forms a multimer, such as a dimer. In some embodiments, the dimer is a homodimer, wherein the two polypeptides of the immunomodulatory protein are the same. In some embodiments, the dimer is a heterodimer, wherein the two polypeptides of the immunomodulatory protein are different.

[0357] In some embodiments, the multimerization domain includes any domain capable of forming stable protein-protein interactions. The multimerization domains can interact via: immunoglobulin sequences (e.g., the Fc domain; see, e.g., International Patent Publication Nos. WO 93 / 10151 and WO 2005 / 063816 US; US Publication No. 2006 / 0024298; US Patent No. 5,457,035); leucine zippers (e.g., from the nuclear transforming proteins fos and jun or the proto-oncogene c-myc or from the general control of nitrogen (GCN4)) (e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz et al., (1989) Science, 243:1695-1699); hydrophobic regions; hydrophilic regions; or free sulfhydryl groups that form intermolecular disulfide bonds between chimeric molecules of homomultimers or heteromultimers. In addition, the multimerization domain can include an amino acid sequence containing a protrusion that is complementary to an amino acid sequence containing a socket, such as described, for example, in US Patent No. 5,731,168; International Patent Publication Nos. WO 98 / 50431 and WO 2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such multimerization regions can be engineered such that the steric interactions not only promote stable interactions but also further promote the formation of heterodimers relative to homodimers in a mixture of chimeric monomers. Generally, the protrusion is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Optionally, a compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0358] The TACI polypeptide sequence (e.g., variant TACI polypeptide sequence) can be joined at any position, but is typically joined to the N-terminus or C-terminus of the multimerization domain via its N-terminus or C-terminus to form a chimeric polypeptide. The linkage can be direct or indirect via a linker. In addition, the chimeric polypeptide can be a fusion protein or can be formed by chemical linkage, 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 the 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, 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 certain 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.

[0359] The polypeptide multimer contains two chimeric proteins that 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 instances, when the multimerization domain is a polypeptide, a gene fusion encoding the TACI polypeptide sequence (e.g., variant TACI polypeptide sequence) and the multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion protein can be expressed in a host cell transformed with the recombinant expression vector and allowed to assemble into a multimer in which the multimerization domains interact to form a multivalent polypeptide. A heterobifunctional linker can be used to effect the chemical linkage of the multimerization domain to the TACI polypeptide (e.g., variant TACI polypeptide).

[0360] The resulting chimeric polypeptides (such as fusion proteins, and multimers formed therefrom) can be purified by any suitable method, such as, for example, affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into a cell, homodimers and heterodimers are formed. The expression conditions can be adjusted such that the formation of heterodimers is favored over the formation of homodimers.

[0361] In some embodiments, the multimerization domain is the Fc region of an immunoglobulin.

[0362] In some embodiments, the multimerization domain is the Fc region of an immunoglobulin (e.g., IgG1), where the fusion protein is TACI-Fc comprising (1) a TACI sequence that comprises or consists of any provided TACI ECD sequence; and (2) an immunoglobulin Fc region. Accordingly, the provided embodiments include TACI-Fc fusion proteins that contain (1) a TACI sequence that contains or consists of any of the foregoing TACI ECD polypeptide sequences, such as variant TACI polypeptides; and (2) an immunoglobulin Fc region.

[0363] In some embodiments, provided herein is a TACI-Fc fusion sequence that contains (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 that contains (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.

[0364] In some embodiments, the TACI-Fc fusion is a variant TACI-Fc fusion containing or consisting of any of the foregoing variant TACI polypeptides and an immunoglobulin Fc region.

[0365] In some embodiments, provided herein is a variant TACI-Fc fusion sequence that comprises (1) a TACI ECD sequence containing CRD1 and CRD2, e.g., a TACI sequence comprising any one of the sequences shown in SEQ ID NO:2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region. In some embodiments, provided herein is a variant TACI-Fc fusion sequence that comprises (1) a TACI ECD sequence containing CRD1 and CRD2, e.g., a TACI sequence consisting of or consisting essentially of any one of the sequences shown in SEQ ID NO:2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region.

[0366] In some embodiments, provided herein are variant TACI-Fc fusion sequences that comprise (1) a TACI ECD sequence that contains CRD2 but lacks the CRD1 domain, such as a TACI sequence that contains any one of the sequences shown in SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and (2) an immunoglobulin Fc region. In some embodiments, provided herein are variant TACI-Fc fusion sequences that comprise (1) a TACI ECD sequence that contains the CRD2 domain but lacks the CRD1 domain, such as a TACI sequence that consists of or consists essentially of any one of the sequences shown in SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and (2) an immunoglobulin Fc region.

[0367] In the provided TACI-Fc embodiments, the immunoglobulin Fc region can be the wild-type Fc of an immunoglobulin, such as IgG1 Fc. In some cases, the Fc region can be a variant Fc that lacks effector function (also referred to as "effectorless Fc"). Exemplary Fc regions and their variants in the provided TACI-Fc fusion proteins are described below.

[0368] In some embodiments, the Fc is murine or human Fc. In some embodiments, the Fc is a mammalian or human IgGl, IgG2, IgG3, or IgG4 Fc region.

[0369] In some embodiments, the Fc region is or comprises any one of the sequences shown in 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 IgG1, such as any of the IgG1s 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 IgG2, such as any IgG2 shown in SEQ ID NO: 138 or 219. In some embodiments, the Fc region is or is derived from IgG4, such as any IgG4 shown in SEQ ID NOs: 139, 140, or 220. In some embodiments, the Fc region in the Fc fusion proteins provided herein can 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% identity to any of the above Fc regions.

[0370] In some embodiments, the Fc is derived from IgG1, such as human IgG1. In some embodiments, the Fc is the IgG1 Fc shown in SEQ ID NO:71, which has the isotype 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 exhibits an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:71. In other embodiments, the Fc is an IgG1 Fc containing the amino acids of the human G1m1 isotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, e.g., as shown in SEQ ID NO:81. Thus, in some cases, the Fc provided herein may contain the amino acid substitutions E356D and M358L to reconstruct the residues of isotype G1 m1. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO:81 or exhibits an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:81.

[0371] In some embodiments, the Fc region has the amino acid sequence shown in SEQ ID NO:81.

[0372] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:81)

[0373] In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the Fc region consists of the amino acid sequence shown in SEQ ID NO:81.

[0374] 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.

[0375] In some embodiments, the Fc is derived from IgG2, such as human IgG2. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 138 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 higher sequence identity to SEQ ID NO: 138. In some embodiments, the Fc region is an IgG2 Fc region comprising the sequence shown in SEQ ID NO: 138. In some embodiments, the Fc region is an IgG2 Fc region comprising the sequence shown in SEQ ID NO: 219.

[0376] In some embodiments, the Fc is derived from IgG4, such as human IgG4. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 139 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 higher sequence identity to SEQ ID NO: 139. In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced with the CH3 domain of human IgG1 and exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 and CH2 domains of human IgG1, or an antibody in which the arginine at position 409 indicated in the EU index proposed by Kabat et al. of human IgG4 is replaced with lysine and exhibits inhibited aggregate formation (see, for example, U.S. Patent No. 8,911,726). In some embodiments, the Fc is an IgG4 containing the S228P mutation, which has been shown to prevent recombination between a therapeutic antibody and endogenous IgG4 by Fab arm exchange (see, for example, Labrijin et al. (2009) Nat. Biotechnol., 27(8): 767-71). In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 140 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 higher sequence identity to SEQ ID NO: 140. In some embodiments, the Fc region is the IgG4 Fc region shown in SEQ ID NO: 140. In some embodiments, the Fc region is the IgG4 Fc region shown in SEQ ID NO: 220.

[0377] In some embodiments, the Fc region is a variant Fc region in which the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or render the Fc inert with respect to Fc effector functions. Exemplary non-effector or inert mutations include those described herein.

[0378] In some embodiments, the Fc region contains one or more modifications that alter (e.g., reduce) one or more of its normal functions. Generally, in addition to having antigen-binding ability (the primary function of immunoglobulins), the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC). In addition, the FcRn sequence present in the Fc region increases the in vivo half-life by binding to the FcRn receptor in the body to play a role in regulating the IgG level in serum. In some embodiments, such functions can be reduced or altered in the Fc for use with the provided Fc fusion proteins.

[0379] In some embodiments, one or more amino acid modifications can be introduced into the Fc region, thereby generating Fc region variants. In some embodiments, the Fc region variants have reduced effector functions. There are many examples of changes or mutations in the Fc sequence that can alter effector functions. For example, WO 00 / 42072, WO2006019447, WO2012125850, WO2015 / 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 those publications are specifically incorporated herein by reference.

[0380] In some embodiments, the provided immunomodulatory protein comprises an Fc region that exhibits reduced effector function, making it an ideal candidate for applications in which the in vivo half-life of the immunomodulatory protein is important but certain effector functions (e.g., 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, an Fc receptor (FcR) binding assay can be performed to ensure that the immunomodulatory protein lacks FcR binding (and thus potentially lacks ADCC activity), but retains FcRn binding ability. The main cells that mediate ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing the ADCC activity of a target molecule are described in U.S. Patent 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); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Effector cells suitable for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the immunomodulatory protein cannot bind C1q and thus lacks CDC activity. See, e.g., the 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, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. 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, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0381] Immunomodulatory proteins with reduced effector function include those antibodies having substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region according to EU numbering (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0382] In some embodiments, the Fc region of the immunomodulatory protein has an Fc region in which any one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as numbered according to EU numbering) are replaced with amino acids different from those in the native Fc region. Alterations of such Fc regions include, for example, alterations such as deglycosylation mutations (N297A and N297Q), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / 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, as described in Current Opinion in Biotechnology (2009) 20(6), 685-691; alterations such as G236R / L328R, L235G / G236R, N325A / L328R, and N325L / L328R, as described in WO 2008 / 092117; amino acid insertions at positions 233, 234, 235, 237 (as numbered according to EU numbering); and alterations at the sites described in WO 2000 / 042072.

[0383] Certain Fc variants with improved or reduced FcR binding are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, WO2006019447, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0384] In some embodiments, there are provided immunomodulatory proteins comprising a variant Fc region that contains one or more amino acid substitutions that increase the half-life and / or improve binding to the neonatal Fc receptor (FcRn). US2005 / 0014934A1 (Hinton et al.) or WO2015107026 describe antibodies with increased half-life and improved binding to FcRn. These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 of the Fc region according to EU numbering, such as those having a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826).

[0385] In some embodiments, the Fc region of the immunomodulatory protein contains one or more amino acid substitutions according to EU numbering of C220S, C226S, and / or C229S. In some embodiments, the Fc region of the immunomodulatory protein contains one or more amino acid substitutions of R292C and V302C. See also 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, which relate to other examples of Fc region variants.

[0386] In some embodiments, changes are made to the Fc region that result in reduced C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0387] In some embodiments, a variant Fc region comprising one or more amino acid modifications (such as 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 isotype 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 the amino acids of the human G1m1 isotype, such as residues Asp (D) and Leu (L) at positions 356 and 358, for example as shown in SEQ ID NO:81. Thus, in some cases, the variant Fc is derived from the amino acid sequence shown in SEQ ID NO:81.

[0388] In some embodiments, the Fc region lacks the C-terminal lysine at position 232 corresponding to the wild-type or unmodified Fc shown in SEQ ID NO:71 or 81 (corresponding to K447del according to EU numbering).

[0389] In some embodiments, the variant Fc region comprises the C5S amino acid modification of the wild-type or unmodified Fc region numbered according to SEQ ID NO:71 (corresponding to C220S according to EU numbering).

[0390] In some embodiments, the Fc region is a variant Fc containing at least one amino acid substitution, which is N82G according to the numbering of SEQ ID NO:71 (corresponding to N297G according to EU numbering). In some embodiments, the Fc also contains at least one amino acid substitution, namely R77C or V87C according to the numbering of SEQ ID NO:71 (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region also comprises the C5S amino acid modification numbered according to 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: N297G and one or more of the following amino acid modifications according to EU numbering C220S, R292C or V302C (corresponding to N82G of reference SEQ ID NO:71 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.

[0391] In some embodiments, the variant Fc contains the amino acid substitutions L234A / L235E / G237A according to EU numbering. In some embodiments, the variant Fc contains the amino acid substitutions A330S / P331S according to EU numbering. In some embodiments, the variant Fc contains the amino acid substitutions L234A / L235E / G237A / A330S / P331S (Gross et al. (2001) Immunity 15:289). In some embodiments, the variant Fc contains the sequence shown in SEQ ID NO:175. In some embodiments, the variant Fc contains the sequence shown in SEQ ID NO:176. In some embodiments, the Fc region used in the constructs provided herein may further lack the C-terminal lysine residue.

[0392] In some embodiments, the Fc region is a variant Fc that includes the 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 substituting the cysteine residue at position 220 according to EU numbering with a serine residue (C220S). 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 isotypes shown in SEQ ID NO:71 or SEQ ID NO:81, respectively. In some embodiments, the Fc region may further lack the C-terminal lysine residue. In some embodiments, the variant Fc region contains one or more amino acid modifications C220S, L234A, L235E, or G237A, e.g., the Fc region contains the sequence shown in SEQ ID NO:73, 75, 83, or 136. In some embodiments, the variant Fc contains the sequence shown in SEQ ID NO:73. In some embodiments, the variant Fc contains the sequence shown in SEQ ID NO:75. In some embodiments, the variant Fc contains the sequence shown in SEQ ID NO:83. In some embodiments, the variant Fc contains the sequence shown in SEQ ID NO:136.

[0393] In some embodiments, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:73.

[0394] EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:73)

[0395] In some embodiments, the Fc region is IgG1 Fc, but does not contain the 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).

[0396] In some embodiments, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:221.

[0397] DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:221)

[0398] In some embodiments, the Fc region is a variant Fc region comprising one or more amino acid modifications C220S, E233P, L234V, L235A, G236del or S267K, such as the Fc region comprising the sequence shown in SEQ ID NO:134. In some embodiments, the Fc region lacks the C-terminal lysine at position 232 corresponding to the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del of EU numbering). In some embodiments, the Fc region comprises the sequence shown in SEQ ID NO:137.

[0399] In some embodiments, the Fc region is a variant Fc region comprising one or more amino acid modifications C220S, R292C, N297G, V302C. In some embodiments, the Fc region lacks the C-terminal lysine at position 232 corresponding to the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del of EU numbering). An exemplary variant Fc region is shown in SEQ ID NO:135.

[0400] 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 at position 232 corresponding to the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del of EU numbering). An exemplary variant Fc region is shown in SEQ ID NO:137.

[0401] Examples of such Fc regions contained in the immunomodulatory polypeptides are shown in Table 2.

[0402]

[0403]

[0404] 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 the sequence shown in any one of SEQ ID NOs in Table 2.

[0405] For example, the variant Fc region can be an effectorless Fc that exhibits reduced effector activity compared to the wild-type IgG1 shown in SEQ ID NO:71 or SEQ ID NO:81. In some embodiments, the variant Fc comprises the amino acid sequence shown in any one of SEQ ID NO: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 higher sequence identity with any one of SEQ ID NO:75, 82, 83, 134, 73, 135, 136 or 137. In some embodiments, the variant Fc has the sequence shown in SEQ ID NO:73. In an embodiment, when produced and expressed from a cell, the provided immunomodulatory protein (e.g., TACI-Fc fusion) is a homodimer containing two identical polypeptide chains.

[0406] In some embodiments, the immunomodulatory protein comprises a first immunomodulatory Fc fusion polypeptide and a second immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are different. In some embodiments, the first Fc polypeptide fusion comprises an Fc region and one or more variant TACI polypeptide sequences, and the second polypeptide fusion comprises 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.

[0407] In some embodiments, the Fc domain of one or both of the first and second immunomodulatory Fc fusion polypeptides comprises a modification (e.g., a substitution) such that the interface of the Fc molecule is modified to favor and / or promote heterodimerization. Methods for promoting Fc chain heterodimerization include mutagenesis of the Fc region, such as by including a set of "knob-into-hole" mutations or mutations that affect Fc electrostatic steering, to favor attractive interactions between different polypeptide chains. In some embodiments, the Fc region of the heterodimer molecule can additionally contain one or more other Fc mutations, such as any of the mutations described above. In some embodiments, the heterodimer molecule contains an Fc region with mutations that reduce effector function. In some embodiments, such Fc regions contain mutations C220S, L234A, L235E, and / or G237A according to EU numbering. In some embodiments, any of the above mutations in the Fc backbone can be made in an isotype 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 backbone can be made in an isotype containing residues Asp (D) and Leu (L) at positions 356 and 358 according to EU numbering.

[0408] In some embodiments, the modification comprises introducing a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide such that the protrusion can be positioned in the cavity to promote the complexation of the first and second Fc-containing polypeptides. The amino acids targeted for replacement and / or modification to create a protrusion or cavity in the polypeptide are typically interface amino acids that interact with or contact one or more amino acids in the interface of the second polypeptide.

[0409] In some embodiments, the first polypeptide modified to contain a protruding (stalk) amino acid comprises replacing a native 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 thus be positioned in a complementary cavity (socket) in the adjacent interface of the second polypeptide. Most commonly, the replacing amino acid has a larger side chain volume than the original amino acid residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify those ideal replacing amino acids that give rise to the protrusion. In some embodiments, the replacing 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 instances, the original residue identified for replacement is an amino acid residue having a small side chain, such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0410] In some embodiments, the second polypeptide modified to contain a cavity (socket) is a polypeptide that comprises replacing a native or original amino acid with an amino acid having at least one side chain that is recessed from the interface of the second polypeptide and can thus accommodate a corresponding protrusion from the interface of the first polypeptide. Most commonly, the replacing amino acid has a smaller side chain volume than the original amino acid residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify those ideal replacing residues for forming the cavity. Generally, the replacing residues used to form the cavity are naturally occurring amino acids and include, for example, alanine (A), serine (S), threonine (T), and valine (V). In some instances, the original amino acid identified for replacement is an amino acid having a large side chain, such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0411] For example, the CH3 interface of human IgG1 involves masking located from each surface Sixteen residues on each domain on the four anti-parallel β-strands (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. Patent No. 5,731,168). Modifications to the CH3 domain for creating a protrusion or a cavity have been described, for example, in U.S. Patent No. 5,731,168, International Patent Applications WO98 / 50431 and WO 2005 / 063816, and Ridgway et al., (1996) Prot. Engin., 9:617-621. In some instances, modifications to the CH3 domain for creating a protrusion or a cavity generally target residues located on the two central anti-parallel β-strands. The aim is to minimize the risk that the resulting protrusion can be accommodated by protruding into the surrounding solvent rather than by the complementary cavity in the partner CH3 domain.

[0412] In some embodiments, the heterodimeric molecule contains a T366W mutation in the CH3 domain of the "stalk chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "socket chain". In some cases, additional inter-chain disulfide bonds between the CH3 domains can also be used (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing a Y349C mutation in the CH3 domain of the "stalk" or "socket" chain and an E356C or S354C mutation in the CH3 domain of the other chain. In some embodiments, the heterodimeric molecule contains S354C, T366W mutations in one of the two CH3 domains, and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. For example, the stalk Fc may contain the sequence shown in SEQ ID NO:89 (containing S354C and T366W), and the socket Fc as shown in SEQ ID NO:90 (containing the mutations Y349C, T366S, L368A, and Y407V). In some embodiments, the heterodimeric molecule contains 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 contains 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 contains 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 stalk-socket technologies are known in the art, such as those described in EP 1 870 459 A1.

[0413] In some embodiments, an Fc variant containing a CH3 protrusion (stalk) or cavity (socket) modification can engage a multi-domain immunomodulatory polypeptide at any position, but typically via its N-terminus or C-terminus, engaging the N-terminus or C-terminus of one or more TACI polypeptide sequences (such as variant TACI polypeptide sequences), such as for forming a fusion polypeptide. The linkage can be direct or indirect via a linker. Typically, the stalk and socket molecules are produced by co-expressing a first immunomodulatory polypeptide linked to an Fc variant containing a CH3 protrusion modification and a second immunomodulatory polypeptide linked to an Fc variant containing a CH3 cavity modification.

[0414] Exemplary sequences of the pestle and mortar Fc polypeptides are shown in SEQ ID NO:128 and 129, respectively. In some embodiments, the C-terminal lysine at position 232 of the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del of EU numbering) is absent in the pestle or mortar Fc region. Exemplary sequences of the pestle and mortar Fc polypeptides are shown in SEQ ID NO:89 and 90, respectively.

[0415] In some embodiments, a single polypeptide of a multi-domain polypeptide or a single polypeptide of a single-domain polypeptide is linked to a multimerization domain to form an immunomodulatory protein that is a trimer, tetramer, or pentamer. In some embodiments, the single polypeptides of such a molecule 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.

[0416] 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 instances, COMP is or contains the amino acid sequence 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 higher sequence identity to SEQ ID NO:146.

[0417] 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 shown in SEQ ID NO:147 (e.g., amino acids 343-375 of full-length VASP, Uniprot accession number P50552) or a sequence having about 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:147.

[0418] In some embodiments, the TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) is conjugated to a multimerization domain (e.g., an Fc region) via a linker (such as a peptide linker). In some embodiments, the length of the peptide linker can be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue, but the length does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue.

[0419] In some embodiments, the linker is (single-letter amino acid code): GGGGS ("4GS"; SEQ ID NO:77) or a multimer of the 4GS linker, such as a repeat sequence of 2, 3, 4, or 5 4GS linkers. 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 may further include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or its multimer). In some embodiments, the linker (single-letter amino acid code) is GSGGGGS (SEQ ID NO:74) or GGGGSAA (SEQ ID NO:80). In some instances, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO:133). In some instances, the linker is as shown in SEQ ID NO:194 or 195.

[0420] In some embodiments, the TACI polypeptide (such as a variant TACI polypeptide) is directly linked to the Fc sequence. In some embodiments, the TACI polypeptide (such as a 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., a variant TACI polypeptide) and 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 peptide linker has at least one amino acid residue, but the length does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. Exemplary linkers include any linker described herein.

[0421] 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 two identical polypeptide fusions forms a covalent disulfide bond, resulting in a dimer molecule containing two TACI polypeptides (e.g., two variant TACI polypeptides).

[0422] In some embodiments, there is provided a TACI-Fc fusion protein comprising, in order, a TACI polypeptide (e.g., any of those described above), a linker, and an Fc region. In some embodiments, each TACI polypeptide of the TACI-Fc fusion protein is a truncated wild-type TACI polypeptide, such as any of the polypeptides described. In some embodiments, the TACI polypeptide of the TACI-Fc fusion is as shown in SEQ ID NO:13. The linker can be any linker as 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 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.

[0423] 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.

[0424] SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:171)

[0425] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:172.

[0426] SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:172)

[0427] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:196 and encodes the sequence shown in SEQ ID NO:207.

[0428] In some embodiments, the TACI polypeptide is a variant TACI polypeptide. In some embodiments, a variant TACI-Fc fusion protein is provided that sequentially comprises a variant TACI polypeptide (e.g., any of those described above), a linker, and an Fc region. In some embodiments, the TACI polypeptides of the TACI Fc fusion protein are all variant TACI polypeptides, such as any of the polypeptides described. In some embodiments, the variant TACI of the variant TACI Fc fusion is shown as any one of SEQ ID NO:2-12, 21, 22, or 101-120. In some embodiments, the variant TACI of the variant TACI Fc fusion is shown as any one of SEQ ID NO: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 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.

[0429] In some embodiments, the TACI-Fc fusion protein has the amino acid sequence shown as any one of SEQ ID NO:167-170, 200, or 222-237.

[0430] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 167.

[0431] SLSCRKEQGEYYDHLLRDCISCASICGQHPKQCADFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:167)

[0432] In some embodiments, the TACI-Fc fusion is encoded by the sequence shown in SEQ ID NO: 211.

[0433] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 168.

[0434] SLSCRKEQGEYYDHLLRDCISCASICGQHPKQCADFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:168)

[0435] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 169.

[0436] SLSCRKEEGKFYDHLLQDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:169)

[0437] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:170.

[0438] SLSCRKEEGKFYDHLLQDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:170)

[0439] In some embodiments, the TACI-Fc fusion protein contains multiple copies of the TACI polypeptide sequence (e.g., 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 linked or may be indirectly linked via a linker (such as including any of the peptide linkers described). In such instances, one of the TACI polypeptide sequences is joined or linked to the Fc region, such as joined or linked 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 individually joined to the N-terminus or C-terminus of the Fc region. The linkage to the Fc region can be direct or indirect (via a linker, such as a peptide linker, including any of the linkers described above).

[0440] In some embodiments, the TACI polypeptide sequence (e.g., variant TACI polypeptide sequence) may be arranged in tandem in the fusion protein (hereinafter referred to as a "tandem" Fc fusion construct). 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, which 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, thereby producing a dimer molecule containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0441] In some embodiments, provided are TACI-Fc fusion proteins that sequentially comprise a TACI polypeptide (e.g., any of those described above), a linker, another TACI polypeptide (e.g., any of those described above), and an Fc region. In some embodiments, each TACI polypeptide of the TACI-Fc fusion protein is a truncated wild-type TACI polypeptide, such as any of the polypeptides described. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is as shown in SEQ ID NO:13. In some embodiments, each TACI polypeptide of the TACI-Fc fusion protein is a variant TACI polypeptide, such as any of the polypeptides described. In some embodiments, each TACI polypeptide in the TACI-Fc fusion is a variant TACI shown in any one of SEQ ID NO:2-12, 21, 22, or 101-120. In some embodiments, each TACI polypeptide in the TACI-Fc fusion is a variant TACI shown in any one of SEQ ID NO:14-20, 23-35, 92-100, or 177-192. The linker can be any of the linkers described. In some embodiments, the linker is GSGGGGS (SEQ ID NO:74). The Fc region can be any of the Fc regions 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.

[0442] In some embodiments, the TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) can be separated by the Fc region in the fusion protein, wherein the Fc region is located between two TACI polypeptide sequences (hereinafter referred to as a "barbell-shaped" 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, interactions between the Fc regions of two identical polypeptide fusions form covalent disulfide bonds, resulting in a dimeric molecule containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0443] In some embodiments, provided are TACI-Fc fusion proteins comprising, in order, a TACI polypeptide (e.g., any of those described above); a linker; an Fc region; a linker; and another TACI polypeptide (e.g., any of those described above). In some embodiments, each TACI polypeptide of the TACI-Fc fusion protein is a truncated wild-type TACI polypeptide, such as any of the polypeptides described. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is as shown in SEQ ID NO:13. In some embodiments, each TACI polypeptide of the TACI-Fc fusion protein is a variant TACI polypeptide, such as any of the polypeptides described. In some embodiments, each TACI polypeptide in the TACI-Fc fusion is a variant TACI shown in any 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 shown in any of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linkers can be any of the linkers described and can 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 of the Fc regions 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 a variant Fc 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.

[0444] In some embodiments, provided are TACI-Fc fusion proteins that are dimers formed by linking two identical TACI polypeptides (e.g., variant TACI polypeptides) to an Fc domain. In some embodiments, any of the provided TACI-Fc fusion polypeptides, e.g., the same species (also referred to as copies) of variant TACI-Fc fusion polypeptides 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 the same. To produce a homodimeric Fc molecule, the Fc region is an Fc region capable of forming a homodimer with a matching Fc region by co-expressing a single Fc region in a cell. In some embodiments, dimerization is mediated by covalent disulfide bonds formed between the Fc regions of the polypeptide fusion.

[0445] Nucleic acid molecules encoding immunomodulatory proteins are also provided. In some embodiments, to produce an immunomodulatory protein, the nucleic acid molecule encoding the immunomodulatory protein is inserted into a suitable expression vector. The resulting immunomodulatory protein can be expressed in a host cell transformed with the expression, wherein assembly between the Fc domains occurs through intermolecular disulfide bonds formed between the Fc moieties, thereby producing a dimer, such as a bivalent immunomodulatory protein.

[0446] Nucleic acid molecules encoding TACI-Fc fusion proteins (such as variant TACI-Fc fusion proteins) are also provided. In some embodiments, to produce an Fc fusion protein, the nucleic acid molecule encoding the TACI-Fc fusion protein (such as variant TACI-Fc fusion protein) is inserted into a suitable expression vector. The resulting TACI-Fc fusion protein, such as variant TACI-Fc fusion protein, can be expressed in a host cell transformed with the expression vector, wherein assembly between the Fc domains occurs through intermolecular disulfide bonds formed between the Fc moieties to obtain a dimer, such as a bivalent TACI-Fc fusion protein. The resulting Fc fusion protein can be easily purified by affinity chromatography through a Protein A or Protein G column. To generate a heterodimer, additional purification steps may be required. For example, when two nucleic acids encoding different immunomodulatory proteins are transformed into cells, the formation of heterodimers must be achieved biochemically because immunomodulatory proteins carrying Fc domains will also be expressed as disulfide-linked homodimers. Thus, homodimers can be reduced under conditions that favor the disruption of intermolecular disulfide bonds but not intramolecular 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 this mixture are separated by chromatographic techniques. Alternatively, using the described pestle and mortar structure method, the formation of this type of heterodimer can be shifted by genetically engineering and expressing an immunomodulatory protein containing an Fc fusion molecule that contains one or more TACI variants.

[0447] In an embodiment, when produced and expressed from cells, the provided immunomodulatory proteins, such as TACI-Fc (such as variant TACI-Fc), are homodimers containing two identical polypeptide chains. Figure 8A and Figure 8B The structure of exemplary TACI-Fc fusion proteins provided herein is described.

[0448] This document provides the TACI(26)-Fc_73 homodimer of two identical variant TACI-Fc fusion proteins, which contain the variant of the cysteine-rich domain 2 (CRD2) of TACI shown in SEQ ID NO:26, and are designed to neutralize the B cell-stimulating activities of APRIL and BAFF. The TACI(26)-Fc_73 homodimer is a dimer composed of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, and each chain carries a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO:167.

[0449] This document provides the TACI(26)-Fc_81 homodimer of two identical variant TACI-Fc fusion proteins, which contain the variant of the cysteine-rich domain 2 (CRD2) of TACI shown in SEQ ID NO:26, and are designed to neutralize the B cell-stimulating activities of APRIL and BAFF. The TACI(26)-Fc_81 homodimer is a dimer composed of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, and each chain carries a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO:168.

[0450] This document provides the TACI(27)-Fc_73 homodimer of two identical variant TACI-Fc fusion proteins, which contain the variant of the cysteine-rich domain 2 (CRD2) of TACI shown in SEQ ID NO:27, and are designed to neutralize the B cell-stimulating activities of APRIL and BAFF. The TACI(27)-Fc_73 homodimer is a dimer composed of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, and each chain carries a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO:169.

[0451] This document provides the TACI(27)-Fc_81 homodimer of two identical variant TACI-Fc fusion proteins, which contain the variant of the cysteine-rich domain 2 (CRD2) of TACI shown in SEQ ID NO:27, and are designed to neutralize the B cell-stimulating activities of APRIL and BAFF. The TACI(27)-Fc_81 homodimer is a dimer composed of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, and each chain carries a variant TACI CRD2 domain human Fc fusion as shown in SEQ ID NO:170.

[0452] In some embodiments, the provided TACI-Fc (e.g., variant TACI-Fc) fusion proteins, such as their homodimers, exhibit an IC for neutralizing BAFF of less than 400 pM50 。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 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. In some embodiments, the IC for neutralizing BAFF 50 is or is 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 between any of the foregoing values.

[0453] In some embodiments, the provided TACI-Fc (e.g., variant TACI-Fc) fusion protein, such as its homodimer, exhibits an IC for neutralizing APRIL that is less than 400 pM 50 。In 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 10 pM, 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 for neutralizing APRIL 50is for 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 between any of the foregoing values.

[0454] III. Nucleic Acids, Vectors and Methods for Producing Polypeptides or Cells

[0455] Provided herein are isolated or recombinant nucleic acids, collectively referred to as "nucleic acids", which encode 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 to express 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 DNA and include mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleic acids provided herein are generally DNA molecules and are generally double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA and hybrid DNA / RNA nucleic acids or combinations thereof containing any nucleotide sequence of the invention are also provided.

[0456] In some cases, a heterologous (non-native) signal peptide can be added to the nucleic acid encoding the immunomodulatory protein. For example, this may be desirable in the case of expressing a TACI fusion protein that does not contain 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 interleukin-2 (IL-2) or CD33), serum albumin (e.g., HSA or albumin), the human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen) or other signal peptides that can be effectively expressed and, in some aspects, secrete proteins from cells. Exemplary signal peptides include any of the signal peptides described in Table 3.

[0457]

[0458]

[0459] In some embodiments, the immunomodulatory protein contains a signal peptide upon expression, and the signal peptide (or a portion thereof) is cleaved from the immunomodulatory protein upon secretion.

[0460] Also provided herein are recombinant expression vectors and recombinant host cells for producing immunomodulatory proteins (such as the TACI fusion proteins provided herein).

[0461] In any of the above embodiments, the nucleic acid encoding the immunomodulatory polypeptide provided herein can be introduced into cells using recombinant DNA and cloning techniques. To this end, recombinant DNA molecules encoding the immunomodulatory polypeptide are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the peptide-encoding sequence can be excised from the DNA using appropriate restriction enzymes. Alternatively, DNA molecules can be synthesized using chemical synthesis techniques such as the phosphoramidite method. In addition, combinations of these techniques can also be used. In some cases, recombinant or synthetic nucleic acids can be generated by polymerase chain reaction (PCR). The DNA insert encoding the immunomodulatory protein can be cloned into an appropriate transduction / transfection vector known to those skilled in the art. Expression vectors containing the nucleic acid molecule are also provided.

[0462] In some embodiments, the expression vector is capable of expressing the immunomodulatory protein in a suitable cell under conditions suitable for protein expression. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding an immunomodulatory protein, which is operably linked to an appropriate expression control sequence. Methods for achieving such an effective linkage are well known, whether before or after insertion of the DNA molecule into the vector. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, initiation signals, termination signals, capping signals, polyadenylation signals, and other signals involved in controlling transcription or translation.

[0463] In some embodiments, the expression of the immunomodulatory protein is controlled by a promoter or enhancer to control or regulate the expression. The promoter is operably linked to a portion of the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein.

[0464] The resulting recombinant expression vector having the DNA molecule thereon is used to transform a suitable host. Such transformation can be carried out using methods well known in the art. In some embodiments, the nucleic acid provided herein further comprises a nucleotide sequence encoding a secretion peptide or signal peptide, which is operably linked to the nucleic acid encoding the immunomodulatory polypeptide, so as to recover the resulting soluble immunomodulatory polypeptide from the culture medium, host cell, or periplasm of the host cell. In other embodiments, appropriate expression control signals are selected to allow membrane expression of the immunomodulatory polypeptide. In addition, commercially available kits and contract manufacturing companies can also be utilized to manufacture the engineered cells or recombinant host cells provided herein.

[0465] In some embodiments, the resulting expression vector having the DNA molecule thereon is used to transform (such as transduce) a suitable cell. The introduction can be carried out using methods well known in the art. Exemplary methods include methods for transferring nucleic acids encoding receptors, including via viruses (such as retroviruses or lentiviruses), transduction, transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred into the cell by a lentiviral or retroviral transduction method.

[0466] Any of a number of publicly available and well-known mammalian host cells, including mammalian T cells or APCs, can be used to produce the polypeptide or engineer the cell. The choice of cell depends on a number 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, transformation efficiency, ease of peptide recovery, expression characteristics, biosafety, and cost. A balance of these factors must be achieved with the understanding that not all cells may be equally effective for the expression of a particular DNA sequence.

[0467] 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 CCL 34), 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 murine embryonic cells (NIH-3T3; ATCC CRL 1658).

[0468] 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 cultured suspension (such as in cultured suspension CHO cells, e.g., CHO-S cells). In some instances, the cell line is a CHO cell line lacking DHFR (DHFR-), such as DG44 and DUXB11. In some embodiments, the cell lacks glutamine synthetase (GS), e.g., CHO-S cells, CHOK1 SV cells, and CHOZN((R))GS- / - cells. In some embodiments, the CHO cells (such as suspension CHO cells) can be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

[0469] In some embodiments, the host cell can also be a prokaryotic cell, such as Escherichia coli. The transformed recombinant host is cultured under conditions for expressing the polypeptide and then purified to obtain the soluble protein. The recombinant host cell can be cultured under conventional fermentation conditions to express the desired polypeptide. Such fermentation conditions are well known in the art. Finally, the polypeptides provided herein can be recovered and purified from the recombinant cell culture by any of a number of 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. A protein refolding step can be used as needed to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification step.

[0470] In some embodiments, the recombinant vector is a viral vector. Exemplary recombinant viral vectors include lentiviral vector genomes, poxviral vector genomes, vaccinia viral vector genomes, adenoviral vector genomes, adeno-associated viral vector genomes, herpes viral vector genomes, and alphaviral vector genomes. The viral vector can be a live, attenuated, replication conditional, or replication defective, non-pathogenic (defective), replication-competent viral vector, and / or modified to express a heterologous gene product, such as the variant immunomodulatory polypeptides provided herein. The vector used to produce the virus can also be modified to alter the attenuation of the virus, including any method that increases or decreases the transcriptional or translational load.

[0471] Exemplary viral vectors that can be used include modified vaccinia virus vectors (see, for example, 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. Patent Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615, and 7,368,116); adenovirus vectors or adeno-associated virus vectors (see, for example, 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. Patent 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 retroviral vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), amphotropic retrovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, 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, NIH Res. 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, for example, 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 Therapy18:483,2007; 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; WO2016 / 011083; McWilliams et al., J.Virol.77:11150,2003; Powell et al., J.Virol.70:5288,1996) or any, its variants, and / or vectors that can be used to generate any of the above viruses. In some embodiments, the recombinant vector may include regulatory sequences, such as promoter or enhancer sequences, which can regulate the expression of the viral genome (such as in the case of RNA viruses) in a packaging cell line (see, for example, U.S. Patent Nos. 5,385,839 and 5,168,062).

[0472] In some aspects, the nucleic acid or expression vector includes a nucleic acid sequence encoding an immunomodulatory protein, which is operably linked to a suitable expression control sequence. Methods for achieving such an effective linkage are well known, whether before or after the nucleic acid sequence encoding the immunomodulatory protein is inserted into the vector. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, initiation signals, termination signals, cap signals, polyadenylation signals, and other signals involved in controlling transcription or translation. The promoter may be operably linked to a portion of the nucleic acid sequence encoding the immunomodulatory protein.

[0473] 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", in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pp. 163-181 (John Wiley & Sons, Inc. 1996)). A useful combination of a promoter and an enhancer is provided by the myeloproliferative sarcoma virus promoter and the human cytomegalovirus enhancer.

[0474] Alternatively, if a prokaryotic promoter is regulated by a eukaryotic promoter, a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter can be used to control the production of immunomodulatory proteins in mammalian cells (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

[0475] Expression vectors can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, particle-mediated delivery, electroporation, etc. Transfected cells can be selected and propagated to provide recombinant host cells comprising an expression vector stably integrated into the genome of the host cell. Techniques for introducing vectors into eukaryotic cells and for selecting such stable transformants using dominant selectable markers have been described, for example, in Ausubel (1995) and Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).

[0476] For example, a suitable selectable marker is a gene conferring resistance to the antibiotic neomycin. In this case, selection is carried out in the presence of neomycin-like drugs such as G-418. The selection system can also be used to increase the expression level of a gene of interest, a process called "amplification". Amplification is carried out by culturing the transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to select cells that produce a high level of the introduced gene product. A suitable amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes can also be used (e.g., hygromycin resistance, multidrug resistance, puromycin acetyltransferase). Alternatively, markers introducing an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, class I MHC, placental alkaline phosphatase can be used to sort transfected cells from untransfected cells by techniques such as FACS sorting or magnetic bead separation.

[0477] In some embodiments, the polypeptides provided herein can also be prepared by synthetic methods. Solid-phase synthesis is the preferred technique for preparing individual peptides as it is the most cost-effective method for preparing small peptides. For example, well-known solid-phase synthesis techniques include the use of protecting groups, linkers, and solid supports, as well as specific protecting and deprotecting reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid-phase peptide synthesis. The peptides can then be assembled into the polypeptides provided herein.

[0478] IV. Pharmaceutical Compositions

[0479] Compositions containing any of the provided immunomodulatory proteins described herein (e.g., TACI-Fc fusion proteins) are provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the TACI-Fc fusion protein, 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 of the uses described in Section VI for treating such diseases or disorders. Also provided is a method of 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 of the diseases or disorders described in Section VI.

[0480] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. For example, the pharmaceutical composition may contain one or more excipients for adjusting, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0481] In some embodiments, the pharmaceutical composition is 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 before administration.

[0482] In some embodiments, the pharmaceutical composition is liquid, such as an immunomodulatory protein (e.g., TACI-Fc fusion protein) dissolved in an aqueous solution (such as normal saline or Ringer's solution). In some embodiments, the pH of the pharmaceutical composition is between about 4.0 and about 8.5 (such as between about 4.0 and about 5.0, between about 4.5 and about 5.5, between about 5.0 and about 6.0, between about 5.5 and about 6.5, between about 6.0 and about 7.0, between about 6.5 and about 7.5, between about 7.0 and about 8.0, or between about 7.5 and about 8.5).

[0483] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, binder, coating agent, preservative, lubricant, flavoring agent, sweetening agent, coloring agent, solvent, buffer, chelating agent or 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 polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose or cellulose. Examples of pharmaceutically acceptable coatings include hydroxypropyl methylcellulose (HPMC), shellac, zein, gli...

Claims

1. A method of treating an autoantibody-related disease or disorder in a subject, the method comprising administering to the subject a TACI-Fc fusion protein, the TACI-Fc fusion protein being a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide, the polypeptide comprising one or more amino acid substitutions selected from K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13, and wherein the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 80 mg to from or about 480 mg once every four weeks (Q4W).

2. A method of treating an autoantibody-related disease or disorder in a subject, the method comprising administering to the subject a TACI-Fc fusion protein, the TACI-Fc fusion protein being a homodimer of two polypeptides of the formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide, which comprises one or more amino acid substitutions selected from K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13, and wherein the TACI-Fc fusion protein is administered subcutaneously at a dose of from or about 24 mg to from or about 480 mg once every four weeks (Q4W), once every eight weeks (Q8W), or once every twelve weeks (Q12W).

3. The method of claim 1, wherein the variant TACI polypeptide comprises the amino acid substitutions K77E, F78Y, and Y102D.

4. The method of any one of claims 1-3, wherein the dose is from or about 80 mg to from or about 240 mg Q4W.

5. The method of any one of claims 1-4, wherein the dose is from or about 80 mg Q4W.

6. The method of any one of claims 1-4, wherein the dose is from or about 240 mg Q4W.

7. The method of claim 2, wherein the dose is from or about 24 mg to from or about 240 mg once every four weeks (Q4W), once every eight weeks (Q8W), or once every twelve weeks (Q12W).

8. The method of claim 2 or claim 3, wherein: (i) the dose is from or about 24 mg Q4W; (ii) the dose is from or about 24 mg Q8W; (iii) the dose is from or about 24 mg Q12W; (iv) the dose is from or about 80 mg Q8W; (v) the dose is from or about 80 mg Q12W; (vi) the dose is from or about 240 mg Q8W; (vii) the dose is from or about 240 mg Q12W.

9. The method of any one of claims 1-8, wherein the autoantibody-related disease or disorder is selected from rheumatic diseases or disorders, kidney (renal) diseases or disorders, blood diseases or disorders, skin diseases or disorders, or neurological diseases or disorders.

10. The method of any one of claims 1-9, wherein the autoantibody-related disease or disorder is a rheumatic disease or disorder.

11. The method according to any one of claims 1-10, wherein the autoantibody-related disease or disorder is Sjögren's syndrome.

12. The method according to any one of claims 1-10, wherein the autoantibody-related disease or disorder is systemic lupus erythematosus (SLE).

13. The method according to any one of claims 1-12, wherein the TACI-Fc fusion protein reduces the amount of circulating immunoglobulin G (IgG).

14. The method according to claim 13, wherein the circulating IgG is reduced by at least 10% relative to the baseline of the subject, optionally by about 35% relative to the baseline of the subject.

15. The method according to any one of claims 1-14, wherein the TACI-Fc fusion protein does not cause severe hypogammaglobulinemia in the subject.

16. The method according to any one of claims 1-15, wherein in a plurality of subjects treated by the method, the TACI-Fc fusion protein causes severe hypogammaglobulinemia in less than 5% of the treated subjects, optionally less than 3% of the subjects, and more optionally less than 1% of the treated subjects.

17. The method according to claim 15 or claim 16, wherein severe hypogammaglobulinemia is characterized by circulating IgG < 3 g / L, optionally circulating IgG < 1.5 g / L or more optionally circulating IgG < 1.0 g / L.

18. The method according to any one of claims 1-12 and 14, wherein administration of the TACI-Fc fusion protein does not reduce the circulating IgG of the subject to < 1.5 g / L.

19. The method according to any one of claims 1-12 and 14, wherein administration of the TACI-Fc fusion protein does not reduce the circulating IgG of the subject to > 1.0 g / L.

20. A method of treating systemic lupus erythematosus (SLE), the method comprising: a) selecting a subject diagnosed with SLE for administration of a TACI-Fc fusion protein; and b) administering a TACI-Fc fusion protein to the selected subject, wherein: the TACI-Fc fusion protein is a homodimer of two polypeptides of 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 the TACI-Fc fusion protein is administered subcutaneously once every four weeks at a dose of from or about 80 mg to from or about 480 mg.

21. The method according to claim 20, wherein the dose is from or about 80 mg to from or about 240 mg Q4W.

22. The method according to claim 20 or claim 21, wherein the dose is from or about 80 mg Q4W.

23. The method according to claim 20 or claim 21, wherein the dose is from or about 240 mg Q4W.

24. The method according to any one of claims 12 - 23, wherein the systemic lupus erythematosus is mild to moderate systemic lupus erythematosus or moderate to severe systemic lupus erythematosus.

25. The method according to any one of claims 12 - 24, wherein if the subject has had active SLE for ≥6 months at the time of screening, the subject is selected for treatment.

26. The method according to any one of claims 12 - 25, wherein if the SLE is characterized by one or more of the following at the time of screening, the subject is selected for treatment: (i) If having high anti - dsDNA or low complement (C) levels, the combined SELENA - SLEDAI score ≥8 or combined SELENA - SLEDAI ≥6; (ii) Urine total protein - to - creatinine ratio (proteinuria) ≤6 g / g; (iii) BILAG score of grade A for ≥1 organ; (iv) BILAG score of grade B for ≥2 organs; and (v) Physician's Global Assessment (PGA) score ≥1.

0.

27. The method according to any one of claims 12 - 26, wherein the subject is receiving standard therapy for treating the SLE.

28. The method according to claims 12 - 27, wherein if the subject is receiving a stable standard treatment regimen at the time of screening or at the time of administering the TACI - Fc fusion protein, the standard treatment regimen is characterized by stable use of standard therapy for treating SLE, optionally wherein the stable use is stable use of standard therapy for at least 30 days, the subject is selected for treatment.

29. The method according to any one of claims 12 - 27, wherein the TACI - Fc fusion protein is administered to the subject in combination with standard therapy for treating the SLE.

30. The method according to any one of claims 27 - 29, wherein the standard therapy comprises one or more of corticosteroids, antimalarials (such as hydroxychloroquine), non - steroidal anti - inflammatory drugs (NSAIDs), or immunosuppressants or immunomodulators, or any combination thereof, optionally wherein the immunosuppressant or immunomodulator is selected from the group consisting of azathioprine, mycophenolate (such as mycophenolate mofetil or mycophenolate sodium), cyclophosphamide, methotrexate, leflunomide, tacrolimus, cyclosporine, and any combination of the foregoing.

31. The method according to any one of claims 27 - 30, wherein the standard therapy comprises corticosteroids, and the administration of the corticosteroids is gradually reduced after administering the TACI - Fc fusion protein.

32. The method according to any one of claims 12 - 29, wherein the SLE is severe SLE.

33. The method according to any one of claims 12 - 32, wherein if the subject is characterized by one or more of the following at the time of screening, the subject is selected for treatment: (i) Severe lupus nephritis, optionally defined as urinary protein >6 g / 24 hours or serum creatinine >2.5 mg / dL or 221 μmol / L; (ii) Requiring hemodialysis; (iii) received high-dose corticosteroids for ≥14 days within the past 2 months, optionally wherein the high-dose corticosteroids were treated with prednisone >100 mg / day or an equivalent; and (iv) central nervous system diseases caused by SLE or not caused by SLE within the past 2 months; optionally wherein the central nervous system disease is epilepsy, psychosis, organic brain syndrome, cerebrovascular accident, encephalitis, or central nervous system vasculitis.

34. The method according to any one of claims 1-9, wherein the autoantibody-related disease or disorder is a kidney (renal) disease or disorder.

35. The method according to any one of claims 1-9 and 32, wherein the autoantibody-related disease or disorder is glomerulonephritis.

36. A method of treating glomerulonephritis, the method comprising: a) selecting a subject diagnosed with glomerulonephritis for administration of a TACI-Fc fusion protein; and b) administering a TACI-Fc fusion protein to the selected subject, 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; and The TACI-Fc fusion protein is administered subcutaneously once every four weeks at a dose of or about 80 mg to or about 480 mg.

37. The method according to claim 36, wherein the dose is or about 80 mg to or about 240 mg Q4W.

38. The method according to claim 36 or claim 37, wherein the dose is or about 80 mg Q4W.

39. The method according to claim 36 or claim 37, wherein the dose is or about 240 mg Q4W.

40. The method according to any one of claims 36-39, wherein if the subject has active glomerulonephritis at the time of screening, the subject is selected for treatment.

41. The method according to any one of claims 36-40, wherein the glomerulonephritis is selected from IgA nephropathy, lupus nephritis, and primary membranous nephropathy.

42. The method according to any one of claims 36-41, wherein the glomerulonephritis is IgA nephropathy, and if the subject is characterized by one or both of the following at the time of screening, the subject is selected for treatment: (i) the subject was diagnosed with IgA nephropathy ≤5 years before the screening; and (ii) the total urine protein to creatinine ratio ≥0.75 g / g (proteinuria).

43. The method according to any one of claims 36-41, wherein the glomerulonephritis is IgA nephropathy, and if the subject is characterized by one or more of the following at the time of screening, the subject is selected for treatment: (i) the subject was diagnosed with IgA nephropathy ≤5 years before the screening; (ii) the total urine protein to creatinine ratio ≥0.75 g / g (proteinuria); and (iii) Elevated galactose-deficient IgA1 (Gd-IgA1).

44. The method according to claim 43, wherein the TACI-Fc fusion protein reduces Gd-IgA1.

45. The method according to claim 44, wherein the reduction of Gd-IgA1 is more than 50%.

46. The method according to any one of claims 36-41, wherein the glomerulonephritis is lupus nephritis, and the lupus nephritis is characterized by class III (active focal), class IV (diffuse), and / or class V (lupus membranous nephropathy).

47. The method according to any one of claims 36-41 and 46, wherein the glomerulonephritis is lupus nephritis, and the subject is selected for treatment if at the time of screening the subject is characterized by one or more of the following: (i) The subject was diagnosed with lupus nephritis class II-V within ≤ 3 years before the screening; (ii) ≥ 1 g / g total urinary protein to creatinine ratio (proteinuria); (iii) Active urinary sediment; (iv) Positive for anti-double-stranded DNA and / or anti-nuclear antibody (ANA), optionally wherein the anti-double-stranded DNA is positive at a titer ≥ 30 IU / mL and the ANA is positive at a titer ≥ 1:80; (v) A stable standard treatment plan, characterized in that Stable use of standard therapy for treating the SLE, optionally wherein the stable use is stable use of the standard therapy for at least 30 days; and (vi) Received stable background immunosuppression, optionally wherein the stable background immunosuppression is receiving a stable dose of MMF ≥ 1 g / day for at least 8 weeks before screening or at the time of administration of the TACI-Fc fusion protein, with or without corticosteroids.

48. The method according to any one of claims 36-41, wherein the glomerulonephritis is primary membranous nephropathy.

49. The method according to any one of claims 36-41 and 48, wherein the glomerulonephritis is primary membranous nephropathy (pMN), and the subject is selected for treatment if at the time of screening the subject is characterized by one or more of the following: (i) The subject was diagnosed with pMN ≤ 5 years before the screening; (ii) Total urinary protein to creatinine ratio ≥ 3.5 g / g (proteinuria); and (iii) Positive anti-PLA2R1 and / or positive anti-THSD7A antibody.

50. The method according to any one of claims 36-49, wherein the subject is selected for treatment if at the time of screening or at the time of administration of the TACI-Fc fusion protein the subject has received treatment with an angiotensin-converting enzyme (ACE) inhibitor and / or an angiotensin II receptor blocker (ARB), optionally wherein the subject has received the maximum recommended dose of the ACE inhibitor or ARB.

51. The method according to any one of claims 36-50, wherein the subject is selected for treatment if at the time of screening or at the time of administration of the TACI-Fc fusion protein the subject's blood pressure is stable.

52. The method according to any one of claims 1-9, wherein the autoantibody-related disease or disorder is a blood disease or disorder.

53. The method according to any one of claims 1-9 and 52, wherein the autoantibody-related disease or disorder is autoimmune cytopenia.

54. A method of treating autoimmune cytopenia, the method comprising: a) selecting a subject diagnosed with autoimmune cytopenia for administration of a TACI-Fc fusion protein; and b) administering a TACI-Fc fusion protein to the selected subject, wherein: the TACI-Fc fusion protein is a homodimer of two polypeptides of 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 the TACI-Fc fusion protein is administered subcutaneously once every four weeks at a dose of or about 80 mg to or about 480 mg.

55. The method according to claim 54, wherein the dose is or about 80 mg to or about 240 mg Q4W.

56. The method according to claim 54 or claim 55, wherein the dose is or about 80 mg Q4W.

57. The method according to claim 54 or claim 55, wherein the dose is or about 240 mg Q4W.

58. The method according to any one of claims 53-57, wherein if the subject has active cytopenia at the time of screening, the subject is selected for treatment.

59. The method according to any one of claims 53-58, wherein the autoimmune cytopenia is selected from immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA).

60. The method according to any one of claims 53-59, wherein the autoimmune cytopenia is ITP, and if the subject is characterized by one or more of the following at the time of screening, the subject is selected for treatment: (i) the subject has been diagnosed with ITP for ≥ 3 months prior to the screening; (ii) platelet count < 30,000 / μL; and (iii) has received ≥ 2 prior treatments for treating the ITP, optionally ≥ 4 prior treatments for treating the ITP.

61. The method according to any one of claims 53-59, wherein the autoimmune cytopenia is AIHA, and the AIHA is warm-type AIHA (wAIHA) or cold-type AIHA (cold agglutinin disease, CAD).

62. The method according to any one of claims 53-59 and 61, wherein the autoimmune cytopenia is wAIHA or CAD, and if the subject is characterized by one or more of the following at the time of screening, the subject is selected for treatment: (i) the subject has been diagnosed with wAIHA or CAD for ≥ 3 months prior to the screening; (ii) Hemoglobin (Hb) < 9 g / dL; and (iii) having received ≥ 1 prior treatment for treating said AIHA, optionally ≥ 2 prior treatments for treating said AIHA.

63. The method according to claim 62, wherein the autoimmune cytopenia is wAIHA.

64. The method according to claim 62, wherein the autoimmune cytopenia is CAD.

65. The method according to claims 53 - 64, wherein if the subject is receiving stable immunosuppression at the time of screening or at the time of administration of the TACI-Fc fusion protein, optionally wherein the TACI-Fc fusion protein is co-administered in combination with the stable immunosuppression to the subject, then the subject is selected for treatment.

66. The method according to claim 65, wherein: the stable immunosuppression comprises the use of a stable dose of steroids, optionally corticosteroids, for at least two weeks prior to the time of screening or at the time of administration of the TACI-Fc fusion protein; and / or the stable immunosuppression comprises the use of a stable dose of azathioprine, MMF, or a calcineurin inhibitor, optionally cyclosporine, for at least four weeks prior to the time of screening or at the time of administration of the TACI-Fc fusion protein.

67. The method according to any one of claims 53 - 66, wherein the subject is not characterized as having secondary cytopenia (such as a systemic autoimmune disease or malignancy) or Evans syndrome.

68. The method according to any one of claims 53 - 66, wherein the subject is characterized as having secondary cytopenia (such as a systemic autoimmune disease or malignancy) or Evans syndrome.

69. The method according to any one of claims 1 - 9, wherein the autoantibody-related disease or disorder is a skin disease or disorder.

70. The method according to any one of claims 1 - 9 and 69, wherein the autoantibody-related disease or disorder is an autoimmune bullous skin disease.

71. A method of treating an autoimmune bullous (vesicular) skin disease, the method comprising: a) selecting a subject diagnosed with an autoimmune bullous (vesicular) skin disease for administration of a TACI-Fc fusion protein; and b) administering a TACI-Fc fusion protein to the selected subject, wherein: the TACI-Fc fusion protein is a homodimer of two polypeptides of formula TACI-linker-Fc, wherein TACI is a variant TACI polypeptide comprising the amino acid substitutions K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO:13; and the TACI-Fc fusion protein is administered subcutaneously once every four weeks at a dose of from or about 80 mg to from or about 480 mg.

72. The method according to claim 71, wherein the dose is from or about 80 mg to from or about 240 mg Q4W.

73. The method according to claim 71 or claim 72, wherein the dose is from or about 80 mg Q4W.

74. The method according to claim 71 or claim 72, wherein the dose is or about 240 mg Q4W.

75. The method according to any one of claims 70 - 74, wherein the subject is selected for treatment if the subject has an active blistering disease at the time of screening.

76. The method according to any one of claims 70 - 75, wherein the autoimmune bullous (blistering) skin disease is selected from pemphigus vulgaris, pemphigus foliaceus, or bullous pemphigoid.

77. The method according to any one of claims 70 - 76, wherein the autoimmune bullous (blistering) skin disease is pemphigus vulgaris or pemphigus foliaceus, and the subject is selected for treatment if the subject is characterized by one or both of the following at the time of screening: (i) Pemphigus disease area index (PDAI) ≥ 15; and (ii) Positive for anti - Dsg1 antibody or positive for anti - Dsg3 antibody.

78. The method according to claim 77, wherein the autoimmune bullous (blistering) skin disease is pemphigus vulgaris.

79. The method according to claim 78, wherein the autoimmune bullous (blistering) skin disease is pemphigus foliaceus.

80. The method according to any one of claims 70 - 77, wherein the autoimmune bullous (blistering) skin disease is pemphigoid, and the subject is selected for treatment if the subject is characterized by one or both of the following at the time of screening: (i) IgA antibody; and (ii) Positive for anti - Bp180 antibody or positive for anti - Bp230 antibody.

81. The method according to any one of claims 70 - 80, wherein the subject is selected for treatment if the subject is receiving stable immunosuppression at the time of screening or at the time of administration of the TACI - Fc fusion protein.

82. The method according to claim 81, wherein the TACI - Fc fusion protein is co - administered to the subject simultaneously with a stable immunosuppressant.

83. The method according to claim 81 or claim 82, wherein: the stable immunosuppression includes the use of a stable dose of steroids, optionally corticosteroids, for at least two weeks prior to the time of screening or at the time of administration of the TACI - Fc fusion protein; and / or the stable immunosuppression includes the use of a stable dose of azathioprine, MMF, or a calcineurin inhibitor, optionally cyclosporine, for at least four weeks prior to the time of screening or at the time of administration of the TACI - Fc fusion protein.

84. The method according to any one of claims 70 - 83, wherein the subject is not characterized by having a secondary disease (e.g., paraneoplastic).

85. The method according to any one of claims 1 - 9, wherein the autoantibody - related disease or disorder is a neurological disease or disorder.

86. The method according to any one of claims 1 - 9 and 85, wherein the autoantibody - related disease or disorder is encephalitis.

87. A method of treating encephalitis, the method comprising: a) Select subjects diagnosed with encephalitis for administration of the TACI-Fc fusion protein; and b) Administer the TACI-Fc fusion protein to the selected subjects, 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 the amino acid substitutions K77E, F78Y, and Y102D in the amino acid sequence shown in SEQ ID NO: 13; and The TACI-Fc fusion protein is administered subcutaneously once every four weeks at a dose of from or about 80 mg to from or about 480 mg.

88. The method according to claim 87, wherein the dose is from or about 80 mg to from or about 240 mg Q4W.

89. The method according to claim 87 or claim 88, wherein the dose is from or about 80 mg Q4W.

90. The method according to claim 87 or claim 88, wherein the dose is from or about 240 mg Q4W.

91. The method according to any one of claims 86-90, wherein the encephalitis is autoimmune encephalitis.

92. The method according to any one of claims 86-91, wherein the encephalitis is limbic encephalitis.

93. The method according to any one of claims 1-92, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 12 to 72 weeks.

94. The method according to any one of claims 1-93, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks or longer.

95. The method according to any one of claims 1-94, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 12 weeks.

96. The method according to any one of claims 1-94, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 16 weeks.

97. The method according to any one of claims 1-94, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 24 weeks.

98. The method according to any one of claims 1-94, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 48 weeks.

99. The method according to any one of claims 1-98, wherein the variant TACI polypeptide is as shown in SEQ ID NO:

26.

100. The method according to any one of claims 1-98, wherein the linker is a GS linker having a length between 5 and 20 amino acids.

101. The method according to any one of claims 1-100, wherein the linker 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), GGGSSA (SEQ ID NO:80) or GSGGGGSGGGGS (SEQ ID NO:194) or a combination thereof.

102. The method according to any one of claims 1-101, wherein the linker is as shown in SEQ ID NO:

74.

103. The method according to any one of claims 1-102, wherein the Fc is an IgG1 Fc domain.

104. The method according to any one of claims 1-103, wherein the Fc is a variant IgG1 Fc that exhibits a reduced binding affinity for Fc receptors and / or reduced effector function compared to the wild-type IgG1 Fc domain.

105. The method according to claim 104, wherein the variant IgG1 Fc domain comprises one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G and V302C numbered according to the EU numbering.

106. The method according to claim 104 or claim 105, wherein the variant IgG1 Fc comprises the amino acid substitutions L234A, L235E and G237A numbered according to the EU numbering.

107. The method according to any one of claims 103-106, wherein the Fc comprises the amino acid substitution C220S, wherein the residue is numbered according to the EU index of Kabat.

108. The method according to any one of claims 103-107, wherein the Fc lacks the hinge sequence EPKSS or EPKSC.

109. The method according to any one of claims 103-108, wherein the Fc region comprises K447del, wherein the residue is numbered according to the EU index of Kabat.

110. The method according to claims 1-107 and 109, wherein the Fc comprises the amino acid sequence shown in SEQ ID NO:

73.

111. The method according to any one of claims 1-107, 109 and 110, wherein the TACI-Fc fusion protein is as shown in SEQ ID NO:

167.

112. The method according to claims 1-103, 107-110, wherein the Fc comprises the amino acid sequence shown in SEQ ID NO:

81.

113. The method according to any one of claims 1-103, 107-110 and 111, wherein the TACI-Fc fusion protein is as shown in SEQ ID NO:

168.

114. The method according to any one of claims 1-113, wherein the TACI-Fc fusion protein is provided in a formulation comprising an acetate buffer having a pH of from about 4.0 to about 6.0, proline at a concentration of from or about 1% to about 10%, and a surfactant at a concentration of from about 0.005 to about 0.05% (w / v).

115. The method according to claim 114, wherein the pH of the formulation is about 5.

2.

116. The method according to claim 114 or claim 115, wherein the acetate buffer comprises acetate at a concentration of from or about 5 mM to or about 15 mM.

117. The method according to any one of claims 114-116, wherein the acetate buffer comprises acetate at a concentration of from or about 10 mM.

118. The method according to any one of claims 114-117, wherein the concentration of proline is from about 2% to about 5%.

119. The method according to any one of claims 114-117, wherein the concentration of proline is from or about 3%.

120. The method according to any one of claims 114-119, wherein the concentration of the surfactant is from about 0.01 to about 0.025% (w / v), optionally from or about 0.015% (w / v).

121. The method according to any one of claims 114-120, wherein the surfactant is polysorbate 80.

122. The method according to any one of claims 114-121, wherein the amount of the TACI-Fc fusion protein in the formulation is from about 50 mg to about 100 mg.

123. The method according to any one of claims 114-122, wherein the amount of the TACI-Fc fusion protein in the formulation is from or about 80 mg.

124. The method according to any one of claims 114-123, wherein the concentration of the TACI-Fc fusion protein is between about 50 mg / mL and about 200 mg / mL.

125. The method according to any one of claims 114-120, wherein the concentration of the TACI-Fc fusion protein is from or about 100 mg / mL.

126. The method according to any one of claims 1-125, wherein the B cell immune response or activity of the subject is reduced.

127. The method according to any one of claims 1-126, wherein the number of mature and total circulating B cells in the subject is reduced.

128. The method according to any one of claims 1-127, wherein the circulating serum immunoglobulins of the subject are reduced.

129. The method according to any one of claims 1-128, wherein one or more of B cell maturation, differentiation and / or proliferation are reduced or inhibited.

130. The method according to any one of claims 1-129, wherein the circulating levels of APRIL or BAFF protein in the subject are reduced, optionally wherein the APRIL or BAFF protein is APRIL homotrimer, BAFF homotrimer, APRIL / BAFF heterotrimer or BAFF 60-mer.

131. The method according to any one of claims 1-130, wherein the subject is a human.

132. The method according to claim 131, wherein the subject is an adult subject, optionally 18 years of age or older, optionally 18-65 years of age.

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