Mutant TACI-FC fusion proteins for use in the treatment of autoantibody-mediated diseases

Abstract

Provided herein are therapeutic methods and uses comprising immunomodulatory TACI-Fc fusion proteins that exhibit neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers). The provided TACI-Fc proteins may comprise a variant domain of transmembrane activator and CAML interactor (TACI). The methods and uses provide therapeutic utility for a variety of immune diseases, disorders, or conditions, such as B-cell mediated diseases, disorders, or conditions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 63 / 378,361, filed October 4, 2022; U.S. Provisional Application No. 63 / 382,094, filed November 2, 2022; U.S. Provisional Application No. 63 / 383,243, filed November 10, 2022; U.S. Provisional Application No. 63 / 385,948, filed December 2, 2022; U.S. Provisional Application No. 63 / 483,936, filed February 8, 2023; U.S. Provisional Application No. 63 / 486, filed February 24, 2023; all of which are entitled "METHODS AND USES OF TACI-FC FUSION IMMUNOMODULATORY PROTEIN." This application claims priority to U.S. Provisional Application No. 946, filed March 21, 2023, U.S. Provisional Application No. 63 / 491,526, filed April 21, 2023, U.S. Provisional Application No. 63 / 497,691, filed May 16, 2023, U.S. Provisional Application No. 63 / 502,611, filed May 30, 2023, and U.S. Provisional Application No. 63 / 505,053, filed May 30, 2023, and U.S. Provisional Application No. 63 / 581,609, filed September 8, 2023, the contents of which are incorporated by reference in their entireties.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (761612004340SEQLIST.xml; size: 345,811 bytes; and creation date: September 27, 2023) are incorporated herein by reference in their entirety.

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

[0004] background Modulation of immune responses by intervening in processes involving the interaction between soluble ligands and their receptors has become of growing medical interest. Currently, biologics used to enhance or suppress immune responses are generally limited to antibodies (e.g., anti-PD-1 antibodies) or soluble receptors for single cell surface molecules (e.g., CTLA-4-Fc). There is a need for improved therapeutic agents that can modulate immune responses, particularly B cell immune responses. Embodiments that meet this need are provided. Summary of the Invention

[0005] In some aspects, provided herein are methods for treating an autoantibody-associated 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, where TACI is a variant TACI polypeptide that contains one or more amino acid substitutions selected from the group consisting of 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 once every four weeks (Q4W) at a dose of 80 mg or about 80 mg to 480 mg or about 480 mg. In some aspects, provided herein are methods for treating an autoantibody-associated 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, where TACI is a variant TACI polypeptide comprising one or more amino acid substitutions selected from the group consisting of 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 once every four weeks (Q4W), once every eight weeks (Q8W), or once every 12 weeks (Q12W) at a dose of 24 mg or about 24 mg to 480 mg or about 480 mg.

[0006] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E, F78Y, and Y102D.

[0007] In some embodiments, the dose is from 80 mg or about 80 mg to 240 mg or about 240 mg Q4W. In some embodiments, the dose is 80 mg or about 80 mg Q4W. In some embodiments, the dose is 240 mg or about 240 mg Q4W. In some embodiments, the dose is 24 mg or about 24 mg to 240 mg 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 24 mg or about 24 mg Q4W. In some embodiments, the dose is 24 mg or about 24 mg Q8W. In some embodiments, the dose is 24 mg or about 24 mg Q12W. In some embodiments, the dose is 80 mg or about 80 mg Q8W. In some embodiments, the dose is 80 mg or about 80 mg Q12W. In some embodiments, the dose is 240 mg or about 240 mg Q8W. In some embodiments, the dose is 240 mg or about 240 mg Q12W.

[0008] In some embodiments, the autoantibody-associated disease or disorder is selected from the group consisting of a rheumatic disease or disorder, a renal (kidney) disease or disorder, a blood disease or disorder, a skin disease or disorder, or a neurological disease or disorder. In some embodiments, the autoantibody-associated disease or disorder is a rheumatic disease or disorder. In some embodiments, the autoantibody-associated disease or disorder is Sjogren's disease. In some embodiments, the autoantibody-associated disease or disorder is systemic lupus erythematosus (SLE).

[0009] In some embodiments, the TACI-Fc fusion protein reduces a subject's risk of developing hypogammaglobulinemia or severe hypogammaglobulinemia. In some embodiments, hypogammaglobulinemia is characterized by circulating IgG≦7 g / L. In some embodiments, severe hypogammaglobulinemia is characterized by circulating IgG<3 g / L. In some embodiments, severe hypogammaglobulinemia is characterized by circulating IgG<1.5 g / L. In some embodiments, severe hypogammaglobulinemia is characterized by 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, circulating IgG is reduced by about 35% from the subject's baseline.

[0010] In some aspects, provided herein is a method for 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, where 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 once every four weeks at a dose of 80 mg or about 80 mg to 480 mg or about 480 mg.

[0011] In some embodiments, the dose is from 80 mg or about 80 mg to 240 mg or about 240 mg Q4W. In some embodiments, the dose is 80 mg or about 80 mg Q4W. In some embodiments, the dose is 240 mg or about 240 mg Q4W.

[0012] 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.

[0013] In some embodiments, a subject is selected for treatment if, at the time of screening, the subject has had active SLE for 6 months or more.

[0014] In some embodiments, subjects are selected for treatment if, at screening, their SLE is characterized by one or more of the following: (i) a hybrid SELENA-SLEDAI score of ≧8, or a hybrid SELENA-SLEDAI score of ≧6 if high anti-dsDNA levels or low complement (C) levels are present; (ii) a urine total protein-to-creatinine ratio of ≦6 g / g (proteinuria); (iii) a BILAG score of A grade in one or more organs; (iv) a BILAG score of B grade in two or more organs; and (v) a Physician Global Assessment (PGA) score of ≧1.0.

[0015] In some embodiments, the subject is receiving standard therapy for treating SLE.

[0016] In some embodiments, a 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 of care regimen characterized by stable use of standard of care to treat SLE. In some embodiments, stable use is stable use of standard of care for at least 30 days.

[0017] In some embodiments, the TACI-Fc fusion protein is administered to the subject in combination with a standard of care for treating SLE.

[0018] In some embodiments, the standard of care includes one or more corticosteroids, antimalarials (e.g., hydroxychloroquine), nonsteroidal anti-inflammatory drugs (NSAIDs), or immunosuppressants or immunomodulators, or any combination thereof.

[0019] In some embodiments, the immunosuppressant or immunomodulatory agent is selected from the group including azathioprine, mycophenolate (e.g., mycophenolate mofetil or mycophenolate sodium), cyclophosphamide, methotrexate, leflunomide, tacrolimus, cyclosporine, and combinations of any of the foregoing.

[0020] In some embodiments, the standard of care includes corticosteroids, and the administration of the corticosteroids is tapered after administration of the TACI-Fc fusion protein.

[0021] In some embodiments, a subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) severe lupus nephritis, as defined by urine protein >6g / 24 hours or serum creatinine >2.5mg / dL, or 221 μmol / L; (ii) the need for hemodialysis; (iii) high-dose corticosteroids administered for 14 days or more in the past two months, e.g., high-dose corticosteroids are treatment with prednisone >100mg / day or equivalent; and (iv) central nervous system disease, with or without SLE, in the past two months. In some aspects, the central nervous system disease is epilepsy, psychosis, organic brain syndrome, cerebrovascular accident, encephalitis, or central nervous system vasculitis.

[0022] In some embodiments, the autoantibody-associated disease or disorder is a renal (kidney) disease or disorder. In some embodiments, the autoantibody-associated disease or disorder is glomerulonephritis. In some aspects, provided herein are methods of treating glomerulonephritis, the methods 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, where TACI is a variant TACI polypeptide comprising the 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 once every four weeks at a dose of at or about 80 mg to at or about 480 mg.

[0023] In some embodiments, the dose is from 80 mg or about 80 mg to 240 mg or about 240 mg Q4W. In some embodiments, the dose is 80 mg or about 80 mg Q4W. In some embodiments, the dose is 240 mg or about 240 mg Q4W.

[0024] In some embodiments, the subject is selected for treatment if, at screening, the subject has active glomerulonephritis, hi some embodiments, the glomerulonephritis is selected from the group consisting of IgA nephropathy, lupus nephritis, and primary membranous nephropathy.

[0025] In some embodiments, the glomerulonephritis is IgA nephropathy, and the subject is selected for treatment if, at screening, the subject is characterized by one or both of the following: (i) the subject was diagnosed with IgA nephropathy within five years prior to screening; and (ii) a urine total protein to creatinine ratio of ≧0.75 g / g (proteinuria). In some embodiments, the glomerulonephritis is IgA nephropathy, and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject was diagnosed with IgA nephropathy within five years prior to screening; (ii) a urine total protein to creatinine ratio of ≧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, Gd-IgA1 is reduced by more than 50%.

[0026] 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).

[0027] In some embodiments, the glomerulonephritis is lupus nephritis, and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject has been diagnosed with lupus nephritis class II-V within three years prior to screening; (ii) a urine total protein-to-creatinine ratio of ≧1 g / g (proteinuria); (iii) an active urinary sediment; (iv) positive anti-dsDNA antibodies and antinuclear antibodies (ANA), e.g., anti-dsDNA positivity is a titer of ≧30 IU / mL, and ANA positivity is a titer of ≧1:80; and (v) a stable standard of care regimen characterized by stable use of standard of care to treat SLE, e.g., stable use is stable use of standard of care for at least 30 days; and (v) received stable basal immunosuppression, e.g., stable basal immunosuppression is a stable dose of ≧1 g / day of MMF, with or without corticosteroids, for at least 8 weeks prior to screening or administration of the TACI-Fc fusion protein.

[0028] In some embodiments, the glomerulonephritis is primary membranous nephropathy.

[0029] In some embodiments, the glomerulonephritis is primary membranous nephropathy (pMN), and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject was diagnosed with pMN within 5 years prior to screening; (ii) a urine total protein-to-creatinine ratio of ≧3.5 g / g (proteinuria); and (iii) positive anti-PLA2R1 antibodies or positive anti-THSD7A antibodies.

[0030] In some embodiments, a subject is selected for treatment if, at the time of screening or administration of the TACI-Fc fusion protein, the subject was receiving treatment with an angiotensin-converting enzyme (ACE) inhibitor and / or angiotensin II receptor blocker (ARB), e.g., the subject was receiving the maximum recommended dose of ACE inhibitor or ARB treatment.

[0031] In some embodiments, a subject is selected for treatment if the subject has stable blood pressure at the time of screening or administration of the TAC-Fc fusion protein.

[0032] In some embodiments, the autoantibody-associated disease or disorder is a hematological disease or disorder. In some embodiments, the autoantibody-associated disease or disorder is an autoimmune cytopenia.

[0033] In some aspects, provided herein is a method for 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, where 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 once every four weeks at a dose of 80 mg or about 80 mg to 480 mg or about 480 mg.

[0034] In some embodiments, the dose is from 80 mg or about 80 mg to 240 mg or about 240 mg Q4W. In some embodiments, the dose is 80 mg or about 80 mg Q4W. In some embodiments, the dose is 240 mg or about 240 mg Q4W.

[0035] In some embodiments, a subject is selected for treatment if the subject has active cytopenia at the time of screening.

[0036] In some embodiments, the autoimmune cytopenia is selected from the group consisting of immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA).

[0037] In some embodiments, the autoimmune cytopenia is ITP, and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject was diagnosed with ITP more than three months prior to screening; (ii) a persistent platelet count <30,000 / μL; and (iii) received four or more prior therapies to treat ITP.

[0038] In some embodiments, the autoimmune cytopenia is AIHA, and the AIHA is warm AIHA (wAIHA) or cold AIHA (cold agglutinin disease, CAD).

[0039] In some embodiments, the autoimmune cytopenia is wAIHA or CAD, and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject was diagnosed with wAIHA or CAD more than three months prior to screening; (ii) a persistent hemoglobin (Hb) < 9 g / dL; and (iii) received two or more prior therapies to treat AIHA.

[0040] In some embodiments, the autoimmune cytopenia is AIHA. In some embodiments, the autoimmune cytopenia is CAD.

[0041] In some embodiments, a subject is selected for treatment if the subject is undergoing stable immunosuppression at the time of screening or administration of the TACI-Fc fusion protein.

[0042] In some embodiments, the TACI-Fc fusion protein is administered to the subject in combination with the concomitant administration of stable immunosuppression.

[0043] In some embodiments, stable immunosuppression includes a stable dose of steroids, e.g., 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.

[0044] In some embodiments, the subject is not characterized by having a secondary cytopenia (e.g., a systemic autoimmune disease or malignancy) or Evans syndrome.

[0045] In some embodiments, the autoantibody-associated disease or disorder is a skin disease or disorder. In some embodiments, the autoantibody-associated disease or disorder is an autoimmune bullous dermatosis.

[0046] In some aspects, provided herein is a method for treating autoimmune bullous (blistering) dermatopathy, the method comprising: a) selecting a subject diagnosed with autoimmune bullous (blistering) dermatopathy 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, where 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 80 mg or about 80 mg to 480 mg or about 480 mg once every four weeks.

[0047] In some embodiments, the dose is from 80 mg or about 80 mg to 240 mg or about 240 mg Q4W. In some embodiments, the dose is 80 mg or about 80 mg Q4W. In some embodiments, the dose is 240 mg or about 240 mg Q4W.

[0048] In some embodiments, the subject is selected for treatment if the subject has an active blistering disease at screening. In some embodiments, the autoimmune bullous (blistering) dermatosis is selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, or bullous pemphigoid.

[0049] In some embodiments, the autoimmune bullous (blistering) dermatopathy is pemphigus vulgaris or pemphigus foliaceus, and the subject is selected for treatment if, at screening, the subject is characterized by one or both of the following: (i) Pemphigus International Severity Scale (PDAI) ≧15; and (ii) anti-Dsg1 antibody positivity or anti-Dsg3 antibody positivity.

[0050] In some embodiments, the autoimmune bullous (blistering) dermatopathy is pemphigus vulgaris. In some embodiments, the autoimmune bullous (blistering) dermatopathy is pemphigus foliaceus.

[0051] In some embodiments, the autoimmune bullous (blistering) dermatopathy is pemphigoid, and the subject is selected for treatment if, at screening, the subject is characterized by one or both of the following: (i) IgA antibodies; and (ii) anti-Bp180 antibody positivity or anti-Bp230 antibody positivity.

[0052] In some embodiments, a subject is selected for treatment if the subject is undergoing stable immunosuppression at the time of screening or administration of the TACI-Fc fusion protein.

[0053] In some embodiments, the TACI-Fc fusion protein is administered to the subject in combination with the concomitant administration of stable immunosuppression.

[0054] In some embodiments, stable immunosuppression includes a stable dose of steroids, e.g., 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.

[0055] In some embodiments, the subject is not characterized as having a secondary disease (eg, paraneoplastic).

[0056] In some embodiments, the autoantibody-associated disease or disorder is a neurological disease or disorder, hi some embodiments, the autoantibody-associated disease or disorder is encephalitis.

[0057] In some aspects, provided herein is a method for 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, where 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 once every four weeks at a dose of 80 mg or about 80 mg to 480 mg or about 480 mg.

[0058] In some embodiments, the dose is from 80 mg or about 80 mg to 240 mg or about 240 mg Q4W. In some embodiments, the dose is 80 mg or about 80 mg Q4W. In some embodiments, the dose is 240 mg or about 240 mg Q4W.

[0059] In some embodiments, the encephalitis is autoimmune encephalitis, hi some embodiments, the encephalitis is limbic encephalitis.

[0060] 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, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or more weeks. 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.

[0061] In some embodiments of any of the provided methods, instead of administering the TACI-Fc fusion protein to the subject Q4W, alternative embodiments contemplate administering the TACI-Fc fusion protein to the subject Q8W or Q12W. In some such embodiments of any of the provided methods, the subject is administered the TACI-Fc fusion protein at a dose of 240 mg or about 240 mg to 480 mg or about 480 mg Q8W, e.g., 240 mg or about 240 mg Q8W, 320 mg or about 320 mg Q8W, or 480 mg or about 480 mg Q8W. In some such embodiments of any of the provided methods, the subject is administered a dose of TACI-Fc fusion protein from 240 mg or about 240 mg to 480 mg or about 480 mg Q12W, e.g., 240 mg or about 240 mg Q12W, 320 mg or about 320 mg Q12W, or 480 mg or about 480 mg Q12W.

[0062] In some embodiments, the variant TACI polypeptide is set forth in SEQ ID NO:26.

[0063] In some embodiments, the linker is a GS linker between 5 and 20 amino acids in length. In some embodiments, the linker is selected from GSGGS (SEQ ID NO:76), GGGGS (GS; SEQ ID NO:77), GSGGGGS (SEQ ID NO:74), GGGGSGGGGS (2xGGGGS; SEQ ID NO:78), GGGGSGGGGGSGGGGGS (3xGGGGS; SEQ ID NO:79), GGGSGGGGSGGGGSGGGGS (4xGGGGS, SEQ ID NO:84), GGGSGGGGSGGGGSGGGGSGGGGGS (5xGGGGS, SEQ ID NO:91), GGGGSSA (SEQ ID NO:80), or GSGGGGSGGGGS (SEQ ID NO:194), or a combination thereof. In some embodiments, the linker is set forth in SEQ ID NO:74.

[0064] In some embodiments, the Fc is an IgG1 Fc domain, hi some embodiments, the Fc is a variant IgG1 Fc that exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a wild-type IgG1 Fc domain.

[0065] 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 EU numbering. In some embodiments, the variant IgG1 Fc comprises amino acid substitutions L234A, L235E, and G237A according to EU numbering.

[0066] In some embodiments, the Fc comprises the amino acid substitution C220S, where residues are numbered according to the EU index of Kabat.

[0067] In some embodiments, the Fc lacks the hinge sequence EPKSS or EPKSC.

[0068] In some embodiments, the Fc region comprises K447del, where residues are numbered according to the EU index of Kabat. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 73.

[0069] In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO:167.

[0070] In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:81.

[0071] In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO:168.

[0072] In some embodiments, the TACI-Fc fusion protein is provided in a formulation comprising an acetate buffer having a pH of about 4.0 to about 6.0, proline at a concentration of 1% or about 1% to about 10%, and a surfactant at a concentration of about 0.005 to about 0.05% (w / v).

[0073] In some embodiments, the formulation has a pH of about 5.2.

[0074] In some embodiments, the acetate buffer comprises acetate at a concentration of at or about 5 mM to at or about 15 mM, hi some embodiments, the acetate buffer comprises acetate at a concentration of at or about 10 mM.

[0075] In some embodiments, proline is at a concentration of about 2% to about 5%. In some embodiments, proline is at a concentration of 3% or about 3%.

[0076] In some embodiments, the surfactant is at a concentration of about 0.01 to about 0.025% (w / v), e.g., 0.015% (w / v) or about 0.015% (w / v). In some embodiments, the surfactant is polysorbate 80.

[0077] In some embodiments, the amount of TACI-Fc fusion protein in the formulation is about 50 mg to about 100 mg, hi some embodiments, the amount of TACI-Fc fusion protein in the formulation is at or about 80 mg.

[0078] In some embodiments, the concentration of the TACI-Fc fusion protein is between about 50 mg / mL and about 200 mg / mL, hi some embodiments, the concentration of the TACI-Fc fusion protein is at or about 100 mg / mL.

[0079] In some embodiments, the immune response or activity of B cells is reduced in the subject, hi some embodiments, the number of mature B cells and total circulating B cells is reduced in the subject.

[0080] In some embodiments, circulating serum immunoglobulins are reduced in the subject.

[0081] In some embodiments, one or more of B cell maturation, differentiation, and / or proliferation is reduced or inhibited.

[0082] In some embodiments, the circulating level of APRIL or BAFF protein is reduced in the subject. In some embodiments, the APRIL or BAFF protein is an APRIL homotrimer, a BAFF homotrimer, an APRIL / BAFF heterotrimer, or a BAFF 60-mer.

[0083] In some embodiments, the subject is a human.

[0084] In some embodiments, the subject is an adult subject, hi some embodiments, the subject is 18 years of age or older, e.g., 18-65 years of age. [Brief explanation of the drawings]

[0085] [Figure 1] A schematic diagram of a functional inhibition assay involving recombinant APRIL and BAFF by TACI is shown. In the assay, Jurkat cells were transduced with a luciferase-based NF-κB reporter and stably expressed mouse or human TACI on the cell surface. After activation by recombinant APRIL or BAFF, endogenous NF-κB transcription factors bind to DNA response elements that control transcription of the firefly luciferase gene. Luciferase expression can be monitored, for example, by detection using Bio-Glo™ reagents and measurement using a Cytation 3 reader. [Figure 2]Exemplary human TACI TD Fc fusion molecules are shown for blockade of signaling involving human APRIL (top panel) and BAFF (bottom panel). TACI TD Fc fusions were incubated with APRIL or BAFF for 20 minutes (with shaking at room temperature) and then added to wells containing 150,000 Jurkat / TACI / NFκB-luciferase cells for 5 hours. [Figure 3A] 1 shows the function of exemplary TACI TD Fc fusion molecules for blocking APRIL (top panel of figure) or BAFF (bottom panel of figure). [Figure 3B] Human TACI TD Fc fusion molecules for blockade of mouse APRIL (left panel) and BAFF (right panel) mediated signaling are shown. [Figure 4A] Human TACI TD Fc fusion molecules for blockade of human APRIL (upper panel) and BAFF (lower panel) mediated signaling compared to TACI 13-118-Fc, TACI 30-110-Fc, and belimumab. [Figure 4B] Figure 1 shows the evaluation of APRIL inhibitory activity and binding by various TACI Fc fusion proteins and the affinity-optimized TACI variant 26TACI CRD2-Fc. Figure 2 shows the inhibition of APRIL and BAFF by the indicated TACI variants and 26TACI CRD2-Fc, as assessed in a TACI / Jurkat / NF-κB reporter assay. Increased inhibitory activity is indicated by decreased luciferase production. [Figure 4C] Figure 1 shows the evaluation of BAFF inhibitory activity and binding by various TACI Fc fusion proteins and the affinity-optimized TACI variant 26TACI CRD2-Fc. SPR sensorgrams using medium-density 26TACI CRD2-Fc and telitacicept are shown in black, and the results of nonlinear least-squares regression analysis of the data are shown in orange. Telitacic was supplied by Clinigen. [Figure 5A]1 shows an exemplary human TACI TD Fc fusion molecule 26TACI CRD2-Fc for blocking BAFF-mediated signaling compared to belimumab, BION-1301, and WT TACI-Fc molecules, including WT TACI 30-110 (atacicept) and WT TACI 13-118-Fc (telitacicept). [Figure 5B] 1 shows an exemplary human TACI TD Fc fusion molecule 26TACI CRD2-Fc for blocking APRIL-mediated signaling compared to belimumab, BION-1301, and WT TACI-Fc molecules, including WT TACI 30-110 (atacicept) and WT TACI 13-118-Fc (telitacicept). [Figure 5C] 1 shows an exemplary human TACI TD Fc fusion molecule 26TACI CRD2-Fc for blocking BAFF+APRIL combination-mediated signaling compared to belimumab, BION-1301, and WT TACI-Fc molecules, including WT TACI 30-110 (atacicept) and WT TACI 13-118-Fc (telitacicept). [Figure 5D] 26 shows that 26TACI CRD2-Fc inhibits APRIL more potently than comparative molecules. 26 shows the inhibition of APRIL by 26TACI CRD2-Fc, and the comparative molecules shown were evaluated in a TACI / Jurkat / NF-κB reporter assay. [Figure 5E] 26 shows that TACI CRD2-Fc inhibits BAFF more potently than comparative molecules. 26 shows the inhibition of BAFF by TACI CRD2-Fc, and the comparative molecules shown were evaluated in a TACI / Jurkat / NF-κB reporter assay. [Figure 5F] 26TACI CRD2-Fc inhibits APRIL and BAFF more potently than comparative molecules. ... [Figure 5G]Figure 1 shows inhibition of BAFF multimers by 26TACI CRD2-Fc in a TACI / Jurkat / NF-κB assay compared to comparator molecules. Figure 2 shows inhibition of BAFF 60-mers by 26TACI CRD2-Fc, TACI 30-110-Fc, telitacicept, belimumab, and an anti-APRIL mAb based on the VIS649 mAb sequence. Log(agonist) vs. response in GraphPad Prism. Curve fit with constraints: Hillslope=-1, F=50. [Figure 5H] Figure 1 shows inhibition of BAFF / APRIL heterotrimer by 26TACI CRD2-Fc in a TACI / Jurkat / NF-κB assay compared to a comparator molecule. Figure 2 shows inhibition of heterotrimeric BAFF / APRIL by 26TACI CRD2-Fc, TACI 30-110-Fc, telitacicept, belimumab, and an anti-APRIL mAb based on the VIS649 mAb sequence. Log(agonist) vs. response in GraphPad Prism. Curve fit with constraints: Hillslope=-1, F=50. [Figure 5I] Figure 1 shows inhibition of BAFF / APRIL heterotrimer by 26TACI CRD2-Fc in a TACI / Jurkat / NF-κB assay compared to a comparator molecule. Figure 2 shows inhibition of heterotrimeric BAFF / APRIL by 26TACI CRD2-Fc, TACI 30-110-Fc, telitacicept, belimumab, and an anti-APRIL mAb based on the VIS649 mAb sequence. Log(agonist) vs. response in GraphPad Prism. Curve fit with constraints: Hillslope=-1, F=50. [Figure 5J] Figure 1 shows inhibition of BAFF multimers by 26TACI CRD2-Fc in a TACI / Jurkat / NF-κB assay compared to comparator molecules. Figure 2 shows inhibition of homotrimeric BAFF by 26TACI CRD2-Fc, TACI 30-110-Fc, telitacicept, belimumab, and an anti-APRIL mAb based on the VIS649 mAb sequence. Log(agonist) vs. response in GraphPad Prism. Curve fit with constraints: Hillslope=-1, F=50. [Figure 5K] 1 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) compared to WT TACI-Fc (telitacicept). [Figure 6A] Figure 1 shows an analysis of parameters evaluated in the NZB / NZW mouse model of human SLE. Proteinuria scores were assessed starting at 20 weeks of age. [Figure 6B]

[0023] Figure 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Mean percent change in body weight was assessed starting at 20 weeks of age. [Figure 6C]

[0023] Figure 1 shows an analysis of parameters evaluated in the NZB / NZW mouse model of human SLE. Survival was assessed starting at 20 weeks of age. [Figure 6D] Figure 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Serum was analyzed for anti-double stranded DNA IgG titers (**** for Fc by Student's t-test, p<0.0001 for anti-dsDNA IgG). [Figure 6E] 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Serum was analyzed for blood urea nitrogen (BUN) (*** for Fc by Student's t-test, p=0.0008 for BUN). [Figure 6F]

[0023] Figure 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Kidneys were processed and analyzed by histology in duplicate periodic acid-Schiff (PAS) stained sections. Individual component and total histology scores are shown. [Figure 6G] 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Frozen kidneys were also sectioned and stained for immunohistochemical analysis of mouse IgG glomerular deposits. [Figure 6H] 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Frozen kidneys were also sectioned and stained for immunohistochemical analysis of complement C3 glomerular deposition. [Figure 6I] 1 shows an analysis of parameters evaluated in the NZB / NZW mouse model of human SLE. Histological scores ± SEM are shown. [Figure 6J] Figure 1 shows an analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Sialadentis as assessed by submandibular gland histology score. [Figure 6K] Analysis of parameters assessed in the NZB / NZW mouse model of human SLE. Renal IgG deposit scores (mean + SD) assessed by IHC from right kidneys at termination (left), and representative IHC (10x) of renal IgG deposits from Fc control or 26TACI CRD2-Fc treated mice are shown (left). [Figure 6L] 1 shows an analysis of parameters evaluated in the NZB / NZW mouse model of human SLE. Serum was analyzed for blood urea nitrogen (BUN). [Figure 7] The ability of TACI mutations (K77E / F78Y / Y102D) to inhibit APRIL (left panel) and BAFF (right panel) mediated signaling, as quantified by luciferase production in Jurkat / NF-κB / TACI cells, is shown. [Figure 8A] 1 shows a schematic diagram of an exemplary TACI-Fc fusion protein. 2 shows an exemplary TACI-Fc fusion protein containing two cysteine-rich quasi-repeats (CRDs). [Figure 8B] 1 shows a schematic diagram of an exemplary TACI-Fc fusion protein. 2 shows an exemplary TACI-Fc fusion protein containing one cysteine-rich quasi-repeat (CRD, e.g., CRD2). [Figure 9]

[0023] An exemplary sequence alignment is shown to identify corresponding residues within a sequence compared to a reference sequence. A "*" symbol between two aligned amino acids indicates that the aligned amino acids are identical. A "-" symbol indicates a gap in the alignment. Exemplary, non-limiting positions for amino acid substitutions described herein are shown in bold. Based on the alignment of two similar sequences that share identical residues, one skilled in the art can use the conserved identical amino acid residues as a guide to identify "corresponding" positions within a sequence by comparison with the reference sequence.

[0024] An exemplary alignment of the reference TACI extracellular domain sequence set forth in SEQ ID NO: 122 (including the complete extracellular domain, including CRD1 and CRD2 and the initial methionine residue) with the TACI extracellular domain sequence set forth in SEQ ID NO: 13 (including only a single CRD, CRD2). Alignment of identical residues shows, for example, that amino acid residue E7 of SEQ ID NO: 13 corresponds to residue E74 of SEQ ID NO: 122, amino acid residue K10 of SEQ ID NO: 13 corresponds to residue K77 of SEQ ID NO: 122, amino acid residue Y12 of SEQ ID NO: 13 corresponds to Y79 of SEQ ID NO: 122, amino acid residue L15 of SEQ ID NO: 13 corresponds to L82 of SEQ ID NO: 122, amino acid residue R17 of SEQ ID NO: 13 corresponds to R84 of SEQ ID NO: 122, and amino acid residue D16 of SEQ ID NO: 13 corresponds to D85 of SEQ ID NO: 122. It is within the level of ordinary skill in the art to perform similar alignments between two similar protein sequences to identify corresponding residues, including based on the examples and explanations herein. [Figure 10](Figure 10A) Analysis of parameters evaluated using the mouse keyhole limpet hemocyanin (KLH) model. Serum KLH IgM OD levels were evaluated as the primary response. (Figure 10B) Analysis of parameters evaluated using the mouse keyhole limpet hemocyanin (KLH) model. Serum KLH IgM OD levels were evaluated as the secondary response. (Figure 10C) Analysis of parameters evaluated using the mouse keyhole limpet hemocyanin (KLH) model. Similarly, serum anti-KLH IgG1 OD levels were evaluated as the primary response. (Figure 10D) Analysis of parameters evaluated using the mouse keyhole limpet hemocyanin (KLH) model. Similarly, serum anti-KLH IgG1 OD levels were evaluated as the secondary response. [Figure 11A]

[0033] Figure 1 shows the analysis of harvested spleens evaluated from the mouse keyhole limpet hemocyanin (KLH) immunization model. Spleens were processed and analyzed by weight. [Figure 11B] Analysis of harvested spleens evaluated from the mouse keyhole limpet hemocyanin (KLH) immunization model is shown. Spleens were processed and analyzed by total cell count. [Figure 11C] Figure 1 shows that 26TACI CRD2-Fc has a stronger effect on splenocytes than WT TACI-Fc in KLH-immunized mice. The total number of splenocytes was enumerated by flow cytometry. [Figure 12A]

[0023] Figure 1 shows analysis of spleens assessed for cell subtype populations. Spleen composition in the mouse keyhole limpet hemocyanin (KLH) model is shown, with results showing B cell subset numbers compared to group means. [Figure 12B] 1 shows an analysis of spleens assessed for cell subtype populations. 2 shows an analysis of spleens assessed for cell subtype phenotype composition in the mouse keyhole limpet hemocyanin (KLH) model, with results shown for germinal center B cell and plasma cell numbers. [Figure 12C]Figure 1 shows the analysis of spleens evaluated for cell subtype populations. Figure 2 shows the analysis of spleens evaluated for cell subtype phenotype composition in the mouse keyhole limpet hemocyanin (KLH) model, with results for plasma cell numbers. Individual mice are shown plotted for splenic plasma cells. [Figure 12D] Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., T1 B cells) on day 20, enumerated by flow cytometry, are shown. [Figure 12E] Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., B cells) on day 20, enumerated by flow cytometry, are shown. [Figure 12F] Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., T2 B cells) on day 20, enumerated by flow cytometry, are shown. [Figure 12G] Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., GC cells) on day 20, enumerated by flow cytometry, are shown. [Figure 12H] Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., FOL B cells) on day 20, enumerated by flow cytometry, are shown. [Figure 12I]Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., MZ B cells) on day 20, enumerated by flow cytometry, are shown. [Figure 12J] Figure 1 shows that 26TACI CRD2-Fc affects splenic B and T cell subsets more potently than WT TACI-Fc in KLH-immunized mice. The total numbers of the indicated splenic B cell subsets (i.e., plasma cells) on day 20, as enumerated by flow cytometry, are shown. [Figure 13A]Gating diagram for quantifying B cell subsets and plasma cells in mouse spleens is shown. Cells were gated away from debris in an FSC-A / SSC-A dot plot. This gate was then analyzed on an FSC-H / FSC-A dot plot, followed by an SSC-H / SSC-W dot plot, with cells gated along the established diagonal to exclude doublet cell populations. CD45+ / LiveDead Aqua viability-negative cells were gated from the SSC-H / SSC-W singlet gate to identify live CD45+ cells. The live CD45± cells were then analyzed on a B220 / Gr1 dot plot. B220+ / Gr1- cells were then analyzed on a GL7 / CD95 dot plot to identify GL7+ / CD95+ GC B cells. B220+ / Gr1- cells were also analyzed on a CD138 / CD19 dot plot to identify CD19+ cells, which were then analyzed on a CD23 / CD19 dot plot. CD23+ / CD19+ cells were further analyzed by CD21 / IgM expression to identify CD21+ / IgM+ follicular (FOL) B cells and CD21br / IgMbr T2 B cells. CD23- / CD19+ cells were also analyzed by CD21 / IgM dot plot to identify CD21- / IgMbr type 1 transitional (T1) B cells and CD21br / IgMbr MZ B cells. The live CD45+ cell gate was also analyzed by B220 / CD19 dot plot to identify B220+ / lo / CD19+ B cells. B220lo / CD138+ plasma cells were gated from the B220+ / lo / CD19+ gate. FSC-A = forward scatter area, SSC-A = side scatter area, H = height, W = width. [Figure 13B]Gating diagram for quantifying CD4+ T cells in mouse spleens is shown. Cells were gated away from debris in an FSC-A / SSC-A dot plot. This gate was then analyzed on an FSC-H / FSC-A dot plot, followed by an SSC-H / SSC-W dot plot, with cells gated along the established diagonal to exclude doublet cell populations. CD45+ / LiveDead Aqua viability-negative cells were gated from the SSC-H / SSC-W singlet gate to identify live CD45+ cells. The live CD45± cells were then gated on a B220 / CD3 dot plot. CD3+ T cells were then analyzed on a CD4 / CD8 dot plot. CD4+ T cells were analyzed on a PD1 / CXCR5 dot plot to identify PD1+CXCR5+ T cells. T cells = T follicular T cells, FSC-A = forward scatter area, SSC-A = side scatter area, H = height, W = width. [Figure 14A] Figure 1 shows the number of T cells in the mouse keyhole limpet hemocyanin (KLH) model. Figure 2 shows splenic CD3+ cells. [Figure 14B] Figure 1 shows the number of T cells in the mouse keyhole limpet hemocyanin (KLH) model. Figure 2 shows splenic CD8+ cells. [Figure 14C] Figure 1 shows the number of T cells in the mouse keyhole limpet hemocyanin (KLH) model. Figure 2 shows splenic CD4+ cells. [Figure 14D] This shows the number of T cells in a mouse keyhole limpet hemocyanin (KLH) model. This shows splenic follicular helper T cells. [Figure 14E] Splenic T cells were also enumerated by flow cytometry. Individual mice are plotted, and the mean ± SD is shown as the horizontal line and error bars, respectively. GC = germinal center, T1 = type 1 transitional B cells, T2 = type 2 transitional B cells, FOL = follicular, MZ = marginal zone. [Figure 14F]Splenic T cells were also enumerated by flow cytometry. Individual mice are plotted, and the mean ± SD is shown as the horizontal line and error bars, respectively. GC = germinal center, T1 = type 1 transitional B cells, T2 = type 2 transitional B cells, FOL = follicular, MZ = marginal zone. [Figure 14G] The total number of TFH cells / spleen of KLH-induced or naive mice on day 20, enumerated by flow cytometry, is shown, and individual mice are plotted. [Figure 15] 1 shows Tcm and Tem cell populations in the mouse keyhole limpet hemocyanin (KLH) model. [Figure 16A] Figure 1 shows the overall incidence of sialadenitis in diabetes-prone mice after treatment with test molecules. [Figure 16B] 1 shows the extent of sialadenitis in diabetes-prone mice after treatment with test molecules. [Figure 17A] Figure 1 shows the overall incidence of insulitis in diabetes-prone mice after treatment with test molecules. [Figure 17B] 1 shows the extent of insulitis in diabetes-prone mice after treatment with test molecules. [Figure 18] 1 shows serum immunoglobulin (IgM, IgA, and IgG) concentrations for exemplary test molecules in a pharmacokinetic / pharmacodynamic study following a single intravenous infusion in male Sprague Dawley rats. [Figure 19] 1 shows serum immunoglobulin (IgM, IgA, and IgG) concentrations for exemplary test molecules in a pharmacokinetic / pharmacodynamic study following a single intravenous infusion in male Sprague Dawley rats. [Figure 20A] 1 shows individual animal serum concentration versus time profiles of exemplary test molecules administered to cynomolgus monkeys in a PK / PD model. [Figure 20B]

[0023] Figure 1 shows individual animal serum concentration versus time profiles of exemplary test molecules administered to cynomolgus monkeys in a PK / PD model. The results for atacicept shown are based on published data (Carbonatto et al. (2008) Toxicol Sci 105:200-210). [Figure 21A] 1 shows serum IgM, IgA, and IgG levels in animals administered exemplary test molecules. 1 shows PK / PD of IgM, IgA, and IgG in a cynomolgus monkey PK / PD model. [Figure 21B]

[0033] Figure 1 shows serum IgM, IgA, and IgG levels in animals administered exemplary test molecules. Serum IgM, IgA, and IgG levels (mean + range) in each treatment group measured by ELISA at various time points are shown and plotted as a percentage of baseline serum concentrations obtained from serum collected on day -8. [Figure 22] Absolute cell counts are shown for animals dosed with exemplary test molecules in the cynomolgus monkey PK / PD model. [Figure 23] 1 shows the % of cells from baseline for animals dosed with exemplary test molecules in the cynomolgus monkey PK / PD model. [Figure 24] Absolute numbers or relative percentages of expanded T cell animals administered exemplary test molecules in the cynomolgus monkey PK / PD model are shown. [Figure 25] (Figure 25A) Predicted human PK profile after repeated IV administration every 4 weeks in a two-compartment PK model. (Figure 25B) Predicted human PK profile after repeated IV administration every 2 weeks in a two-compartment PK model. [Figure 26A] 1 shows inhibition of class-switched memory B cells. [Figure 26B] Shows inhibition of plasma cells. [Figure 26C] Shown is inhibition of immunoglobulin secretion. [Figure 26D] Shown is inhibition of immunoglobulin secretion. [Figure 26E] Shown is inhibition of immunoglobulin secretion. [Figure 26F] CD19+ B cells were activated with rhCD40L and re-cultured with exogenous APRIL, BAFF, and 26TACI CRD2-Fc or the indicated comparison molecules. Cells were stained and analyzed by flow cytometry to identify class-switched memory B cells (IgD, IgM, CD27+). [Figure 26G] CD19+ B cells were activated with rhCD40L and re-cultured with exogenous APRIL, BAFF, and 26TACI CRD2-Fc or the indicated comparison molecules. Cells were stained and analyzed by flow cytometry to identify plasma cells (IgM, IgD, CD38+, CD319+). [Figure 26H] After 7 days, supernatants were collected and IgM secretion was quantified by multiplex analysis. Statistical differences between group medians were determined using the Kruskal-Wallis test and the uncorrected Dunn's test. A p value of <0.05 was considered statistically significant. [Figure 26I] After 7 days, supernatants were collected and IgA secretion was quantified by multiplex analysis. Statistical differences between group medians were determined using the Kruskal-Wallis test and the uncorrected Dunn's test. A p value of <0.05 was considered statistically significant. [Figure 26J] After 7 days, supernatants were collected and IgG1 secretion was quantified by multiplex analysis. Statistical differences between group medians were determined using the Kruskal-Wallis test and the uncorrected Dunn's test. A p value of <0.05 was considered statistically significant. [Figure 26K] After 7 days, supernatants were collected and IgG2 secretion was quantified by multiplex analysis. Statistical differences between group medians were determined using the Kruskal-Wallis test and the uncorrected Dunn's test. A p value of <0.05 was considered statistically significant. [Figure 26L] After 7 days, supernatants were collected and IgG3 secretion was quantified by multiplex analysis. Statistical differences between group medians were determined using the Kruskal-Wallis test and the uncorrected Dunn's test. A p value of <0.05 was considered statistically significant. [Figure 26M] After 7 days, supernatants were collected and IgG4 secretion was quantified by multiplex analysis. Statistical differences between group medians were determined using the Kruskal-Wallis test and the uncorrected Dunn's test. A p value of <0.05 was considered statistically significant. [Figure 27] (Figure 27A) shows the levels of plasma cells in the bone marrow of a mouse model of CIA administered with the test molecule. (Figure 27B) shows the levels of plasma cells in the spleen of a mouse model of CIA administered with the test molecule. (Figure 27C) shows the levels of plasma cells in the lymph nodes of a mouse model of CIA administered with the test molecule. [Figure 28] 1 shows the number of plasma cells in bone marrow smears from cynomolgus monkeys administered an exemplary TACI-Fc fusion protein. [Figure 29A] 1 shows the dose-dependent serum concentration over time profiles for animals administered an exemplary TACI-Fc fusion protein in a cynomolgus monkey one-month GLP toxicity study. [Figure 29B] 1 shows the % of cells from baseline for animals administered an exemplary TACI-Fc fusion protein in a cynomolgus monkey 1-month GLP toxicity study. [Figure 30A] 1 shows serum IgA, IgG, IgM, and IgE levels in animals administered exemplary TACI-Fc fusions in a cynomolgus monkey one-month GLP toxicity study. [Figure 30B] 1 shows serum IgA, IgG, IgM, and IgE levels in animals administered exemplary TACI-Fc fusions in a 6-month GLP toxicity study. [Figure 31] Analysis of harvested spleens evaluated from a murine chronic graft-versus-host disease (cGVHD) model. Spleens were processed and analyzed for weight and total cell count. [Figure 32] 1 shows an analysis of spleens derived from a mouse chronic graft-versus-host disease model, where the cell population composition was assessed, and the results of CD45+ cell count and B220+ B cell count are shown. [Figure 33]Analysis of spleens assessed for cell subtype population composition is shown, with results for CD4+ and CD8+ T cell subset counts shown. [Figure 34] 1 shows the number of CD4+ T cell subsets in a cGVHD model. [Figure 35A] The numbers of B220+ B cells and CD1dhiCD5+ B-1 cells in the cGVHD model are shown. [Figure 35B] The numbers of type 1 transitional (T1) and type 2 transitional (T2) B cells in a cGVHD model are shown. [Figure 36A] Follicular and marginal zone (MZ) B cells in a cGVHD model. [Figure 36B] Figure 1 shows the number of germinal center (GC) B cells and plasma cells in a cGVHD model. [Figure 37] 1 shows the numbers of early plasma cells, plasmablasts, and long-lived plasma cells (LL-PC) in a cGVHD model. [Figure 38A] 1 shows renal IgG immune complex deposits in the kidney as measured by immunohistochemical staining with a fluorescently labeled antibody specific for mouse IgG. [Figure 38B] Representative IHC (20x) of Fc control, TACI CRD2-Fc (DAPI overlay at bottom right), or renal IgG deposits from naive mice are shown. [Figure 39] Analysis of anti-dsDNA autoantibody serum titers at 8 and 13 weeks is shown. [Figure 40] (FIG. 40A) Analysis of anti-dsDNA autoantibody serum titers in the H-2bm12 mouse model of autoantibody-associated glomerulonephritis over a 56-day or 8-week period. (FIG. 40B) Analysis of anti-dsDNA autoantibody serum titers in the H-2bm12 mouse model of autoantibody-associated glomerulonephritis at 8 weeks. [Figure 41] 1 shows renal IgG immune complex deposits in the kidney as measured by immunohistochemical staining with a fluorescently labeled antibody specific for mouse IgG. [Figure 42]1 shows serum IgA, IgM, and IgG (IgG1, IgG2b, and IgG3) levels in animals administered exemplary TACI-Fc fusions in a mouse model of autoantibody-associated glomerulonephritis. [Figure 43A] 1 shows the levels of anti-SRBC IgG1 in animals administered 26TACI CRD2-Fc compared to BAFF and APRIL specific biologics. [Figure 43B] 1 shows the levels of plasma cells in animals treated with 26TACI CRD2-Fc compared to BAFF and APRIL specific biologics. [Figure 43C] 26 shows that TACI CRD2-Fc exhibits enhanced immunosuppressive activity in a mouse SRBC immunization model compared to the inhibitors telitacicept and BAFF alone or APRIL alone. Serum anti-SRBC Ig concentrations measured on day 15 are shown. Data are presented as mean ± SD. [Figure 43D] 26 shows that TACI CRD2-Fc exhibits enhanced immunosuppressive activity in a mouse SRBC immunization model compared to the inhibitors telitacicept and BAFF alone or APRIL alone. Serum anti-SRBC Ig concentrations measured on day 15 are shown. Data are presented as mean ± SD. [Figure 43E] 26 shows that TACI CRD2-Fc exhibits enhanced immunosuppressive activity in a mouse SRBC immunization model compared to the inhibitors telitacicept and BAFF alone or APRIL alone. Serum anti-SRBC Ig concentrations measured on day 15 are shown. Data are presented as mean ± SD. [Figure 43F] 26 shows that TACI CRD2-Fc exhibits enhanced immunosuppressive activity in a mouse SRBC immunization model compared to the inhibitors telitacicept and BAFF alone or APRIL alone. Serum anti-SRBC Ig concentrations measured on day 15 are shown. Data are presented as mean ± SD. [Figure 43G]This shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared with the inhibitors telitacicept and BAFF alone or APRIL alone in a mouse SRBC immunization model. Total germinal center (GC) B cell numbers per spleen were enumerated by flow cytometry, and values ​​for individual mice were plotted. Data are presented as median ± interquartile range. [Figure 43H] This shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared with the inhibitors telitacicept and BAFF alone or APRIL alone in a mouse SRBC immunization model. Total CD4+ TNF-α cells / spleen were enumerated by flow cytometry, and values ​​for individual mice were plotted. Data are presented as median ± interquartile range. [Figure 43I] This shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared with the inhibitors telitacicept and BAFF alone or APRIL alone in a mouse SRBC immunization model. Total plasma cell (PC) numbers per spleen were enumerated by flow cytometry, and values ​​for individual mice were plotted. Data are shown as mean ± SD. [Figure 43J] This shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to the inhibitors telitacicept and BAFF alone or APRIL alone in a mouse SRBC immunization model. Total plasmablast (PB) numbers per spleen were enumerated by flow cytometry, and values ​​for individual mice were plotted. Data are shown as mean ± SD. [Figure 43K] This shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared with telitacicept and BAFF alone or APRIL alone in a mouse SRBC immunization model. The percentage of long-lived plasma cells (LL-PC) in the bone marrow was also determined by flow cytometry, and data for individual mice were plotted. Data are shown as mean ± SD. [Figure 43L]This shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to the inhibitors telitacicept and BAFF alone or APRIL alone in a mouse SRBC immunization model. The percentage of total plasma cells (PCs) in the bone marrow was also determined by flow cytometry, and data for individual mice were plotted. Data are shown as mean ± SD. [Figure 44A] 1 shows individual serum concentration versus time profiles of the 26TACI CRD2-Fc fusion molecule in a human cohort administered 26TACI CRD2-Fc by the IV route. 26TACI CRD2-Fc serum concentrations over 56 days are shown. [Figure 44B] 1 shows individual serum concentration versus time profiles of the 26TACI CRD2-Fc fusion molecule in a human cohort administered 26TACI CRD2-Fc by the SC route. 26TACI CRD2-Fc serum concentrations over 56 days are shown. [Figure 44C] 1 shows individual serum concentration versus time profiles of the 26TACI CRD2-Fc fusion molecule in a human cohort administered 26TACI CRD2-Fc by the IV route. 26TACI CRD2-Fc serum concentrations over 112 days are shown. [Figure 44D] 1 shows individual serum concentration versus time profiles of the 26TACI CRD2-Fc fusion molecule in a human cohort administered 26TACI CRD2-Fc by the SC route. 26TACI CRD2-Fc serum concentrations over 112 days are shown. [Figure 45A] 1 shows serum IgA, IgG, IgM levels and their corresponding changes from baseline in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 45B] Serum IgA, IgG, IgM levels and their corresponding changes from baseline in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 45C]1 shows serum galactose-deficient IgA1 (Gd-IgA1) levels and corresponding change from baseline in a human cohort administered 26TACI CRD2-Fc by the IV route. Serum Gd-IgA1 levels over 28 days are shown. [Figure 45D] 1 shows serum galactose-deficient IgA1 (Gd-IgA1) levels and corresponding change from baseline in a human cohort administered 26TACI CRD2-Fc by the SC route. Serum Gd-IgA1 levels over 28 days are shown. [Figure 45E] 1 shows serum galactose-deficient IgA1 (Gd-IgA1) levels and corresponding change from baseline in a human cohort administered 26TACI CRD2-Fc by the IV route. Serum Gd-IgA1 levels over 112 days are shown. [Figure 45F] 1 shows serum galactose-deficient IgA1 (Gd-IgA1) levels and corresponding change from baseline in a human cohort administered 26TACI CRD2-Fc by the SC route. Serum Gd-IgA1 levels over 112 days are shown. [Figure 46] Serum IgA, IgG, or IgM levels and their corresponding changes from baseline are shown in a human cohort administered 80 mg of 26TACI CRD2-Fc SC compared to the levels of the comparators, atacicept (first column from the left), telitacicept (second column), BION 1301 (third column), or sibeprenlimab (fourth column), as determined from published data. [Figure 47A] 1 shows a dose-dependent, on-target 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 by the IV route. [Figure 47B] 1 shows a dose-dependent, on-target 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 by the SC route. [Figure 47C]1 shows the frequency of circulating CD27-IgD+ antibody-secreting cells, including naive B cells, in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 47D] 1 shows the frequency of circulating CD27-IgD+ antibody-secreting cells, including naive B cells, in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 47E] 1 shows the frequency of circulating CD27+IgD− antibody-secreting cells, including memory B cells, in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 47F] 1 shows the frequency of circulating CD27+IgD− antibody-secreting cells, including memory B cells, in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 48A] 1 shows the dose-dependent decrease and duration of free APRIL (pg / mL or % change from baseline) observed up to 28 days post-dose in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 48B] 1 shows the dose-dependent decrease and duration of free APRIL (pg / mL or % change from baseline) observed up to 28 days post-dose in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 48C] 1 shows the dose-dependent decrease and duration of free APRIL (pg / mL or % change from baseline) observed up to 56 days post-dose in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 48D] 1 shows the dose-dependent decrease and duration of free APRIL (pg / mL or % change from baseline) observed up to 56 days post-dose in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 48E] 1 shows the dose-dependent decrease and duration of free BAFF (% change from baseline) observed up to 28 days post-dose in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 48F] 1 shows the dose-dependent decrease and duration of free BAFF (% change from baseline) observed up to 28 days post-administration in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 48G] 1 shows the dose-dependent decrease and duration of free APRIL (pg / mL) observed up to 112 days post-dose in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 48H] 1 shows the dose-dependent decrease in free APRIL (pg / mL) and its duration observed up to 112 days post-administration in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 48I] 1 shows the dose-dependent decrease and duration of free BAFF (pg / mL) observed up to 112 days post-administration in a human cohort administered 26TACI CRD2-Fc by the IV route. [Figure 48J] 1 shows the dose-dependent decrease in free BAFF (pg / mL) and its duration observed up to 112 days post-administration in a human cohort administered 26TACI CRD2-Fc by the SC route. [Figure 49A] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. Anti-SRBC Ig serum concentrations were measured on day 15. Data are presented as median ± interquartile range. [Figure 49B] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. Anti-SRBC Ig serum concentrations were measured on day 15. Data are presented as mean ± SD. [Figure 49C]Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. Anti-SRBC Ig serum concentrations were measured on day 15. Data are presented as mean ± SD. [Figure 49D] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. Anti-SRBC Ig serum concentrations were measured on day 15. Data are presented as mean ± SD. [Figure 49E] 26 shows that TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. The percentage of plasma cells in the bone marrow, as enumerated by flow cytometry, is shown and plotted for individual mice. Data are presented as median ± interquartile range. [Figure 49F] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. The total number of germinal center (GC) B cells / spleen enumerated by flow cytometry is shown, and individual mice are plotted. Data are presented as median ± interquartile range. [Figure 49G] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and WT TACI CRD2-Fc in a mouse SRBC immunization model. The total number of CD4+ T cells / spleen enumerated by flow cytometry is shown and plotted for individual mice. Data are presented as mean ± SD. [Figure 50A] 26 shows that TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. Anti-SRBC Ig serum levels measured on day 15 are shown. Data are presented as mean ± SD. [Figure 50B]26 shows that TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. Anti-SRBC Ig serum levels measured on day 15 are shown. Data are presented as mean ± SD. [Figure 50C] 26 shows that TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. Anti-SRBC Ig serum levels measured on day 15 are shown. Data are presented as mean ± SD. [Figure 50D] 26 shows that TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. Anti-SRBC Ig serum levels measured on day 15 are shown. Data are presented as mean ± SD. [Figure 50E] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. The percentage of plasma cells in bone marrow enumerated by flow cytometry is shown, and individual mice are plotted. No bone marrow was collected from the naive group. Data are presented as mean ± SD. [Figure 50F] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. The total number of germinal center (GC) B cells / spleen enumerated by flow cytometry is shown, and individual mice are plotted. Data are presented as median ± interquartile range. Data are presented as mean ± SD. [Figure 50G] Figure 2 shows that 26TACI CRD2-Fc exhibits improved immunosuppressive activity compared to telitacicept and anti-CD20 antibodies in a mouse SRBC immunization model. The total number of CD4+ T cells / spleen enumerated by flow cytometry is shown, and individual mice are plotted. Data are presented as mean ± SD. [Figure 51A]2 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN) or primary membranous nephropathy (pMN). Urinary protein:creatinine ratios and anti-SRBC Ig serum concentrations measured on days 3, 8, and 15 after administration of TACI CRD2-Fc are shown. [Figure 51B] 2 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN) or primary membranous nephropathy (pMN). Shown are urinary protein:creatinine ratios and anti-SRBC Ig serum concentrations measured on days 3, 8, 15, 22, 29, 43, and 57 after administration of TACI CRD2-Fc. [Figure 51C] 2 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN) or primary membranous nephropathy (pMN). Urinary protein:creatinine ratios measured 12 weeks after administration of TACI CRD2-Fc in patients with IgAN and pMN are shown. [Figure 51D] 1 shows serum IgA, IgG, and IgM in IgAN patients. [Figure 51E] 1 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). Percent UPCR change (mean±SD) from baseline in a single (spot) UPCR test is shown. [Figure 51F] 1 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). Percent change in UPCR from baseline (mean±SD) in the 24-hour UPCR test is shown. [Figure 51G] 1 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). IgA, IgG, and IgM (mg / dL) are shown as percent change (mean ± SD) from baseline over time in IgAN patients receiving 80 mg Q4W or 240 mg Q4W. [Figure 51H] 1 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). Gd-IgA1 is shown as percent change (mean±SD) from baseline over time. [Figure 51I] 1 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). eGFR is shown as change from baseline (mean±SD). [Figure 51J] 1 shows results after administration of 26TACI CRD2-Fc in subjects with IgA nephropathy (IgAN). eGFR is shown as percent change from baseline (mean±SD). [Figure 51K] 1 shows results after administration of 26TACI CRD2-Fc in subjects with primary membranous nephropathy (pMN). Percent change from baseline (mean±SD) in 24-hour UPCR tests is shown. [Figure 51L] 1 shows results after administration of 26TACI CRD2-Fc in subjects with primary membranous nephropathy (pMN). Shown are the changes from baseline (mean ± SD) in circulating levels (RU / mL) of the disease-specific biomarker anti-phospholipase A2 receptor. [Figure 52A] We demonstrate that 26TACI CRD2-Fc provides benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). We demonstrate an RBC-restricted triple fusion protein that can be bound by T cell receptors to initiate the pathogenesis of AIHA. [Figure 52B] We show that 26TACI CRD2-Fc provides benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). HOD autoantibodies in HOD mice were not administered CTLA-4, IL-10R, LAG-3, or PD-1 antibodies (4 days before administration); 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]26 shows that TACI CRD2-Fc provides benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). 26 shows the change in HOD autoantibodies at day 28 in HOD mice administered TACI CRD2-Fc, Fc control, or PBS. [Figure 52D] Figure 2 shows that 26TACI CRD2-Fc provides benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). Figure 3 shows the number of plasma cells per spleen in HOD mice treated with 26TACI CRD2-Fc, Fc control, or PBS. [Figure 52E] 26TACI CRD2-Fc confers benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). Plasma cell counts per spleen and bone marrow are shown for HOD mice treated with 26TACI CRD2-Fc, Fc control, anti-CD20, or PBS. [Figure 52F] 26TACI CRD2-Fc provides benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). Figure 52B shows hematocrit levels in HOD mice before administration of 26TACI CRD2-Fc, Fc control, or PBS (4 Aby), 3 days before administration of 26TACI CRD2-Fc, Fc control, or PBS (day -3), and 15 and 28 days after administration of 26TACI CRD2-Fc, Fc control, or PBS. [Figure 52G] Figure 1 shows that 26TACI CRD2-Fc provides benefit in the HEL-OVA-Duffy (HOD) mouse model of autoimmune hemolytic anemia (AIHA). Figure 1 shows autoantibodies (antiglobulins) bound to red blood cells (RBCs) in HOD mice 4 days before administration of 26TACI CRD2-Fc, Fc control, or PBS, and 9, 15, 23, and 28 days after administration of 26TACI CRD2-Fc, Fc control, or PBS. [Figure 53A]26 shows that TACI CRD2-Fc provides benefit in an experimental autoimmune myasthenia gravis (EAMG) mouse model. A timeline of the EAMG mouse model is shown. [Figure 53B] 26 shows that TACI CRD2-Fc provides benefit in an experimental autoimmune myasthenia gravis (EAMG) mouse model. Mean EAMG clinical scores over time are shown. [Figure 53C] 26 shows that TACI CRD2-Fc provides benefit in an experimental autoimmune myasthenia gravis (EAMG) mouse model. Serum anti-AChR IgG concentrations at day 91 (end of study) are shown. [Figure 53D] 26 shows that TACI CRD2-Fc provides benefit in an experimental autoimmune myasthenia gravis (EAMG) mouse model. Serum Ig isotype concentrations at day 91 (end of study) are shown. [Figure 53E] 26 shows that TACI CRD2-Fc provides benefit in an experimental autoimmune myasthenia gravis (EAMG) mouse model. Serum Ig isotype concentrations at day 91 (end of study) are shown. [Figure 53F] 26 shows that TACI CRD2-Fc provides benefit in an experimental autoimmune myasthenia gravis (EAMG) mouse model. Muscle AChR content at day 91 (end of study) is shown. [Figure 54A] 1 shows the diagnostic plots of the population pharmacokinetic analysis. 2 shows the PK model structure, where Vc refers to the blood system and Vp refers to the tissues. [Figure 54B] Diagnostic plots of the population pharmacokinetic analysis are shown, showing individual predicted concentrations (μg / mL) versus observed concentrations (μg / mL). [Figure 54C] 1 shows diagnostic plots of the population pharmacokinetic analysis, showing population predicted concentrations (μg / mL) versus observed concentrations (μg / mL). [Figure 54D] Diagnostic plots of the population pharmacokinetic analysis are shown, showing time (days) versus conditional weighted residuals. [Figure 54E]Diagnostic plots of the population pharmacokinetic analysis are shown, showing population predictions (μg / mL) versus conditional weighted residuals. [Figure 55] Concentrations of 26TACI CRD2-Fc over 8 weeks following 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 are shown. [Figure 56] 1 shows a simulation demonstrating the concentration of 26TACI CRD2-Fc over 40 weeks after subcutaneous (SC) administration at doses of 24 mg, 80 mg, and 240 mg. Dosing was repeated every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W) for the 24-week period of administration and for 16 weeks after administration. [Figure 57A] PK and PD modeling of circulating APRIL is shown. PK / PD model structure of circulating free APRIL is shown. [Figure 57B] Figure 1 shows PK and PD modeling of circulating immunoglobulins (IgA, IgG, IgM). Figure 2 shows the PK / PD model structures of circulating IgA, IgM, and IgG. [Figure 57C] Figure 1 shows PK and PD modeling of circulating APRIL and free APRIL (pg / mL) over 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] Figure 1 shows PK and PD modeling of circulating immunoglobulin (IgA). IgA (g / L) over 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]Figure 1 shows PK and PD modeling of circulating immunoglobulin (IgG) (g / L) over 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] Figure 1 shows PK and PD modeling of circulating immunoglobulin (IgM). IgM (g / L) over 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 58A] PK / PD simulation of 26TACI CRD2-Fc. Free APRIL and APRIL are shown as percentage change from baseline over 40 weeks after repeated subcutaneous (SC) administration (80 mg or 240 mg) every 4 weeks (Q4W). [Figure 58B] PK / PD simulation of 26TACI CRD2-Fc.APRIL shown as percentage change from baseline over 40 weeks after 24 weeks of repeated subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). [Figure 58C] PK / PD simulation of 26TACI CRD2-Fc. IgA is shown as percentage change from baseline over 40 weeks after 24 weeks of repeated subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). [Figure 58D] PK / PD simulation of 26TACI CRD2-Fc. IgG is shown as percentage change from baseline over 40 weeks after 24 weeks of repeated subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). [Figure 58E]PK / PD simulation of 26TACI CRD2-Fc. IgM is shown as percentage change from baseline over 40 weeks after 24 weeks of repeated subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). [Figure 58F] PK / PD simulations of 26TACI CRD2-Fc are shown. Free APRIL is shown as the percentage change from baseline over 40 weeks after 24 weeks of repeated subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W), and IgA, IgG, and IgM are shown as the percentage change from baseline over 72 weeks after 24 weeks of repeated subcutaneous (SC) administration (24 mg, 80 mg, 240 mg) every 4 weeks (Q4W), every 8 weeks (Q8W), or every 12 weeks (Q12W). [Figure 58G] PK / PD simulation of 26TACI CRD2-Fc. Free IgG and IgG are shown as percentage change from baseline over 72 weeks after repeated subcutaneous (SC) administration (80 mg or 240 mg) every 4 weeks (Q4W). [Figure 59A] PK and PD modeling of circulating BAFF is shown. PK / PD model structure of circulating free BAFF is shown. [Figure 59B] PK and PD modeling of circulating Gd-IgA1. PK / PD model structure of circulating Gd-IgA1. [Figure 59C] PK and PD modeling of circulating BAFF is shown. Free BAFF (pg / mL) over 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 is shown. BLQ = below limit of quantitation. [Figure 59D]Figure 1 shows PK and PD modeling of circulating Gd-IgA1. Gd-IgA1 (pg / mL) over 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. [Figure 60A] PK / PD simulation of 26TACI CRD2-Fc. Free APRIL and APRIL are shown as percentage change from baseline over 40 weeks after repeated subcutaneous (SC) administration (80 mg or 240 mg) every 4 weeks (Q4W). [Figure 60B] PK / PD simulation of 26TACI CRD2-Fc. Free Gd-IgA1 and Gd-IgA1 are shown as percentage change from baseline over 72 weeks after repeated subcutaneous (SC) administration (80 mg or 240 mg) every 4 weeks (Q4W). [Figure 61A] Summary of target coverage in the simulation. Patients with >95% APRIL coverage are shown. [Figure 61B] Figure 1 shows an overview of the target coverage in the simulation, showing the percentage of patients with IgG below 1.5 g / L. [Figure 62A] Figure 1 shows an overview of target coverage in the APRIL / BAFF simulation. A patient with 95% APRIL / BAFF coverage is shown. [Figure 62B] Figure 1 shows an overview of target coverage in the IgG / Gd-IgA1 simulation, predicting a greater than 50% reduction in Gd-IgA1 in patients. [Figure 63A]

[0023] Figure 1 shows proteinuria scores over time in the IFNα-accelerated NZB / W lupus model. Mean proteinuria scores (+SD) for each treatment group over time are shown, with last observation carried forward (LOCF) used for mice terminated before day 50. Study details and methodology are provided in Example 32. [Figure 63B]Figure 1 shows urinary creatinine scores over time in the IFNα-accelerated NZB / W lupus model. Mean creatinine scores (+SD) over time are shown, with LOCF used for mice terminated before day 50. Study details and methodology are provided in Example 32. [Figure 63C] Figure 1 shows proteinuria scores at day 48 in the IFNα-accelerated NZB / W lupus model. Proteinuria scores at day 48 (last measurement before termination) by treatment group are shown. Data are expressed as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences (p<0.05) are reported. [Figure 63D] Figure 1 shows urinary creatinine scores at day 48 in the IFNα-accelerated NZB / W lupus model. Creatinine scores at day 48 by treatment group are shown. Data are expressed as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences (p<0.05) are reported. [Figure 63E] Figure 1 shows proteinuria and urinary creatinine scores on day 48 in an IFNα-accelerated NZB / W lupus model. The ratio of proteinuria to urinary creatinine scores on day 48 is shown. Data are expressed as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences (p<0.05) are reported. [Figure 64A] Figure 1 shows histological analysis of kidneys at termination (day 50) in an IFNα-accelerated NZB / W lupus model. The total glomerular lesion score in the kidneys is shown. Data are presented as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 64B]

[0033] Figure 1 shows histological analysis of kidneys at termination (day 50) in an IFNα-accelerated NZB / W lupus model. Total tubular and interstitial lesion scores in the kidneys are shown. Data are presented as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 64C] Figure 1 shows histological analysis of spleens at termination (day 50) in an IFNα-accelerated NZB / W lupus model. Mean follicle diameter in the spleen is shown. Data are presented as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 64D] Figure 1 shows histological analysis of the spleen at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Spleen follicle scores are shown. Data are presented as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 64E] Figure 1 shows histological analysis of submandibular glands at termination (day 50) in an IFNα-accelerated NZB / W lupus model. The number of inflammatory foci within the submandibular gland is shown. Data are presented as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 64F]Figure 1 shows histological analysis of lacrimal glands at termination (day 50) in an IFNα-accelerated NZB / W lupus model. The number of inflammatory foci within the lacrimal glands is shown. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. Data are presented as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 65A] Figure 1 shows whole blood hemoglobin (HGB) levels at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Hemoglobin concentrations are shown. Data are presented as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 65B]

[0039] Figure 1 shows whole blood hematocrit (HCT) levels at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Hematocrit (%) is shown. Data are presented as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 65C] Figure 1 shows RBC levels in whole blood at termination (day 50) in the IFNα-accelerated NZB / W lupus model. RBC concentrations are shown. Data are presented as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 65D]Figure 1 shows direct antiglobulin test (DAT) levels in whole blood at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Pan-Ig DAT levels (MFI) are shown. MFI: mean fluorescence intensity recorded using flow cytometry. Data are presented as median ± IQR. Study details and methodology are shown in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 65E] Figure 1 shows direct antiglobulin test (DAT) levels in whole blood at termination (day 50) in the IFNα-accelerated NZB / W lupus model. IgA DAT levels (MFI) are shown. MFI: mean fluorescence intensity recorded using flow cytometry. Data are presented as median ± IQR. Study details and methodology are provided in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 66A] Figure 1 shows serum blood urea nitrogen (BUN) levels at termination (day 50) in the IFNα-accelerated NZB / W lupus model. BUN concentrations are shown. Data are presented as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 66B] Figure 1 shows serum anti-double-stranded (ds)DNA IgM levels at termination (day 50) in the IFNα-accelerated NZB / W lupus model. Anti-dsDNA IgM levels are shown. Data are presented as median ± IQR. Study details and methodology are described in Example 32. Statistical differences between groups were determined by Kruskal-Wallis test with Dunn's test uncorrected for multiple comparisons, as described in Example 32. Only significant differences of p<0.05 are reported. [Figure 67A]HE scoring, serum Ig, and B cell frequency in an experimental model of epidermolysis bullosa acquisita (EBA). Mice in the EBA model treated with 26TACI CRD2-Fc, starting when the disease occupied at least 2% of the surface area, had significantly lower dermal HE scores at the end of treatment compared to Fc control-treated mice. P values ​​are shown from the Mann-Whitney test. [Figure 67B-1] Serum Ig in an experimental model of epidermolysis bullosa acquisita (EBA). Serum immunoglobulins of both total and collagen VII-specific isotypes are shown. P values ​​are shown from the Mann-Whitney test. [Figure 67B-2] See the description of Figure 67B-1. [Figure 67C] Figure 1 shows the frequency of B cells in an experimental model of epidermolysis bullosa acquisita (EBA). Figure 2 shows the frequency of COL7vWFA2 (antigen)-specific B cells in lymph nodes of 26TACI CRD2-Fc-treated mice. Statistical significance between the Fc control-treated group and the 26TACI CRD2-Fc-treated group was determined by Mann-Whitney test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 68] Serum levels of anti-GluN1 peptide IgG antibodies at week 4 (before treatment initiation) and at the end (week 10) of treatment are shown. P values ​​are shown for statistical differences between treatment groups, determined using the Mann-Whitney test at each serum dilution. DETAILED DESCRIPTION OF THE INVENTION

[0086] Detailed Description Provided herein are immunomodulating proteins that bind to one or more ligands, for example, produced as soluble factors, to suppress or reduce B cell responses or activity. Among the provided immunomodulating proteins are proteins that bind to and neutralize the activity of BAFF or APRIL ligands, blocking or antagonizing the activity of B cell stimulatory receptors such as TACI or BCMA. The provided immunomodulating proteins may be fusion proteins of the TACI extracellular domain or binding portion thereof (hereinafter, TACI ECD) and a multimerization domain, e.g., immunoglobulin Fc. For example, a TACI-Fc fusion protein is provided herein. In some embodiments, the immunomodulating proteins provided herein can be used to treat diseases, disorders, or conditions associated with dysregulated immune responses, e.g., diseases, disorders, or conditions associated with inflammatory or autoimmune conditions, including inflammatory or autoimmune diseases.

[0087] The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and protect tissue from excessive damage during immune response, for example, during attacks against pathogenic infections.However, in some cases, the immune system may become dysregulated, and abnormal immune responses may be caused against normal body parts or tissues, resulting in autoimmune diseases or conditions or autoimmune symptoms.In other cases, unwanted immune responses may be caused against foreign tissues, such as transplants, resulting in transplant rejection.

[0088] B cells have long been implicated in autoimmune diseases such as systemic lupus erythematosus (SLE) due to their ability to present antigens to autoreactive T cells, secrete proinflammatory cytokines (Lund, Curr Opin Immunol 2008, 20(3):332-338), and differentiate into antibody-secreting cells (ASCs), i.e., plasmablasts and plasma cells (PCs) involved in the production of pathogenic autoantibodies (Banchereau et al., Cell 2016, 165(3):551-565). Therefore, depletion or inhibition of B cells and ASCs is an attractive approach for many rheumatic and other autoimmune disorders.

[0089] Similarly, because B cells are critical mediators of the systemic immune response, they are also involved in autoimmune diseases of the kidney, blood, skin, and nerves.

[0090] In the immunopathogenesis of IgA nephropathy (IgAN), a type of kidney disease, B cells produce small amounts of antibodies (e.g., Gd-IgA1) and plasma cells produce large amounts of autoantibodies (e.g., anti-Gd-IgA1). This leads to the formation of antibody:autoantibody complexes that deposit and accumulate in mesangial cells, activating the alternative and lectin pathways of the complement system, resulting in chronic inflammation, decreased renal function, hematuria, proteinuria, and a decreased glomerular filtration rate (Maixnerova et al., (2022) J Clin Med, 11(10):2810). Therefore, targeting BAFF and APRIL has emerged as a promising approach to reduce the levels of pathogenic autoantibodies (e.g., Gd-IgA1).

[0091] In autoimmune cytopenias, targeting BAFF and APRIL can result in a reduction of pathogenic autoantibodies that cause destruction of platelets in immune thrombocytopenia (ITP) and red blood cells in warm autoimmune hemolytic anemia (wAIHA) and cold autoimmune hemolytic anemia (cAIHA or CAD).

[0092] B cells are known to be important in the pathogenesis of autoimmune diseases with cutaneous manifestations. These include autoimmune blistering diseases, lupus erythematosus, dermatomyositis, systemic sclerosis, psoriasis, pemphigus, and pemphigoid, the latter two of which are particularly driven by autoantibodies (Fetter et al., Cells (2020) 9(12):2627). Traditionally, skin was thought to lack B cells. However, recent data have shown 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 in the skin, autoreactive skin-associated B cells can locally contribute to autoantibody production, cytokine expression, and crosstalk with autoreactive T cells (Fetter et al., Cells (2020) 9(12):2627).

[0093] Autoimmune blistering diseases (ABDs) are characterized by autoantibodies targeting structural skin proteins. Treatments are limited, with rituximab being the only approved biologic for pemphigus vulgaris (Uzawa et al. (2021) Clin Exp Immunol 203:366; Ma et al. (2023) Front Immunol 13:1064007). However, these diseases can be associated with frequent relapses, often accompanied by elevated levels of the cytokine BAFF3. BAFF and its related cytokine, APRIL, play a key role in B cell activation across a broader range of B cells than rituximab, and are elevated in ABDs, correlating with disease activity. BAFF / APRIL inhibition may result in more durable reductions in autoantibodies and improve clinical outcomes.

[0094] In the case of autoantibody-mediated neurological disorders, there is a rapidly expanding and clinically distinct group of central nervous system (CNS) disorders caused by pathogenic autoantibodies. Some of these autoantibodies target neuroglial surface proteins. Autoantigen-specific B cells have consistently been identified in the circulation of patients with neuroglial surface autoantibody (NSAb)-mediated disorders (Sun et al., Nat Rev Neurol, (2020) 16(9):481-492). The efficacy of specific anti-B cell therapies has been documented for the treatment of patients with multiple sclerosis, neuromyelitis spectrum disorders, autoimmune encephalitis and hyperexcitability CNS disorders, autoimmune neuropathies, myasthenia gravis, and inflammatory myopathies (Stathopoulos and Dalakas, Neurotherapeutics, (2022) 19(3):691-710). Even more specifically, myasthenia gravis (MG) is a typical B cell-mediated autoimmune disorder in which the presence of autoantibodies that specifically target components of the acetylcholine receptor (AChR) reduces neuromuscular transmission at the postsynaptic membrane (Yi et al., Muscle Nerve (2018) 57 (2): 172-184).

[0095] Furthermore, BAFF and APRIL play important roles in B cell biology. It has been reported that upregulation of one or both cytokines is associated with clinical parameters of MG, autoimmune encephalitis, NMOSD, MS, and other autoantibody-associated neurological disorders (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). While therapeutic agents targeting B cell pathways, including BAFF and APRIL, have shown promising clinical potential in the treatment of myasthenia gravis (MG) and other autoantibody-associated neurological disorders, safer and more effective therapies remain needed. Targeting BAFF and APRIL can reduce the levels of pathogenic autoantibodies (e.g., anti-NMDAR, anti-AChR, anti-MOG) and autoantibodies against proteins at the neuromuscular junction or other sites of neuron-neuron or neuron-tissue interaction.

[0096] Therefore, depletion or inhibition of B cells and ASCs is an attractive approach for many renal, hematological, cutaneous, and neurological autoimmune disorders.

[0097] Key modulators of B cell development, differentiation, and survival include the tumor necrosis factor (TNF) family cytokines, B cell-activating factor (BAFF / TNFSF13B) and proliferation-inducing ligand (APRIL / TNFSF13), which are primarily expressed by myeloid cells and signal through multiple receptors. BAFF binds with varying affinities to B cell-expressed BAFF-R (TNFRSF13C), transmembrane activating factor and calcium-regulated cyclophilin ligand interactor (TACI; TNFRSF13B), and B cell maturation antigen (BCMA; TNFRSF17), whereas APRIL binds to TACI and BCMA (Samy, et al., Int Rev Immunol, 2017, 36(1):3-19), heparin sulfate proteoglycans (HSPGs) (Ingold, et al., J Exp Med, 2005, 201(9):1375-1383), such as the syndecan-like 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 by 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, and 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).

[0098] Thus, BAFF and APRIL are TNF superfamily members that bind to both TACI and BCMA receptors on B cells. BAFF also binds to a third receptor, the BAFF receptor (BAFF-R). Both BAFF and APRIL can bind to and activate BCMA and TACI, and BAFF also binds to and activates BAFF-R (Xu et al. 2020 Cancers (Basel) 12(4):1045). Collectively, BAFF and APRIL support the development, differentiation, and survival of B cells, particularly plasmablasts and plasma cells, and are involved in the pathogenesis of B cell-related autoimmune diseases. BAFF and APRIL are initially expressed as transmembrane proteins, primarily in stromal and myeloid cells (Smulski et al. Front. Immunol. 2018 9:2285), and can be cleaved to release soluble cytokines. BAFF circulates as a homotrimer, a 60-mer, or a heterotrimer containing two APRIL and one BAFF or two BAFF and one APRIL protomer. APRIL circulates as either a homotrimer or a heterotrimer and can localize to the intracellular matrix or cell surface via interactions with heparin sulfate proteoglycans.

[0099] Despite structural similarities and involvement of common signaling pathways, APRIL and BAFF play nonredundant roles in B cell regulation, due in part to differences in receptor expression during overlapping stages of B cell development. BAFF plays a key role early in B cell development when BAFF-R is expressed, whereas APRIL plays a key role in the function of differentiated ASCs that express TACI, BCMA, and HSPGs (e.g., syndecan-1 / CD138).

[0100] Expression of BAFF and APRIL increases under pro-inflammatory conditions (Smulski et al. 2018), and elevated serum levels of these cytokines correlate with disease severity in patients with B cell-mediated 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 the development, differentiation, and activation of B cells and plasma cells. For example, activation of BAFF-R contributes to the survival and maturation of transitional and naive B cells, whereas TACI is involved in T cell-independent B cell responses to specific antigens, B cell regulation, and immunoglobulin (Ig) class switch recombination. BCMA, which is upregulated in activated B cells, is important for the long-term survival of plasma cells.

[0101] In some embodiments, 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. In particular, inhibiting or attenuating immune responses, such as B cell responses, may be desirable to reduce or prevent unwanted inflammation, autoimmune symptoms, and / or transplant rejection. However, therapeutic approaches that attempt to modulate the interaction between ligands that mediate immune responses and their receptors are not entirely satisfactory. In some cases, therapies that intervene in and alter the immunomodulatory effects of immune cell, e.g., B cell, activation are constrained by spatial orientation requirements and size limitations imposed by the region of the immunological synapse. In some embodiments, existing therapeutics, including antibody drugs, may not be able to simultaneously interact with multiple target proteins involved in regulating these interactions. For example, soluble receptors and antibodies generally bind competitively (e.g., to one target species at a time) and therefore lack the ability to simultaneously bind multiple targets. Furthermore, differences in pharmacokinetics between drugs that independently target one of these receptors can make it difficult to adequately maintain desired blood concentrations of a combination of drugs targeting two different targets throughout the course of treatment.

[0102] Inhibitors of BAFF and / or APRIL are being investigated in clinical trials for the treatment of various autoimmune or other B cell-related diseases. The BAFF inhibitor, belimumab (Benlysta®), is approved for the treatment of SLE (Benlysta Product Information, 2020), and single-pathway inhibitors of APRIL (e.g., BION1301 and VIS649) are currently being evaluated in phase 2 trials [NCT04684745; NCT04287985].

[0103] Among several B cell-targeting strategies, blockade of BAFF or APRIL has shown clinical promise. Belimumab is an anti-BAFF antibody approved for the treatment of SLE (Hahn, N Engl J Med, 2013, 368(16):1528-1535) and SLE-associated lupus nephritis (LN) (Asif et al., Curr Opin Nephrol Hypertens, 2022). However, clinical remission, as assessed by the lupus low disease activity status (LLDAS) or complete renal response (CRR), is achieved in only a small proportion of patients (12-4% and 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 remain needed. Other BAFF / APRIL-targeting antibodies include ianalumab, 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, American Society of Hematology, 2016) and sibeprenlimab (VIS649) (Myette et al., Kidney Int, 2019, 96(1):104-116).These antibodies have shown promising pharmacodynamic activity in phase 1 clinical trials (Barratt, American Society of Nephrology, 2021; Mathur et al., Kidney Int Rep, 2022,7(5):993-1003), but are more limited by inhibiting either BAFF or APRIL alone (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, a hybrid Fc fusion protein of WT TACI and WT BAFF-R (Zhou et al., Signal Transduct Target Ther, 2019, 4:19), also shares the limitation of inhibiting BAFF alone.

[0104] Co-neutralization of BAFF and APRIL dramatically reduces B cell function, including antibody production, whereas inhibition of either BAFF or APRIL alone mediates relatively limited effects. Fc fusions of the wild-type (WT) extracellular domain of TACI with the Fc domain of IgG1 (e.g., atacicept and telitacicept) are in clinical development and target both BAFF and APRIL. Atacicept (Samy et al., Int Rev Immunol, 2017, 36(1):3-19) and telitacicept (Shi et al., Immunopharmacol Immunotoxicol, 2021, 1-8) are soluble WT TACI extracellular domain (ECD) Fc fusion proteins that potently inhibit BAFF and modestly inhibit APRIL signaling.

[0105] These BAFF / APRIL dual antagonists have been shown to inhibit the survival of immature and mature B cells and plasma cells while sparing B cell precursors and memory B cells (Coggollo et al. 2015 Drug Des Devel Ther. 9:1331-9; Samy et al. 2017; Zhao et al. 2016 J Clin Pharmacol. 56:948-959). Serum IgG, IgM, and IgA levels, as well as the numbers of mature and total circulating B cells, are both reduced (Coggollo et al. 2015; Chen et al. 2014 Clin Pharmacokinet. 53:1033-44; Chen et al. 2016 Br J Clin Pharmacol. 82:41-52; Zhao et al. 2016). When directly compared with the inhibition of either BAFF or APRIL alone in nonclinical studies, dual inhibitors demonstrated stronger pharmacodynamic (PD) effects and greater modification of disease models (Ramanujam et al. 2006 J Clin Invest. 116:724-34; Benson et al. 2008 J Immunol. 180:3655-3659; Haselmeyer et al. 2017 Eur J. Immunol. 47:1075-1085; Samy et al. 2017). Atacicept and telitacicept have shown promising clinical potential in certain autoimmune diseases, such as systemic lupus erythematosus (SLE) and IgA nephropathy, although long-term and / or complete disease remission has not yet been clearly demonstrated. For example, atacicept and telitacicept have both shown clinical activity in SLE (Merrill et al., Arthritis Rheumatol, 2018, 70(2):266-276; Dhillon, Drugs, 2021; Shi et al., Immunopharmacol Immunotoxicol, 2021, 1-8).However, atacicept formally failed to meet the primary endpoint in a pivotal trial (Merrill et al., Arthritis Rheumatol, 2018, 70(2):266-276), and it appears that it is no longer being actively developed for SLE (Vera, Therapeutics Provides Business Update and Reports Second Quarter 2022 Financial Results, 2022). In contrast, telitacicept was conditionally approved in China for the treatment of SLE based on a Phase 2b trial and recently reported positive confirmatory Phase 3 results. However, most subjects still appear to relapse within the first 6 months of treatment (Wu et al., American College of Rheumatology, 2019).

[0106] B cell-targeted therapies have shown promising therapeutic potential but are not entirely satisfactory. To date, cotargeting of B cell antigens (BAFF) and APRIL has only been attempted with the development of the wild-type TACI-Fc molecules atacicept and telitacicept. However, the affinity of wild-type TACI-Fc for APRIL is likely suboptimal, far below that achieved by anti-APRIL mAbs, with K values ​​ranging from 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 telitacicept) shows considerable promise as a therapeutic agent, but its utility appears to be hindered by its low to moderate affinity for APRIL. Thus, although these molecules likely neutralize BAFF sufficiently, their inefficient blockade of APRIL activity clearly leaves room for improvement. These findings provide clinical validation of the BAFF / APRIL pathway for SLE but also suggest that further refinement of the drug design of atacicept and telitacicept, perhaps particularly by improving APRIL inhibition, may offer a unique opportunity to realize more effective yet safer treatment options.

[0107] Among the provided embodiments are those that provide improved neutralizing activity and suppression or reduction of B cell responses. In some embodiments, the improved activity involves increased or improved binding or interaction of the provided immunomodulatory proteins (e.g., TACI-Fc fusion proteins) with BAFF and / or APRIL. The provided immunomodulatory proteins block or antagonize the interaction of BAFF or APRIL, e.g., a BAFF or APRIL homotrimer, a BAFF / APRIL heterotrimer, or a BAFF 60-mer, with their cognate B cell stimulatory receptors, thereby neutralizing the activity of the BAFF and / or APRIL ligand. In some embodiments, the provided immunomodulatory proteins reduce one or more B cell responses or activities, including the ability of B cells to produce immunoglobulins. In some embodiments, the provided immunomodulatory proteins (e.g., TACI-Fc fusion proteins) reduce circulating serum immunoglobulins when administered to a subject. In some embodiments, the provided immunomodulatory proteins reduce one or more of B cell maturation, differentiation, and proliferation. In provided aspects, such activity is improved or superior to that achieved by a WT TACI-Fc fusion protein (e.g., telitacicept or atacicept). In some embodiments, provided immunomodulatory proteins (TACI-Fc fusion proteins) are candidate therapeutics for the treatment of multiple autoimmune and inflammatory diseases, particularly B-cell-related diseases, e.g., SLE, SjS, and other connective tissue diseases.

[0108] Provided embodiments include methods and uses of specific Fc-fusion proteins of the TACI variant TNF receptor domain (TD, i.e., CRD2) that simultaneously inhibit the cytokines BAFF and APRIL. Provided embodiments relate to the identification of variant TACI polypeptides engineered after random mutagenesis to have improved affinity for APRIL and / or BAFF and directed evolution of the second cysteine-rich domain (CRD2) of TACI spanning residues 68-110. As shown herein, affinity maturation involves five rounds of alternating selection between APRIL and BAFF, with simultaneous reduction in the concentration of the selection reagent to maintain selective pressure. Results demonstrated variant TACI polypeptides with significantly improved 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 (substitutions or mutations) that improve the binding affinity of the protein for BAFF and / or APRIL. Notably, some provided embodiments result in combined improved inhibition of BAFF and APRIL. Thus, the immunomodulating proteins provided provide effective and long-lasting disease suppression in the treatment of autoimmune or inflammatory diseases, including severe B-cell-related autoimmune diseases such as SLE.

[0109] For example, provided embodiments are based on the discovery that directing evolution by affinity engineering of the TNFR domain (TD) of the TACI ectodomain facilitated the development of molecules with improved affinity for APRIL and / or BAFF. Thus, affinity engineering generates variant TACIs containing variant TNFR domains (vTDs). Fusion of such molecules to immunoglobulin Fc results in immunomodulatory proteins that suppress B cell activity and responses. For example, when reformatted as soluble Fc fusion proteins, the output of affinity-matured TACI variants exhibits inhibition of APRIL and BAFF, as shown herein in TACI-dependent reporter assays, with lower IC than wild-type TACI-Fc and belimumab controls. 50Furthermore, results from the animal models evaluated demonstrated rapid and significant reductions in key lymphocyte subsets, including plasma cells, germinal center B cells, and follicular helper T cells. Furthermore, the tested variant molecules demonstrated improved activity in mouse models, including significant reductions in autoantibodies and sialadenitis in a spontaneous SjS model, inhibition of glomerular IgG deposition in a bm12-induced lupus model, and potent suppression of anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, sialadenitis, renal lesions, and renal immune complex deposition in a NZB / W lupus model. Furthermore, compared with wild-type TACI-Fc, the tested TACI-Fc fusions demonstrated significant and sustained reductions in serum IgM, IgG, and IgA antibody titers in mice. The findings herein demonstrate that these immunomodulatory proteins consistently demonstrate potent immunosuppressive activity and efficacy in vitro and in vivo, and appear to be superior to existing and / or approved immunomodulatory agents such as belimumab, abatacept, atacicept, or telitacicept. Thus, such biologics may be attractive development candidates for the treatment of severe autoimmune and / or inflammatory diseases, including B-cell-related disorders, such as SLE, Sjögren's syndrome, and other connective tissue disorders.

[0110] Furthermore, observations herein demonstrate that TACI-Fc fusion proteins exhibit high serum exposure when administered to mice and cynomolgus monkeys. The favorable, higher serum exposure and stronger immunosuppressive activity achieved by the provided TACI-Fc fusion proteins support their use at lower clinical doses and / or less frequent administration (or longer administration intervals) than existing wild-type TACI-Fc therapeutics. For example, existing wild-type TACI-Fc therapeutics, such as telitacicept and atacicept, must be administered at least once a week. Reducing the administration frequency may provide better symptom control in treated subjects, improve compliance with the administration regimen, enhance patient quality of life or patient satisfaction, and / or reduce the overall cost of receiving treatment. Furthermore, reducing the dose even to a more regular frequency, such as once a week, may alleviate certain side effects.

[0111] In certain embodiments, the provided TACI-Fc fusion proteins are intended for treating SLE and other autoantibody-associated rheumatic diseases with high unmet medical needs. In SLE, treatment options are hampered by the complex etiology and heterogeneity of the disease, suggesting that multiple pathways or aspects of B cell development and differentiation may need to be simultaneously inhibited to enable a durable response. While B cell depleting agents such as rituximab / ocrelizumab / obinutuzumab (anti-CD20) and obexelimab (anti-CD19) have shown favorable clinical efficacy in the setting of certain autoimmune diseases, this has not translated to SLE, and rituximab has failed to demonstrate 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). One potential limitation of these therapeutics is that CD20 and CD19 are not expressed on all ASCs or LL-PCs, and only early stage B cells (including pro / pre, immature, mature, and memory B cells) are depleted, leaving mostly pathogenic plasmablasts and PCs (Lee et al., Nat Rev Drug Discov, 2021, 20(3):179-199; Arbitman et al., J Autoimmun, 2022, 102873).

[0112] Targeting or co-targeting BAFF and / or APRIL is an alternative to ADCC-mediated B cell depletion. Preclinical studies have demonstrated that starving B cells of these two key B cell survival and differentiation factors significantly reduces all B cell subsets beyond the immature T1 developmental stage, including LL-PCs, without affecting CD19+CD20+ pro- / pre-B cell precursors (Gross et al., Immunity, 2001, 15(2):289-302). Inhibition of ASCs can dramatically impact pathogenic antibody production, thereby potentially reducing disease activity. Early attempts to target the BAFF / APRIL pathway focused on agents such as belimumab, which neutralize only BAFF, but inhibition of both APRIL and BAFF may be required to impact the survival of more differentiated, pathogenic TACI+ / BCMA+ ASCs (Samy et al., Int Rev Immunol, 2017, 36(1):3-19).

[0113] APRIL plays a particularly important role in IgA class switching, production, and glycosylation, as first demonstrated by studies of APRIL knockout mice (Castigli et al., Proc Natl Acad Sci USA, 2004, 101(11):3903-3908). Furthermore, elevated plasma APRIL levels in patients with IgA nephropathy (IgAN) are associated with more severe clinical symptoms, such as high proteinuria and Gd (galactose-deficient) IgA1 levels (Zhai et al., Medicine (Baltimore), 2016, 95(11):e3099), which are important causative factors contributing to the pathogenesis of the disease. Indeed, early studies of BION-1301 and sibeprelimab suggest that inhibition of APRIL alone may mediate significant reductions in IgA (especially IgA) in healthy subjects, and ongoing studies have demonstrated that BION-1301 has impacted proteinuria in patients with IgAN (Barratt et al., J Immunol, 2022, 180(6):3655-3659). However, targeting APRIL alone has its own limitations and would not be expected to impact 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). Neutralization of BAFF leads to downregulation of B cell function, reduced autoantibody production, and inhibition of the formation of tertiary lymphoid structures in the kidney (Samy, et al., Int Rev Immunol, 2017, 36(1):3-19).

[0114] Belimumab was the first approved treatment for SLE and LN (Lee et al., Nat Rev Drug Discov, 2021, 20(3):179-199), highlighting the need for novel therapies. Another advance in SLE treatment was the recent approval of anifrolumab, an anti-type I interferon receptor antibody (Morand et al., N Engl J Med, 2020, 382(3):211-221; Deeks, Drugs, 2021). Anifrolumab targets a different pathophysiology of SLE than B cell modulators by targeting myeloid dendritic cells rather than B cells. However, type I interferons are known to indirectly promote B cell differentiation and loss of tolerance. IFN-regulated gene expression is significantly increased in SLE. However, expression of the interferon gene signature is not predictive of response, highlighting the pleiotropic effects of the IFN system (Morand et al., N Engl J Med, 2020, 382(3):211-221). In contrast, the presence of elevated serum levels of BAFF and APRIL in SLE patients is well established and has been described in numerous studies (Samy, et al., Int Rev Immunol, 2017, 36(1):3-19). High serum BAFF levels also correlate with elevated autoantibody levels, particularly anti-dsDNA Abs (Samy, et al., Int Rev Immunol, 2017, 36(1):3-19).

[0115] The provided TACI-Fc fusion proteins are potential best-in-class BAFF / APRIL inhibitors for SLE and other autoantibody-associated diseases. The provided TACI-Fc fusion proteins exhibit significantly improved ligand affinity, superior to WT TACI-Ig, BAFF, and / or APRIL inhibitors alone, are well-tolerated in healthy adults via IV or SC administration, and have dose-dependent PK / PD. For example, Figure 46 of the present disclosure shows the superiority of the provided TACI-Fc fusion proteins in lowering circulating immunoglobulins (i.e., IgA, IgG, and IgM) compared to current biologics. This indicates that the provided TACI-Fc fusion proteins are suitable for multiple autoantibody-associated inflammatory diseases.

[0116] Furthermore, TACI-Fc fusion proteins are well tolerated at low doses (e.g., 80 mg) to high doses (e.g., 960 mg) without side effects. TACI-Fc fusion proteins are also well tolerated when administered once every four weeks (Q4W). Furthermore, TACI-Fc fusion proteins are effective at low doses (e.g., 80 mg) whether injected SC or IV.

[0117] All published documents, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein contradicts or is inconsistent with a definition set forth in a patent, application, published application, or other published document incorporated by reference herein, the definition set forth herein takes precedence over the definition incorporated by reference herein.

[0118] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0119] I. Definition Unless otherwise defined, all terminology, notations, and other technical and scientific terms or terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0120] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0121] The term "about" as used herein refers to a normal error range for each value, which is readily understood by those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

[0122] The term "affinity modified" as used in the context of a domain of a protein refers to a mammalian protein having an altered amino acid sequence in its extracellular domain or specific binding portion thereof (compared to the corresponding wild-type parent or unmodified domain) such that it has binding activity, such as increased or decreased binding affinity, for at least one of its binding partners (or "counter structure"), compared to the parent wild-type or unmodified (i.e., non-affinity modified domain) protein. In some embodiments, the affinity modified domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences, e.g., amino acid substitutions, from the wild-type or unmodified domain. Increased or decreased 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 increase in binding activity, e.g., affinity, of a protein for its binding partner(s) is at least 10% greater than that of a wild-type control, and in some embodiments, is at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type control value. The binding activity, e.g., affinity, of the protein for at least one of its binding partners is reduced by 90% or less of the control but 10% or more of the wild-type control value, and in some embodiments, by 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type control value but 10% or more. Affinity-modified proteins have altered primary amino acid sequence of the extracellular domain or specific binding portion thereof by substitution, addition, or deletion of amino acid residues. The term "affinity-modified" should not be construed as imposing any conditions on any particular starting composition or method by which the affinity-modified protein is made.Thus, affinity-modified proteins are not limited to wild-type protein domains that are subsequently converted into affinity-modified domains by any particular affinity-modification process. Affinity-modified domain polypeptides can be generated, for example, starting from wild-type mammalian domain sequence information, then modeled in silico for binding with their binding partners, and finally synthesized recombinantly or chemically to obtain the desired affinity-modified domain composition. However, in one alternative, affinity-modified domains can be generated by site-directed mutagenesis of wild-type domains. Thus, affinity-modified TD domains refer to products, but not necessarily products generated by any given process. A variety of techniques are available, including recombinant methods, chemical synthesis, or a combination thereof.

[0123] The term "affinity-modified TD domain" refers to an affinity-modified domain of a member of the tumor necrosis receptor superfamily (TNFRSF) protein, or its TNF ligand, having an altered amino acid sequence of a TNFR domain or TNF domain, respectively, therein. For example, an affinity-modified TD domain of a TNFRSF protein has an altered amino acid sequence of at least one cysteine-rich domain (CRD) within the extracellular domain of the TNFRSF protein, or a specific binding portion thereof (compared to a corresponding wild-type parent or unmodified domain), such that it has binding activity, such as increased or decreased binding affinity, for at least one of its binding partners (or "counterstructures"), compared to a parent wild-type or unmodified protein that includes a non-affinity-modified or unmodified TD domain.

[0124] "Affinity-modified TACI" (also referred to as variant TACI) refers to a TACI protein molecule that antagonizes or blocks the activity of B cell stimulatory receptors. For example, TACI binds to the B cell stimulatory receptors B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), and APRIL and / or BAFF, which are ligands for transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). In certain embodiments, BIM comprises the extracellular domain of TACI, or a portion of the extracellular domain of TACI that includes a TNF receptor family domain (e.g., TD, e.g., CRD) that binds to the cognate ligands APRIL and / or BAFF, and a heterotrimer of APRIL and BAFF. Affinity-modified variants of the extracellular domain of TACI, or portions thereof, may also include another amino acid modification (e.g., an amino acid substitution) in the TD that increases binding affinity for the cognate ligand (e.g., APRIL and / or BAFF, and a heterotrimer of APRIL and BAFF).

[0125] 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), and transmembrane activator and calcium-regulated 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, regulates B cell homeostasis, including B cell survival, B cell maturation, and differentiation, as well as immunoglobulin class switching. B cell stimulating receptors generally comprise an extracellular portion, a transmembrane domain, and a cytoplasmic region, which contains one or more TNF receptor-associated factor (TRAF) binding sites. Recruitment of various TRAF molecules to the cytoplasmic domain can activate various transcription factors, such as NF-κB (e.g., NF-κB1 or NF-κB2), mediating B cell signaling pathways that regulate B cell homeostasis.

[0126] As used herein, "bind," "bound," or grammatical variations thereof, refers to the participation of a molecule in any attractive interaction with another molecule, resulting in a stable association in which the two molecules are in close proximity to one another. 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, e.g., compounds, including drugs.

[0127] As used herein, avidity refers to a characteristic of a molecule, e.g., a polypeptide, with respect to whether and how it binds to one or more binding partners. Avidity can include any measure of the binding of a molecule to a binding partner. Avidity includes the ability to bind to a binding partner(s), the affinity (e.g., high affinity) for binding to a binding partner, the avidity for binding to a binding partner, the strength of binding to a binding partner, and / or the specificity or selectivity for binding to a binding partner.

[0128] The term "binding affinity" as used herein refers to the specific binding affinity of a protein to its binding partner (i.e., its corresponding structure) under specific binding conditions. Binding affinity refers to the strength of the interaction between two or more molecules, such as binding partners, and typically refers to the strength of non-covalent interactions between two binding partners. The increase or decrease in binding affinity of an affinity-modifying domain or an immunomodulating protein containing an affinity-modifying domain to a binding partner is determined by comparing it with the binding affinity of the unmodified domain (e.g., a natural or wild-type TD domain). Methods for determining binding affinity or relative binding affinity, such as solid-phase ELISA immunoassays, ForteBio Octet, Biacore assays, or flow cytometry, are known in the art. 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 may be measured by flow cytometry, for example, based on mean fluorescence intensity (MFI) in a flow binding assay.

[0129] As used herein, the term "binding affinity" refers to the specific binding affinity of a protein for its binding partner (i.e., its corresponding structure) under specific binding conditions. In biochemical kinetics, affinity refers to the cumulative strength of the affinities of multiple individual non-covalent interactions, such as between proteins for their binding partners (i.e., their corresponding structures). Thus, affinity differs from affinity, which describes the strength of a single interaction.

[0130] The term "biological half-life" refers to the amount of time it takes for a substance, such as an immunomodulatory protein, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the substance's excretion, excretion, degradation (e.g., enzymatic degradation / digestion), or absorption and concentration in specific organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the substance's plasma concentration to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derivatize proteins and extend their biological half-life are known in the art and include, but are not limited to, multimerization domains (e.g., Fc immunoglobulin domains), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides, see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0131] The term "cell surface counterpart structure" (or "cell surface binding partner"), as used herein, refers to a counterpart structure (or binding partner) expressed on a mammalian cell. Generally, cell surface binding partners are transmembrane proteins. In some embodiments, the cell surface binding partner is a receptor.

[0132] The term "binding partner" or "corresponding structure" in reference to a protein, e.g., a receptor, a soluble ligand, or an extracellular domain or portion thereof, or an affinity-modified variant thereof, refers to at least one molecule (generally a native mammalian protein) to which the referenced protein specifically binds under specific binding conditions. In some aspects, an affinity-modifying domain, or an immunomodulating protein comprising an affinity-modifying domain, specifically binds to the binding partner of the corresponding domain of the native or wild-type protein, but with increased or attenuated affinity. A "cell surface binding partner" is a binding partner expressed on a mammalian cell. Generally, 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 cells, e.g., mammalian cells that form an immunological synapse, such as immune cells.

[0133] 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 means that the two or more cell surface molecules are present on only one or only the other (but not both) of the two mammalian cells that form the IS.

[0134] The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of amino acid groups with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic 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.

[0135] The term "corresponding to" with respect to a protein position, such as a listing where a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence as set forth in the sequence listing, refers to a nucleotide or amino acid position identified after alignment with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm such as the GAP algorithm. By aligning the sequences, one of skill in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Figure 9 illustrates identifying corresponding residues by aligning two sequences.

[0136] As used herein, a "domain" (typically a sequence of three or more, generally five or seven or more amino acids, e.g., 10 to 200 amino acid residues) refers to a portion of a molecule, such as a protein or encoding nucleic acid, that is structurally and / or functionally distinct and identifiable from other portions of the molecule. For example, a domain includes a portion of a polypeptide chain that can form an independently folded structure within a protein composed of one or more structural motifs and / or a portion of a polypeptide chain that is recognized by a functional activity, such as binding activity. A protein may have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology to related family members, e.g., homology to a motif. In another example, a domain can be distinguished by its function, e.g., its ability to interact with a biomolecule, such as a cognate binding partner. A domain can independently exhibit a biological function or activity, such that a domain, either independently or fused to another molecule, can perform an activity, e.g., binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. While definitions are provided herein for illustrative purposes, it is understood that identifying specific domains by name is well within the skill of one of ordinary skill in the art. Suitable software can be utilized to identify domains, if necessary. It should be understood that reference to amino acids, including specific sequences shown as SEQ ID NOs, used to describe domain structure (e.g., of a TD domain) is for illustrative purposes only and is not intended to limit the scope of the provided embodiments. It is understood that descriptions of polypeptides and their domains are theoretically derived based on homology analysis and alignment with similar molecules. Additionally, in some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g., TD) may also be included in the sequence, such as to ensure proper folding of the domain when expressed. Thus, the exact locus may vary and may not necessarily be the same for each protein.For example, a particular TD domain, such as a particular CRD domain, may be several amino acids longer or shorter (1-10, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

[0137] The terms "ectodomain," "extracellular domain," or "ECD," used interchangeably herein, refer to a region of a membrane protein, such as a transmembrane protein, that is located outside the vesicle membrane (e.g., in the extracellular space) when the full-length form of the membrane protein is expressed from a cell. For purposes herein, reference to an ECD refers to the sequence and domain that make up this region, and it is understood that a protein containing an ECD does not require that the protein be a membrane protein or that the domain reside extracellularly. For example, a soluble immunomodulatory protein may contain the ECD sequence of a membrane protein fused to another moiety, such as a multimerization domain, e.g., an Fc region. Ectodomains often interact with specific ligands or specific cell surface receptors, e.g., via a binding domain that specifically binds to the ligand or cell surface receptor. Examples of binding domains include cysteine-rich domains (CRDs). The ectodomains of members of the TNFR superfamily contain a TD domain (e.g., a CRD domain). Thus, reference to an ECD herein includes the full-length sequence of the ECD of a membrane protein, as well as specific binding fragments thereof that contain a CRD that binds to a ligand or cognate binding partner.

[0138] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a therapeutic composition, e.g., comprising an immunomodulatory protein or an Fc-fusion protein, that, when administered ex vivo (by contacting with cells from a patient) or in vivo (by administration to a patient), either alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in 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, condition, or disorder, such as an immune system disease, condition, or disorder, can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with the disease, condition, or disorder, an amount that prevents the progression of the disease, condition, or disorder, or an amount that improves the patient's physical function. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient. In some embodiments, the patient is a human patient.

[0139] As used herein, a fusion protein refers to a polypeptide encoded by a nucleic acid sequence containing coding sequences for two or more proteins, possibly two, three, four, five, or more proteins, 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 may be adjacent to another protein in the construct or may be separated by a linker polypeptide containing one, two, three, or more, but generally fewer than 20, 15, 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide. An example of a fusion protein according to provided embodiments is an Fc-fusion protein containing an affinity-modifying domain (e.g., a variant of the TACI extracellular domain or a portion thereof containing a CRD) linked to an immunoglobulin Fc domain.

[0140] The term "half-life extending moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of the protein not so conjugated to that moiety. In some embodiments, the half-life is extended by more than 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, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. Half-life refers to the amount of time it takes for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, using an ELISA assay or activity assay. Exemplary half-life extending moieties include an Fc domain, a multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides, see WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).

[0141] The Fc (fragment crystallizable) region or domain (also referred to as an Fc polypeptide) of an immunoglobulin molecule corresponds primarily to the constant region of an immunoglobulin heavy chain and is responsible for various functions, including, in some cases, antibody effector function(s). The Fc domain comprises part or all of the hinge domain of an immunoglobulin molecule, as well as the CH2 and CH3 domains. In some cases, all or part of the Fc hinge sequence may be removed for inclusion in a provided fusion protein. The Fc domain can form a dimer of two polypeptide chains linked by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits a reduction in effector function-promoting activity (e.g., about a 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more reduction). In some embodiments, references to amino acid substitutions in the Fc region are in accordance with the EU numbering system, unless otherwise noted with reference to a specific SEQ ID NO. EU numbering is known and follows the EU index reported in the recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information: http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013) and Kabat, EA et al. Sequences of Proteins of Immunological Interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0142] Immunoglobulin Fc fusions ("Fc fusions"), such as immunomodulatory Fc fusion proteins, are molecules comprising one or more polypeptides operably linked to the Fc region of an immunoglobulin. Fc fusions may comprise, for example, an Fc region operably linked to a TACI extracellular domain, including a CRD, or a portion thereof, including any of the affinity-engineered variants thereof provided. The immunoglobulin Fc region may be indirectly or directly linked to one or more polypeptides. Various linkers are known in the art and can optionally be used to link the Fc to a fusion partner to generate an Fc fusion. Homologous Fc fusions can be dimerized to form Fc fusion homodimers. Non-homologous Fc fusions (e.g., knob-into-hole engineering) can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a murine or human Fc.

[0143] The term "host cell" refers to any cell that can be used to express a protein encoded by a recombinant expression vector. A host cell can be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Exemplary host cells include Chinese hamster ovary (CHO) cells, or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium, or the CHO line DX-B11 (DHFR-deficient).

[0144] As used herein, the term "immunological synapse" or "immune synapse" (abbreviation "IS") refers to the interface between a mammalian cell expressing MHC I (major histocompatibility complex) or MHC II, e.g., an antigen-presenting cell or tumor cell, and a mammalian lymphocyte, e.g., an effector T cell or natural killer (NK) cell.

[0145] As used herein, the term "immunoglobulin" (abbreviated "Ig") is synonymous with the term "antibody" (abbreviated "Ab") and refers to mammalian immunoglobulin proteins, including any of the five human classes: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also includes less-than-full-length immunoglobulins, including any fragment thereof, whether wholly or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, that comprise at least a portion of the variable heavy (VH) and / or variable light (VL) chain regions of the immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, specifically bind to an antigen. An antibody may also include all or a portion of the constant region. Such fragments include antigen-binding fragments (Fab), variable fragments (Fv) comprising VH and VL, single-chain variable fragments (scFv) comprising VH and VL linked together in a single chain, and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments. Thus, reference to antibodies herein is understood to include full-length antibodies and antigen-binding fragments. The term antibody also includes antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules. Bispecific antibodies, homobispecifics, and heterobispecifics are included within the meaning of this term. Antibodies 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., page 71 and chapter 6 of Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994.

[0146] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in substantially complete form, as opposed to an antibody fragment. Full-length antibodies generally include antibodies having 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), as well as a hinge region, e.g., antibodies produced by antibody-secreting B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.), and synthetically produced antibodies having the same domains. Specifically, whole antibodies include those having heavy and light chains comprising an Fc region. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0147] "Antibody fragments" include portions of an intact antibody, including the antigen-binding and / or variable regions of the intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

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

[0148] An "Fv" consists of one heavy-chain and one light-chain variable domain linked by a non-covalent bond. The folding of these two domains results in six complementarity-determining regions (CDRs) (three from the heavy chain and three from the light chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, although in some cases with lower affinity than the entire binding site.

[0149] "dsFv" is V H - VL Refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the pair.

[0150] An "Fd fragment" is a fragment of the variable domain (V H ) and one constant region domain (C H 1) is an antibody fragment containing

[0151] A "Fab fragment" is an antibody fragment resulting from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g., by recombinant methods. A Fab fragment contains only the light chain (V L and C L ) and the variable domain of the heavy chain (V H ) and one constant region domain of the heavy chain (C H 1) and another chain containing

[0152] An "F(ab')2 fragment" is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment of the same structure produced synthetically, e.g., by recombinant methods. An F(ab')2 fragment essentially contains two Fab fragments, with each heavy chain containing a few additional amino acids, including a cysteine ​​residue, that form a disulfide bond linking the two fragments.

[0153] A "Fab' fragment" is a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain).

[0154] An "Fd' fragment" is a fragment of an antibody that contains one heavy chain portion of the F(ab')2 fragment.

[0155] "Fv' fragment" refers to the V fragment of an antibody molecule. H and V L A fragment containing only the domain.

[0156] An "scFv fragment" is a fragment of variable light chains (V) covalently linked by a polypeptide linker in any order. L ) and variable heavy chain (V H ) The linker is of a length such that the two variable domains are bridged without substantial interference. An exemplary linker contains several Glu or Lys residues dispersed throughout to enhance solubility (Gly-Ser). n It is a residue.

[0157] "Diabodies" are dimeric scFvs; diabodies typically have shorter peptide linkers than scFvs and preferentially dimerize.

[0158] As used herein, the term "immune activity" refers to the activity of one or more immune cells, such as T cells or B cells, including, for example, activation, cell survival, cell proliferation, cytokine production (e.g., interferon-gamma), cytotoxic activity, or the ability to activate the NF-κB pathway or other signaling cascades that result in the activation of transcription factors in immune cells. Assays for assessing the immune activity of immunomodulatory proteins can be compared to control proteins with known activity.

[0159] An "immunomodulating protein" or "immunomodulating polypeptide" is a protein that modulates immune activity. "Modulation" or "modulating" an immune response means either enhancing or suppressing immune activity. Such modulation can include any induction, alteration in the degree or extent, or suppression of immune activity of immune cells, such as B cells or T cells. For example, the soluble Fc fusion protein herein can suppress immune activity of B cells. An immunomodulating protein can be a single polypeptide chain or a multimer (dimer or higher order multimer) of at least two polypeptide chains covalently linked to each other, for example, by interchain disulfide bonds. Thus, monomeric, dimeric, and higher order multimeric proteins are within the scope of the defined term. A multimeric protein can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains).

[0160] As used herein, modification refers to a modification of the amino acid sequence or nucleotide sequence of a polypeptide in a nucleic acid molecule, including a change in the amino acid or nucleotide sequence, respectively. The amino acid modification or change can be a deletion, insertion, or replacement (substitution) of an amino acid or nucleotide, respectively. Methods for modifying polypeptides are common to those skilled in the art, such as by using recombinant DNA techniques.

[0161] The term "multimerization domain" refers to a sequence of amino acids that promotes the formation of multimers of two or more polypeptides. A multimerization domain comprises a sequence that promotes stable interaction between a polypeptide molecule and one or more additional polypeptide molecules, each of which comprises a complementary multimerization domain (e.g., a first multimerization domain and a second multimerization domain), which may be the same or different multimerization domains. The interaction between complementary multimerization domains, e.g., the interaction between a first multimerization domain and a second multimerization domain, forms a stable protein-protein interaction, generating a multimer of the polypeptide molecule with the additional polypeptide molecule. In some cases, the multimerization domain is the same and interacts with itself to form a stable protein-protein interaction between the two polypeptide chains. Generally, a polypeptide is bound directly or indirectly to a multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain can be, for example, an immunoglobulin constant region or domain, such as an Fc domain from IgG, including IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM, or portions thereof, and modified forms thereof.

[0162] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides, have similar binding properties, and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide 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 substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0163] As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the association of nucleic acid sequences in a manner or orientation such that the segments are positioned so that they function in concert for their intended purpose. In some embodiments, the terms refer to the linkage of nucleic acids to produce a nucleic acid molecule capable of directing transcription of a given gene and / or to produce a functional desired protein molecule. For example, segments of DNA sequence, e.g., coding sequence and regulatory sequence(s), are linked in a way that allows gene expression when the appropriate molecule (e.g., transcriptional activator protein) is bound to the regulatory sequence.

[0164] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. Pharmaceutical compositions generally comprise an effective amount of an active agent (e.g., an immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is generally a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0165] 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, "peptide" and "oligopeptide" are included within the definition of polypeptide. These terms include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, and the like. These terms also include molecules containing one or more amino acid analogs or non-standard or unnatural amino acids, which may be recombinantly synthesized or expressed using known protein engineering techniques. Additionally, proteins can be derivatized as described herein by well-known organic chemistry techniques.

[0166] The term "purified," as applied to an immunomodulatory protein, or a nucleic acid encoding a protein (e.g., an immunomodulatory protein), generally refers to 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 will form a distinct 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 gives rise to essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or protein is at least about 50% pure, and typically is at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., on a weight percent or molar basis).

[0167] The term "recombinant" indicates that a material (e.g., a nucleic acid or polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. Modifications can be made to material within its natural environment or condition or to material removed from it. For example, a "recombinant nucleic acid" is one created by recombining nucleic acids, e.g., during cloning, affinity engineering, DNA shuffling, or other well-known molecular biological procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by such molecular biological techniques. As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule (e.g., an immunomodulatory protein) expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid or is otherwise modified by genetic engineering, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein, e.g., an immunomodulatory protein provided herein. Transcriptional 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 (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest.

[0168] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing a desired coding sequence (e.g., a sequence encoding an immunomodulatory protein) and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence is also optionally encoded by the recombinant expression vector and operably linked to the coding sequence, thereby allowing the expressed protein to be secreted by the recombinant host cell, such as for its expression as a secretable protein or, if desired, for easy isolation or purification of the immunomodulatory protein from the cell. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which they are introduced. Among these vectors are viral vectors, such as lentiviral vectors.

[0169] The term "sequence identity," as used herein, refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by aligning the sequences and comparing the amino acid sequence at a given position in each sequence. Similarly, nucleic acid sequence identity can be determined by aligning the sequences and comparing the nucleotide sequence at a given position in each sequence. Methods for aligning sequences for comparison are well known in the art, and include GAP, BESTFIT, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, FASTA, and TFASTA. The BLAST algorithm also calculates percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. In some cases, percent sequence identity can be determined as the percentage of amino acid residues (or nucleotide residues) in a candidate sequence that are identical to those in a reference sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity. Reference to sequence identity includes sequence identity over the full length of each of the sequences being compared. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0170] The term "soluble" as used herein with respect to a protein means that the protein is not a membrane protein or is not anchored to a cell membrane. A protein can be constructed as a soluble protein by including only the extracellular domain or a portion thereof, but not the transmembrane domain. In some cases, the solubility of a protein can be improved by linking or conjugating, directly or indirectly via a linker, to an Fc domain or other half-life extending molecule, which can also improve the stability and / or half-life of the protein. In some embodiments, the soluble protein is an Fc fusion protein.

[0171] As used herein, the term "specifically binds" refers to the ability of a protein to bind to a target protein under specified binding conditions with an affinity or avidity that is at least 10 times, but optionally 50, 100, 250, or 500 times, or even at least 1000 times, greater than the average affinity or avidity of the same protein for a random population of peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind to only a single target molecule but may specifically bind to multiple target molecules. In some cases, a specifically binding protein can bind to proteins that share structural conformational similarity with the target protein (e.g., paralogs or orthologs). Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or molecules with substantially similar epitopes to the target molecule (e.g., paralogs) is possible without compromising the specificity of binding determined against a statistically valid population of unique non-targets (e.g., random polypeptides). Thus, the immunomodulatory proteins of the present invention can specifically bind to target molecules in multiple distinct species due to cross-reactivity. Solid-phase ELISA immunoassays, ForteBio Octet, or Biacore measurements can be used to determine the specific binding between two proteins. Generally, the interaction between two binding proteins is approximately 1 x 10 -5 Less than m, often about 1 x 10 -12In certain embodiments of the present disclosure, the interaction between two binding proteins has a dissociation constant (Kd) of about 1×10 -6M , 1×10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, or 1 x 10 -11 It has a dissociation constant of less than M or less.

[0172] As used herein with respect to a protein, the term "specific binding fragment" or "fragment" refers to a polypeptide that is shorter than the full-length protein or a specific domain or region thereof and that specifically binds to a binding partner of the full-length protein or specific domain or region in vitro and / or in vivo. A specific binding fragment refers to a fragment of the full-length extracellular domain of a polypeptide or a fragment of the binding domain of a polypeptide, which still binds to a 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 a fragment of the full-length TNFR domain (TD) (e.g., CRD) thereof, which still binds to a TNFR family member or a binding partner of the CRD of the TNFR family member. In some embodiments, a 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 may have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific binding fragment comprises the CRD1 and / or CRD2 domain. In some embodiments, the specific binding fragment comprises the CRD2 domain.

[0173] As used herein, a "subject" is a mammal, e.g., a human or other animal, typically a human. The subject may be male or female and of any appropriate age, including infant, juvenile, adolescent, adult, and elderly subjects.

[0174] As used herein, "synthetic," for example, with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule produced by recombinant and / or chemical synthesis methods.

[0175] The term "TNF receptor superfamily" or "TNFRSF," as used herein, refers to a group of cell surface cytokine receptors that are all type I (N-terminal extracellular) transmembrane glycoproteins containing one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on common structural features, including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular regions, which are often involved in protein binding to their cognate binding partners or ligands. TNFRSF proteins may have only one or several CRDs (e.g., CRD1, CRD2, etc.). Generally, the ECD or ectodomain of TNFRSF members contains one to six pseudorepeats of CRDs. For example, the BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are usually trimeric or multimeric complexes stabilized by cysteine-cysteine ​​disulfide bonds. The binding of TNFRSF proteins to their ligands promotes a variety of biological activities within the cell, such as the induction of apoptotic cell death or cell survival and proliferation.

[0176] The term "TD" refers to the structural domain(s) of a TNFRSF protein or TNF family ligand. For example, the TD of a TNFRSF protein is a cysteine-rich domain (CRD) module of approximately 40 amino acids containing six conserved cysteines. Therefore, reference to a CRD can also be used synonymously with the term TD for the TD of a TNFRSF protein. The six cysteines are involved in the formation of intrachain disulfide bonds. Because the extracellular domain (ECD) of a TNFRSF member contains one or more CRD domains, the term TD is also used in reference to the ECD of such a protein molecule. Reference to a variant TD (vTD) refers to a variant or modified sequence of a TD.

[0177] The term "trans," with respect to binding to cell surface molecules, refers to binding to two different cell surface molecules, each present on the surface of a different cell. In some embodiments, trans refers to two different cell surface molecules, one of which is present only on one of the two mammalian cells forming the IS, and the second of which is present only on the other of the two mammalian cells forming the IS.

[0178] As used herein, the term "transmembrane protein" refers to a membrane protein that substantially or completely spans a lipid bilayer, such as a lipid bilayer found in a biological membrane, such as a mammalian cell, or in an artificial construct, such as a liposome. A transmembrane protein contains a transmembrane domain ("transmembrane domain") that integrates the transmembrane protein into the lipid bilayer, and this integration is thermodynamically stable under physiological conditions. Transmembrane domains can generally be predicted from their amino acid sequences using any number of commercially available bioinformatics software applications based on the high hydrophobicity of the transmembrane domain relative to the region of the protein that interacts with the aqueous environment (e.g., cytosol, extracellular fluid). Transmembrane domains are often hydrophobic alpha helices that span the membrane. A transmembrane protein may pass through both layers of the lipid bilayer one or more times.

[0179] As used herein, the terms "treating," "treatment," or "therapy" of a disease, condition, or disorder means slowing, halting, or reversing the progression of the disease or disorder, as evidenced by the reduction, arrest, or elimination of any clinical or diagnostic symptoms, by administration of an immunomodulatory protein or engineered cell of the invention, either alone or in combination with another compound described herein. "Treating," "treatment," or "therapy" also means reducing the severity of symptoms in an acute or chronic disease, condition, or disorder, or reducing the relapse rate in the case of, for example, a relapsing or remitting autoimmune disease course or inflammatory condition, or reducing inflammation in the case of the inflammatory aspect of an autoimmune disease or inflammatory condition. As used in the context of the present invention, "preventing," "prophylaxis," or "prevention" of a disease, condition, or disorder refers to the administration of an immunomodulatory protein of the invention, either alone or in combination with another compound, to prevent the onset or development of the disease, condition, or disorder, or some or all of the symptoms of the disease, condition, or disorder, or to reduce the likelihood of developing the disease, condition, or disorder.

[0180] The term "variant" (also referred to as "modified" or "mutant," which can be used interchangeably) as used with respect to a variant protein or polypeptide refers to a protein, such as a mammalian (e.g., human or murine) protein, that is created by human intervention. A variant is a polypeptide that has an amino acid sequence that has been altered or modified, such as by one or more amino acid substitutions, deletions, additions, or a combination thereof, relative to the unaltered or wild-type protein or domains thereof. A variant polypeptide 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, e.g., amino acid substitutions. Variant polypeptides generally exhibit at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the wild-type or unmodified protein, e.g., its mature sequence (lacking the signal sequence) or a corresponding form of a portion thereof, including the extracellular domain or binding domain thereof. Non-naturally occurring and naturally occurring amino acids are included within the scope of permissible substitutions or additions. Variant proteins are not limited to any particular method of production, including, for example, chemical synthesis, recombinant DNA technology, or a combination thereof. The variant proteins of the present invention specifically bind to at least one or more binding partners. In some embodiments, alterations in the amino acid sequence alter (i.e., increase or decrease) binding activity, such as binding affinity or avidity, for one or more binding partners. Thus, variant proteins may be "affinity-modified" proteins as described herein.

[0181] As used herein, the terms "wild-type" or "native" or "native," used interchangeably, are used in reference to biological material, such as nucleic acid molecules, proteins, host cells, etc., that are found in nature and have not been modified by human intervention.

[0182] II. TACI Immunomodulatory Proteins and Variant TACI Polypeptides Provided herein are TACI immunomodulatory proteins, or variants thereof, comprising a portion of the extracellular domain (ECD) of a TACI receptor that binds to at least one TACI cognate binding partner. Also provided herein are variant TACI polypeptides that exhibit altered (e.g., increased) binding activity or affinity 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. Provided TACI immunomodulatory proteins and polypeptides include soluble fusion proteins thereof, in which 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-extending moiety. Thus, in some embodiments, the immunomodulatory protein is a TACI-Fc fusion protein. In some embodiments, provided are TACI-Fc fusion proteins comprising: (1) a TACI polypeptide comprised of the extracellular domain or portion thereof of a TACI receptor, or a variant TACI polypeptide, that binds 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 (eg, via a peptide linker) to the Fc domain.

[0183] TACI is a member of the tumor necrosis factor receptor family, characterized by an extracellular domain (ECD) containing a cysteine-rich pseudo-repeat domain (CRD). TACI is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudo-repeats (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 that is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with subsets of B cells and T cells. The TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family. One ligand is called BAFF (B cell activating factor of the TNF family), also variously referred to as ZTNF4, "Neutrokine-α," "BLyS," "TALL-1," and "THANK" (Yu et al., International Publication No. WO 98 / 18921 (1998); Moore et al., Science 285:269 (1999); Mukhopadhyay et al., J. Biol. Chem. 274:15978 (1999); Schneider et al., J. Exp. Med. 189:1747 (1999); Shu et al., J. Leukoc. Biol. 65:680 (1999)). Another ligand is called APRIL, also variously referred to as "ZTNF2" and "TNRF death ligand-1" (Hahne et al., J. Exp. Med. 188:1185 (1998); Kelly et al., Cancer Res. 60:1021 (2000)). Both ligands are also bound by the B cell maturation receptor (BCMA) (Gross et al., Nature 404:995 (2000)). Binding of the TACI receptor to its ligands BAFF or APRIL stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.

[0184] The amino acid sequence of full-length TACI is set forth in SEQ ID NO: 88. This protein is a type III membrane protein, lacks a signal peptide, and the N-terminal methionine is removed after expression in eukaryotic cells. In some embodiments, the mature TACI protein does not contain the N-terminal methionine set forth in SEQ ID NO: 88. The extracellular domain of TACI (amino acid residues 1-166 of SEQ ID NO: 88, ECD set forth in SEQ ID NO: 122) contains two cysteine-rich domains (CRDs, hereinafter also referred to as tumor necrosis family receptor domains or TDs), each of which exhibits affinity for binding to BAFF and APRIL. The first cysteine-rich domain (CRD1) comprises amino acid residues 34-66 of the sequence set forth in SEQ ID NO: 122. The second cysteine-rich domain (CRD2) corresponds to amino acids 71-104 of the sequence set forth in SEQ ID NO: 122. TACI also contains an approximately 60 amino acid stem region following the second cysteine ​​repeat in the extracellular domain, corresponding to amino acid residues 105-165 of the sequence set forth in SEQ ID NO:122.

[0185] In some embodiments, variant TACI polypeptides provided herein comprise one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "substitutions"), deletions, or additions, in the extracellular domain of a reference TACI polypeptide, e.g., a wild-type or unmodified TACI polypeptide comprising a CRD(s) (hereinafter also referred to as a 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 within a CRD. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "substitutions"), deletions, or additions, are within the CRD1 region. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "substitutions"), deletions, or additions, are within the CRD2 region. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "substitutions"), deletions, or additions, are in amino acids within both the CRD1 and CRD2 regions.

[0186] In some embodiments, the reference (e.g., unmodified) TACI sequence is a wild-type TACI sequence or a portion thereof comprising one or both CRDs. In some embodiments, the reference (e.g., unmodified) TACI is or comprises the extracellular domain (ECD) of TACI or a portion thereof comprising one or both 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 comprise both CRD1 and CRD2. In some embodiments, a variant TACI polypeptide comprises or consists essentially of CRD1 or a specific binding fragment thereof. In some embodiments, a variant TACI polypeptide comprises or consists essentially of CRD2 or a specific binding fragment thereof. 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, optionally, a cytoplasmic domain.

[0187] In some embodiments, the reference (e.g., unmodified) TACI sequence is a mammalian TACI sequence. In some embodiments, the reference (e.g., unmodified) TACI sequence can be a mammalian TACI, including, but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the reference (e.g., unmodified) TACI sequence is human. The extracellular domain of an exemplary human TACI sequence is set forth in SEQ ID NO: 122.

[0188] In some embodiments, the reference (e.g., unmodified) TACI sequence is (i) the sequence of amino acids set forth in SEQ ID NO: 122 or a sequence thereof lacking the N-terminal methionine, (ii) a sequence of amino acids that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 122 and binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer, or (iii) a fragment or portion of (i) or (ii) comprising CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the reference (e.g., unmodified) TACI sequence lacks the N-terminal methionine set forth in SEQ ID NO: 122. TACI extracellular domain (ECD): SEQ ID NO: 122 MSGLGRSRRGGRSRVDQEERFPQGLWTGVAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELRRQRSGEVENNSDNSGRYQGLEHRGSEASPALPGLKLSADQVALVYST

[0189] In some embodiments, the reference (e.g., unmodified) TACI sequence is an extracellular domain sequence of TACI that is a portion of the ECD that includes an N-terminal deletion relative to the sequence of amino acids set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-28, which correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-29, which correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-30, which correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-31, which correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-32, which correspond to the residues set forth in SEQ ID NO: 122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-33, which correspond to the residues set forth in SEQ ID NO: 122.

[0190] In any of the provided embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion that includes a deletion of one or more residues in the stem 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 contiguous C-terminal amino acid residues beginning at residue 105, which corresponds to the residues in the ECD sequence set forth in SEQ ID NO: 122, up to or including amino acid residue 166. 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 ECD sequences are deleted.

[0191] In some embodiments, the reference (e.g., unmodified) TACI sequence includes CRD1 and / or CRD2 (e.g., CRD1 and CRD2 or only CRD2) and an ECD portion having a contiguous sequence of amino acids that includes only a segment or portion of the stem sequence. A suitable stem segment includes one or more amino acids from amino acid residues 105 to 154 of SEQ ID NO: 122. For example, the stem segment may consist of the following with reference to SEQ ID NO: 122: amino acid residue 105, amino acid residues 105 to 106, amino acid residues 105 to 107, amino acid residues 105 to 108, amino acid residues 105 to 109, amino acid residues 105 to 110, amino acid residues 105 to 111, amino acid residues 105 to 112, amino acid residues 105 to 113, amino acid residues 105 to 114, amino acid residues 105 to 115 , amino acid residues 105 to 116, amino acid residues 105 to 117, amino acid residues 105 to 118, amino acid residues 105 to 119, amino acid residues 105 to 120, amino acid residues 105 to 121, amino acid residues 105 to 122, amino acid residues 105 to 123, amino acid residues 105 to 124, amino acid residues 105 to 125, amino acid residues 105 to 126, amino acid residues 105 to 127, amino acid residues 105 to 128, Amino acid residues 105 to 129, amino acid residues 105 to 130, amino acid residues 105 to 131, amino acid residues 105 to 132, amino acid residues 105 to 133, amino acid residues 105 to 134, amino acid residues 105 to 135, amino acid residues 105 to 136, amino acid residues 105 to 137, amino acid residues 105 to 138, amino acid residues 105 to 139, amino acid residues 105 to 140, amino acid residues 105 to 141, amino acid residues 105 to 142, amino acid residues 105 to 143, amino acid residues 105 to 144, amino acid residues 105 to 145, amino acid residues 105 to 146, amino acid residues 105 to 147, amino acid residues 105 to 148, amino acid residues 105 to 149, amino acid residues 105 to 150, amino acid residues 105 to 151, amino acid residues 105 to 152, amino acid residues 105 to 153, and amino acid residues 105 to 154.

[0192] 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 an ECD or portion in which the arginine residue at position 119 is mutated (e.g., R119G). In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or portion in which the glutamine residue at position 121 is mutated (e.g., Q121P). In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or portion in which the arginine residue at position 122 is mutated (e.g., R122Q).

[0193] In some embodiments, the reference TACI sequence is a TACI ECD sequence set forth in International PCT Publication Nos. WO2000 / 067034, WO2002 / 094852, or WO2008 / 154814.

[0194] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence set forth in SEQ ID NO:131. TACI ECD (CRD1 / CRD2): SEQ ID NO: 131 SRVDQEER FPQGLWTGVA MRSCPEEQYW DPLLGTCMSCKTICNHQSQR TCAAFCRSLS CRKEQGKFYD HLLRDCISCA SICGQHPKQCAYFCENKLRS PVNLPPEL

[0195] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence set forth in SEQ ID NO:130. TACI ECD (CRD1 / CRD2): SEQ ID NO: 130 AMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

[0196] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence set forth in SEQ ID NO:1 (encoded by the sequence of nucleotides set forth in SEQ ID NO:36). TACI ECD (CRD1 / CRD2): SEQ ID NO: 1 VAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

[0197] In some embodiments, the reference TACI sequence is a region of the extracellular domain of TACI consisting essentially of only the CRD2 sequence, with the entire CRD1 sequence and substantially all of the stem region deleted or lacking. Previous studies have shown that residues within the stem region may contain protease cleavage sites, but at least CRD1 and CRD2 were thought to be required for full expression and / or binding activity of TACI to its cognate ligand. For example, International PCT Publication No. WO 2002 / 094852 demonstrated that TACI molecules containing CRD1 and CRD2 but lacking the entire amino-terminal region and partial sequence of the stem region exhibited reduced proteolysis upon expression. Other studies have shown that at least a portion of the N-terminal region preceding CRD1 is required for full binding activity of TACI to its cognate ligand. See, e.g., International Publication No. WO 2008 / 154814, in which residues 13-118 or 13-108 of the TACI extracellular domain were determined to be necessary for biological activity while minimizing degradation of TACI during expression. Surprisingly, it has been found herein (e.g., Example 3) that a TACI extracellular domain consisting essentially of only the CRD2 along with a small portion of the stem region exhibits substantially improved cognate binding activity compared to longer TACI molecules containing both CRD1 and CRD2.

[0198] Provided herein are immunomodulatory proteins (e.g., TACI-Fc fusion proteins) that include a TACI polypeptide that is a portion of the TACI extracellular domain (ECD) region, including CRD2, with deletion of the N-terminal region and CRD1, and deletion of one or more residues in the stem portion of the TACI extracellular domain, e.g., relative to the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the portion of the TACI extracellular domain that includes CRD2 includes amino acid residues 71-104, which correspond to the residues set forth in SEQ ID NO: 122. In provided embodiments, the immunomodulatory protein TACI polypeptide includes a deletion of N-terminal amino acid residues 1-66, which correspond to the residues set forth in SEQ ID NO: 122. In provided embodiments, the immunomodulatory protein TACI polypeptide includes a deletion of N-terminal amino acid residues 1-67, which correspond to the residues set forth in SEQ ID NO: 122. In provided embodiments, the immunomodulatory protein TACI polypeptide includes a deletion of N-terminal amino acid residues 1-68, which correspond to the residues set forth in SEQ ID NO: 122. In provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-69, which correspond to the residues set forth in SEQ ID NO: 122. In provided embodiments, the TACI polypeptide of the immunomodulatory protein comprises a deletion of N-terminal amino acid residues 1-70, which correspond to the residues set forth in SEQ ID NO: 122. In any of such embodiments, the TACI polypeptide of the immunomodulatory protein lacks one or more contiguous C-terminal amino acid residues beginning with residue 105, which corresponds to the residues of the ECD sequence set forth in SEQ ID NO: 122, up to or including amino acid residue 166. 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 ECD sequences are deleted.

[0199] In some embodiments, the immunomodulatory proteins (e.g., TACI-Fc fusion proteins) provided herein have a TACI polypeptide that includes a sequence that includes an ECD portion having a continuous sequence of amino acids of the TACI ECD, including CRD2 (e.g., residues 71-104 with respect to SEQ ID NO: 122), but with deletion of the N-terminal region and CRD1, and deletion of one or more residues in the stem portion of the TACI extracellular domain, e.g., relative to the sequence of amino acids set forth in SEQ ID NO: 122. For example, the TACI ECD portion may consist of the following, with reference to the amino acid residues set forth in SEQ ID NO: 122: amino acid residues 67 to 118, amino acid residues 67 to 117, amino acid residues 67 to 116, amino acid residues 67 to 115, amino acid residues 67 to 114, amino acid residues 67 to 113, amino acid residues 67 to 112, amino acid residues 67 to 111, amino acid residues 67 to 110, amino acid residues 67 to 109, amino acid residues 67 to 108, amino acid residues 67 to 107, amino acid residues 67 to 106, amino acid residues 67 to 105, or amino acid residues 67 to 104. In some examples, the TACI ECD portion may consist of the following, with reference to the residues set forth in SEQ ID NO: 122: amino acid residues 68 to 118, amino acid residues 68 to 117, amino acid residues 68 to 116, amino acid residues 68 to 115, amino acid residues 68 to 114, amino acid residues 68 to 113, amino acid residues 68 to 112, amino acid residues 68 to 111, amino acid residues 68 to 110, amino acid residues 68 to 109, amino acid residues 68 to 108, amino acid residues 68 to 107, amino acid residues 68 to 106, amino acid residues 68 to 105, or amino acid residues 68 to 104.In some examples, the TACI ECD portion may consist of the following, with reference to the residues set forth in SEQ ID NO: 122: amino acid residues 69 to 118, amino acid residues 69 to 117, amino acid residues 69 to 116, amino acid residues 69 to 115, amino acid residues 69 to 114, amino acid residues 69 to 113, amino acid residues 69 to 112, amino acid residues 69 to 111, amino acid residues 69 to 110, amino acid residues 69 to 109, amino acid residues 69 to 108, amino acid residues 69 to 107, amino acid residues 69 to 106, amino acid residues 69 to 105, or amino acid residues 69 to 104. In some examples, the TACI ECD portion may consist of the following, with reference to the residues set forth in SEQ ID NO: 122: amino acid residues 70 to 118, amino acid residues 70 to 117, amino acid residues 70 to 116, amino acid residues 70 to 115, amino acid residues 70 to 114, amino acid residues 70 to 113, amino acid residues 70 to 112, amino acid residues 70 to 111, amino acid residues 70 to 110, amino acid residues 70 to 109, amino acid residues 70 to 108, amino acid residues 70 to 107, amino acid residues 70 to 106, amino acid residues 70 to 105, or amino acid residues 70 to 104. In some examples, the TACI ECD portion may consist of, with reference to the residues set forth in SEQ ID NO: 122, the following: 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 may also be a TACI reference sequence according to the immunomodulatory proteins provided herein, which include variant TACI polypeptides modified relative to such TACI reference sequences by one or more amino acid modifications (e.g., substitutions) described herein.

[0200] In particular, among the TACI polypeptides provided herein are TACI ECD sequences having or consisting of the sequence set forth in SEQ ID NO: 13 (encoded by the sequence of nucleotides set forth in SEQ ID NO: 48). In some embodiments, the reference TACI sequence has or consists of the sequence set forth in SEQ ID NO: 13, and the provided variant TACI polypeptides are modified compared to such reference TACI sequence by one or more amino acid modifications (e.g., substitutions) described herein. TACI ECD sequence (CRD2): SEQ ID NO: 13 SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

[0201] In some embodiments, the reference TACI sequence comprises the amino acid sequence set forth in SEQ ID NO: 204. In some embodiments, the reference TACI sequence consists of the amino acid sequence set forth in SEQ ID NO: 204. In some embodiments, the reference TACI sequence comprises the amino acid sequence set forth in SEQ ID NO: 206. In some embodiments, the reference TACI sequence consists of the amino acid sequence set forth in SEQ ID NO: 206. In some embodiments, the reference TACI sequence comprises the amino acid sequence set forth in SEQ ID NO: 215. In some embodiments, the reference TACI sequence consists of the amino acid sequence set forth in SEQ ID NO: 215. In some embodiments, the reference TACI sequence comprises the amino acid sequence set forth in SEQ ID NO: 217. In some embodiments, the reference TACI sequence consists of the amino acid sequence set forth in SEQ ID NO: 217. In some embodiments, the reference TACI sequence comprises the amino acid sequence set forth in SEQ ID NO: 240. In some embodiments, the reference TACI sequence comprises the amino acid sequence set forth in SEQ ID NO: 241. In some embodiments, the reference TACI sequence consists of the amino acid sequence set forth in SEQ ID NO: 241.

[0202] Among the provided TACI polypeptides are variant TACI polypeptides. Also provided are immunomodulatory proteins, such as TACI-Fc fusion proteins, that comprise the provided variant TACI polypeptides. In any of the provided embodiments, the variant TACI sequence has the sequence of a reference (e.g., unmodified) TACI sequence, such as any of those described above, but further comprises another amino acid modification, such as one or more amino acid substitutions. In particular, provided herein are variant TACI polypeptides that include at least one or more affinity-modified TD domains (e.g., CRD1 and / or CRD2) or specific-binding fragments thereof that include one or more amino acid substitutions in the TD of a reference (e.g., unmodified or wild-type) TACI polypeptide, such that the variant TACI polypeptide exhibits modified (e.g., increased) binding activity or affinity for one or both of APRIL and BAFF, compared to the reference (e.g., unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has a binding affinity for APRIL and / or BAFF that differs from that of a reference (e.g., unmodified or wild-type) TACI polypeptide control sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, or Biacore assay. The binding affinity for each of the cognate binding partners is independent, i.e., in some embodiments, the variant TACI polypeptide has increased binding affinity for one or both of APRIL and BAFF, and decreased or unchanged binding affinity for the other of APRIL or BAFF, compared to the reference (e.g., unmodified or wild-type) TACI polypeptide.

[0203] In some embodiments, a variant TACI polypeptide has increased binding affinity for BAFF compared to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, a variant TACI polypeptide has increased binding affinity for APRIL compared to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, a variant TACI polypeptide has increased binding affinity for APRIL and BAFF compared to a reference (unmodified or wild-type) TACI polypeptide. The cognate ligands BAFF and / or APRIL can be mammalian proteins, such as human or mouse proteins. In certain embodiments, the cognate ligands BAFF and / or APRIL are human. In some embodiments, a variant TACI polypeptide having increased or higher binding affinity for APRIL and / or BAFF will have an increased binding affinity of at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 35%, or 50%, compared to a reference (e.g., unmodified or wild-type) TACI polypeptide control. In some embodiments, the increase in binding affinity compared to a reference (e.g., unmodified or wild-type) TACI polypeptide is greater 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. In any of the examples, the reference (e.g., unmodified or wild-type) TACI polypeptide has the same sequence as the variant TACI polypeptide, except that it does not include one or more amino acid modifications (e.g., substitutions).

[0204] In some embodiments, the equilibrium dissociation constant (K) of any of the preceding embodiments for BAFF d ) is 1×10 -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 1×10 -11 M, or 1 x 10 -12In some embodiments, the K of any of the foregoing embodiments for BAFF may be less than M. d is 1×10-9M or approximately 1×10 -9 M, 1 x 10 -10 M, or 1 x 10 -11 M, or approximately 1 x 10 -12 In some embodiments, the K of any of the preceding embodiments for BAFF is less than M. d is 1 x 10 -9 M to 1 x 10 -12 M or approximately 1 x 10 -12M In some embodiments, the K of any of the foregoing embodiments for BAFF is between d is 1 x 10 -9 M or approximately 1 x 10 -9 M, 2 x 10 -9 M or approximately 2 x 10 -9 M, 4 x 10 -9 M or approximately 4 x 10 -9 M, 6 x 10 -9 M or approximately 6 x 10 -9 M, 8 x 10 -9 M or approx. 8 x 10 -9 M, 1 x 10 -10 M or approximately 1 x 10 -10 M, 2 x 10 -10 M or approximately 2 x 10 -10 M, 4 x 10 -10 M or approximately 4 x 10 -10 M, 6 x 10 -10 M or approximately 6 x 10 -10 M, 8 x 10 -10 M or approx. 8 x 10 -10 M, 1 x 10 -11 M or approximately 1 x 10 -11 M, 2 x 10 -11 M or approximately 2 x 10 -11 M, 4 x 10 -11 M or approximately 4 x 10 -11 M, 6 x 10 -11 M or approximately 6 x 10 -11 M, 8 x 10 -11 M or approx. 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10 -12In some embodiments, provided embodiments comprise a variant TACI polypeptide as described above, which has a K d is decreased by about 1.5-fold or more, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more (higher binding affinity).

[0205] In some embodiments, the equilibrium dissociation constant (K) of any of the preceding embodiments for APRIL d ) is 1×10 -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 1×10 -11 M, or 1 x 10 -12 In some embodiments, the K of any of the foregoing embodiments for APRIL may be less than M. d is 1×10-9M or approximately 1×10 -9 M, 1 x 10 -10 M, or 1 x 10 -11 M, or approximately 1 x 10 -12 In some embodiments, the K of any of the foregoing embodiments for APRIL is less than M. d is 1 x 10 -9 M to 1 x 10 -12 M or approximately 1 x 10 -12 In some embodiments, the K of any of the foregoing embodiments for APRIL is between M. d is 1 x 10 -9 M or approximately 1 x 10 -9 M, 2 x 10 -9 M or approximately 2 x 10 -9 M, 4 x 10 -9 M or approximately 4 x 10 -9 M, 6 x 10 -9 M or approximately 6 x 10 -9 M, 8 x 10 -9 M or approx. 8 x 10 -9 M, 1 x 10 -10 M or approximately 1 x 10 -10 M, 2 x 10 -10M or approximately 2 x 10 -10 M, 4 x 10 -10 M or approximately 4 x 10 -10 M, 6 x 10 -10 M or approximately 6 x 10 -10 M, 8 x 10 -10 M or approx. 8 x 10 -10 M, 1 x 10 -11 M or approximately 1 x 10 -11 M, 2 x 10 -11 M or approximately 2 x 10 -11 M, 4 x 10 -11 M or approximately 4 x 10 -11 M, 6 x 10 -11 M or approximately 6 x 10 -11 M, 8 x 10 -11 M or approx. 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10 -12 In some embodiments, provided embodiments comprise a variant TACI polypeptide as described above, having a K d is decreased by about 1.5-fold or more, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more (higher binding affinity).

[0206] A reference (e.g., unmodified or wild-type) TACI sequence need not necessarily be used as the starting composition for generating the variant TACI polypeptides described herein. Thus, the use of the term "modification," such as "substitution," does not imply that the present embodiments are limited to a particular method for generating a variant TACI polypeptide or an immunomodulatory protein comprising the same. Variant TACI polypeptides may be generated, for example, by de novo peptide synthesis and therefore do not necessarily require modification, e.g., "substitution," in the sense of altering a codon encoding the modification, e.g., substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which similarly do not imply a particular method of generation. The means by which variant TACI polypeptides are designed or generated 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 the 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 in any number of public databases and then screened. The National Center for Biotechnology Information provides such information, and its website, like the UniProtKB database mentioned above, is publicly accessible via the Internet.

[0207] Unless otherwise indicated, amino acid modification(s) in variant TACI polypeptides presented throughout this disclosure are designated by amino acid position numbers corresponding to the numbering of positions in the reference ECD sequence set forth in SEQ ID NO: 122. It is within the level of ordinary skill in the art to identify the corresponding positions of modifications, e.g., amino acid substitutions, in a TACI polypeptide, including portions thereof, including its TD (e.g., CRD1 and / or CRD2), such as by aligning a reference sequence (e.g., SEQ ID NO: 1 or 13) with SEQ ID NO: 122. An alignment identifying corresponding residues is illustrated in Figure 9. In listings of modifications throughout this disclosure, the amino acid position is shown in the center, the corresponding reference (e.g., 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, insertions are listed with the amino acid position centered, with the corresponding reference amino acid listed before and after that number, and the insertion of the specified variant amino acid listed after the unaltered (e.g., wild-type) amino acid.

[0208] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, e.g., substitutions, in a reference (e.g., unmodified or wild-type) TACI sequence, such as, for example, any of those described. The one or more amino acid modifications, e.g., substitutions, may be in the ectodomain (extracellular domain) of the reference (e.g., unmodified or wild-type) TACI sequence. In some embodiments, the one or more amino acid modifications, e.g., substitutions, are in the CRD1 domain or a specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g., substitutions, are in the CRD2 domain or a specific binding fragment thereof. In some embodiments of the variant TACI polypeptide, some of the one or more amino acid modifications, e.g., substitutions, are in the CRD1 domain or a specific binding fragment thereof, and some of the one or more amino acid modifications, e.g., substitutions, are in the CRD2 domain or a specific binding fragment thereof.

[0209] In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modification(s), e.g., substitution(s), in the reference TACI sequence. The modifications, e.g., substitutions, may be within the CRD1 domain or the CRD2 domain. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in the CRD1 domain of the reference TACI sequence, or a specific-binding fragment thereof. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in the CRD2 domain of a reference TACI sequence or a specific binding fragment thereof.

[0210] In some embodiments, a variant TACI polypeptide comprising one or more of the described 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 set forth in SEQ ID NO: 122, or a specific binding fragment thereof comprising the CRD1 and / or CRD2 domains. In some embodiments, the specific binding fragment comprises the CRD1 domain, e.g., the specific binding fragment comprises the sequence set forth in amino acids 34-66 of SEQ ID NO: 122. In some instances, the CRD1 domain is the only complete CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment comprises the CRD2 domain, e.g., the specific binding fragment comprises the sequence set forth in amino acids 71-104 of SEQ ID NO: 122. In some instances, the CRD2 domain is the only complete CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment comprises the CRD1 and CRD2 domains, 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 stem domain, e.g., the specific binding fragment comprises a contiguous portion of amino acids 105-165 of SEQ ID NO: 122. In some embodiments of any embodiment, the specific binding fragment of SEQ ID NO: 122 is less than the full-length ECD set forth in SEQ ID NO: 122. In some embodiments, the specific binding fragment is set forth in SEQ ID NO: 1. In some embodiments, the specific binding fragment is set forth in SEQ ID NO: 13. In some embodiments, the specific binding fragment is set forth in SEQ ID NO: 130. In some embodiments, the specific binding fragment is set forth in SEQ ID NO: 131.

[0211] In some embodiments, a variant TACI polypeptide comprising one or more of the described 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, e.g., the amino acid sequence of SEQ ID NO: 1, 13, or 122.

[0212] In some embodiments, a variant TACI polypeptide containing one or more of the described 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.

[0213] In some embodiments, a variant TACI polypeptide containing one or more of the described 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.

[0214] In some embodiments, a variant TACI polypeptide containing one or more of the described 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.

[0215] In some embodiments, a variant TACI polypeptide containing one or more of the described 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.

[0216] In some embodiments, a variant TACI polypeptide containing one or more of the described 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.

[0217] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, e.g., substitutions, in a reference TACI polypeptide or a specific binding fragment thereof corresponding to positions (or 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 based on the numbering of SEQ ID NO: 122. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, e.g., a substitution 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 a conservative amino acid substitution thereof. In some embodiments, the reference TACI polypeptide comprises a CRD1 domain or a CRD2 domain, e.g., the reference TACI polypeptide is set forth in SEQ ID NO: 1 or SEQ ID NO: 122.

[0218] In some embodiments, the amino acid substitutions are only within the CRD2 domain. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, e.g., substitutions, in a reference TACI polypeptide or a specific binding fragment thereof corresponding to positions 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, based on the numbering of SEQ ID NO: 122. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, e.g., a substitution 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 a conservative amino acid substitution thereof. In some embodiments, a reference TACI polypeptide includes only the CRD2 domain and lacks the CRD1 domain of the CRD domains, e.g., the reference TACI polypeptide is set forth in SEQ ID NO: 13. Thus, in some embodiments, a variant TACI polypeptide comprises a portion of the ECD sequence of a TACI polypeptide that includes the CRD2 domain but lacks the CRD1 domain.

[0219] A conservative amino acid modification, e.g., substitution, is any amino acid that falls into the same class of amino acids as the substituted amino acid, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl- 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).

[0220] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 75, relative to the numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 75 enhances binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not comprise the amino acid substitution. In some embodiments, the substituted amino acid is an acidic amino acid or amide, e.g., to a different acidic amino acid or amide compared to a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 75 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 75 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 75 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 75 is glutamine (Gln, Q).

[0221] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 77, relative to the numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 77 enhances binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not comprise the amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 77 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 77 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 77 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 77 is glutamine (Gln, Q).

[0222] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 78, relative to the numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 78 enhances binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not comprise the amino acid substitution. In some embodiments, the substituted amino acid at position 78 is an aromatic amino acid, e.g., to a different aromatic amino acid compared to the 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).

[0223] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 84, relative to the numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 84 enhances binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not comprise 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).

[0224] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 101, relative to the numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 101 enhances binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not comprise 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).

[0225] In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution at position 102, relative to the numbering of SEQ ID NO: 122. In some embodiments, the amino acid substitution at position 102 enhances binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not comprise 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).

[0226] In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution E74V. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution Q75E. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution K77E. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution F78Y. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution Y79F. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution L82H. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution L82P. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution R84G. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution R84L. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution R84Q. In some embodiments, a variant TACI polypeptide comprises at least one amino acid substitution D85V. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution A101D. In some embodiments, the variant TACI polypeptide comprises at least one amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide comprises two or more amino acid substitutions of any two or more of the foregoing. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions that are any of the foregoing conservative amino acid substitutions. In 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, the at least one amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1.In some embodiments, at least one amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, at least one amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, at least one amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO: 131.

[0227] In some embodiments, a variant TACI polypeptide comprises the amino acid substitution E74V. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution Q75E. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution K77E. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution F78Y. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution Y79F. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution L82H. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution L82P. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution R84G. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution R84L. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution R84Q. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution D85V. In some embodiments, a variant TACI polypeptide comprises the amino acid substitution C86Y. In some embodiments, a 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 comprises two or more amino acid substitutions of any two or more of the foregoing. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions that are any of the foregoing conservative amino acid substitutions. In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0228] 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 provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0229] In any embodiment, the variant TACI polypeptide comprises one or more amino acid substitutions from Q75E, K77E, F78Y, R84G, R84Q, A101D, or Y102D, or any combination thereof. In some embodiments, the variant TACI polypeptide comprises any one, two, three, four, five, or six of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide comprises one of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide comprises two of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide comprises three of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide comprises four of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide comprises five of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide comprises six of the above amino acid substitutions.

[0230] In an embodiment of any embodiment, the one or more amino acid substitutions comprise Q75E / R84Q. In an embodiment of any embodiment, the one or more amino acid substitutions comprise Q75E / K77E. In an embodiment of any embodiment, the one or more amino acid substitutions comprise Q75E / F78Y. In an embodiment of any embodiment, the one or more amino acid substitutions comprise Q75E / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise Q75E / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise F77E / F78Y. In an embodiment of any embodiment, the one or more amino acid substitutions comprise K77E / R84Q. In an embodiment of any embodiment, the one or more amino acid substitutions comprise K77E / A101D. In an embodiment of any embodiment, another amino acid substitution comprises K77E / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise F78Y / R84Q. In an embodiment of any embodiment, the one or more amino acid substitutions comprise F78Y / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise F78Y / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise R84Q / A101D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise R84Q / Y102D. In an embodiment of any embodiment, the one or more amino acid substitutions comprise A101D / Y102D. In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described, for example, in the sequence set forth in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131.

[0231] In some embodiments, the variant TACI polypeptide comprises amino acid substitution(s) 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 provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described, for example, in the sequences set forth in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131.

[0232] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E and F78Y (K77E / F78Y). In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0233] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E and Y102D (K77E / Y102D). In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0234] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions F78Y and Y102D (F78Y / Y012D). In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0235] In some embodiments, the variant TACI polypeptide comprises amino acid substitutions K77E, F78Y, and Y102D (K77E / F78Y / Y102D). In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0236] In some embodiments, the variant TACI polypeptide comprises the amino acid substitution Q75E / R84Q. In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is within the reference TACI sequence set forth in SEQ ID NO: 131.

[0237] In some embodiments, the variant TACI polypeptide comprises any of the mutations listed in Table 1. Table 1 also provides exemplary sequences by reference to the SEQ ID NOs of a reference (e.g., unmodified) TACI polypeptide and exemplary variant TACI polypeptides. As indicated, the exact locus or residues corresponding to a given domain may vary depending, for example, on the method used to identify or classify the domain. Also, in some cases, the adjacent N- and / or C-terminal amino acids of a given domain (e.g., a CRD) may also be included in the sequence of the variant TACI polypeptide, such as to ensure proper folding of the domain when expressed. Therefore, it should be understood that the exemplary SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain, such as the ECD domain of a variant TACI polypeptide or a portion thereof comprising only CRD1 / CRD2 or CRD2, may be several amino acids longer or shorter than the amino acid sequence set forth in the respective SEQ ID NO, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0238] In some embodiments, the variant TACI polypeptide comprises any of the mutations (amino acid substitutions) listed in Table 1. In some examples, the mutations (amino acid substitutions) are made in a reference TACI comprising the sequence of amino acids set forth in SEQ ID NO: 122. In some examples, the mutations (amino acid substitutions) are made in a reference TACI comprising the CRD1 and CRD2 domains of TACI, for example, as set forth in SEQ ID NO: 1. In some examples, the mutations (amino acid substitutions) are made in a reference TACI that has been further truncated by deletion of N- and C-terminal amino acid residues to retain the CRD2, for example, as set forth in SEQ ID NO: 13.

[0239] The use of the term "modification," such as "substitution" or "mutation," does not imply that the present embodiments are limited to a particular method of producing an immunomodulatory protein. Variant TACI polypeptides may be produced, for example, by de novo peptide synthesis and, therefore, do not necessarily require modification, e.g., "substitution," in the sense of altering a codon encoding the modification, e.g., substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which similarly do not imply a particular method of production. The means by which vTDs are designed or produced 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 desired specific binding activity, e.g., binding affinity, and / or altered NF-κB regulatory or other functional activity. In some embodiments, vTDs are synthesized de novo using protein or nucleic acid sequences available in any number of public databases and then screened. The National Center for Biotechnology Information provides such information, and its website, like the UniProtKB database, is publicly accessible via the Internet.

[0240] In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence comprising CRD1 and CRD2, such as a variant TACI polypeptide set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120. In some embodiments, the variant TACI polypeptide comprises a polypeptide sequence 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, e.g., at least about 96% identity, 97% identity, 98% identity, or 99% identity, to any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and retains amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide comprises a specific binding fragment of any one of SEQ ID NOs: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a contiguous sequence therein that includes amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0241] In some embodiments, a variant TACI polypeptide consists of, or consists essentially of, a variant TACI extracellular domain (ECD) sequence set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120. In some embodiments, a variant TACI polypeptide consists of, or consists essentially of, a polypeptide sequence exhibiting at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, e.g., at least about 96% identity, 97% identity, 98% identity, or 99% identity, to any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and retains amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or consists essentially of a specific binding fragment of any one of SEQ ID NOs: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds to BAFF, APRIL, or the APRIL / BAFF heterotrimer and includes a contiguous sequence therein that includes amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0242] In some embodiments, a variant TACI polypeptide comprises an extracellular domain (ECD) sequence that includes CRD2 but lacks CRD1 of a reference TACI polypeptide, such as a variant TACI polypeptide set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192. In some embodiments, the variant TACI polypeptide comprises a polypeptide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, e.g., at least about 96% identity, 97% identity, 98% identity, or 99% identity, to any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and retains amino acid modification(s), e.g., substitution(s), that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide comprises a specific binding fragment of any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a contiguous sequence therein that includes amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0243] In some embodiments, the variant TACI polypeptide consists of, or consists essentially of, the sequence set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192. In some embodiments, the variant TACI polypeptide consists of, or consists essentially of, a polypeptide sequence exhibiting at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, e.g., at least about 96% identity, 97% identity, 98% identity, or 99% identity, to any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, and retains amino acid modification(s), e.g., substitution(s), that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or consists essentially of a specific binding fragment of any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and includes a contiguous sequence therein that includes amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI.

[0244] In some embodiments, the variant TACI polypeptide comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the variant TACI polypeptide consists essentially of the sequence set forth in SEQ ID NO: 20. In some embodiments, the variant TACI polypeptide consists of the sequence set forth in SEQ ID NO: 20.

[0245] In some embodiments, the variant TACI polypeptide comprises the sequence set forth in SEQ ID NO: 26. In some embodiments, the variant TACI polypeptide consists essentially of the sequence set forth in SEQ ID NO: 26. In some embodiments, the variant TACI polypeptide consists of the sequence set forth in SEQ ID NO: 26.

[0246] In some embodiments, the variant TACI polypeptide comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the variant TACI polypeptide consists essentially of the sequence set forth in SEQ ID NO: 27. In some embodiments, the variant TACI polypeptide consists of the sequence set forth in SEQ ID NO: 27.

[0247] In some embodiments, the variant TACI polypeptide comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, the variant TACI polypeptide consists essentially of the sequence set forth in SEQ ID NO: 107. In some embodiments, the variant TACI polypeptide consists of the sequence set forth in SEQ ID NO: 107.

[0248] In some embodiments, the variant TACI polypeptide is encoded by a sequence of nucleotides set forth in any of SEQ ID NOs: 37-47, 56, or 57. In some embodiments, the variant TACI polypeptide is encoded by a sequence of nucleotides 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, e.g., at least about 96% identity, 97% identity, 98% identity, or 99% identity, to any one of SEQ ID NOs: 37-47, 56, or 57, and retains amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI. Also provided herein are nucleic acids comprising a sequence set forth in any one of SEQ ID NOs: 37-47, 56, or 57, or a sequence exhibiting at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 37-47, 56, or 57.

[0249] In some embodiments, the variant TACI polypeptide is encoded by a sequence of nucleotides set forth in any of SEQ ID NOs: 49-55 or 58-70. In some embodiments, the variant TACI polypeptide is encoded by a sequence of nucleotides 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, e.g., at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 49-55 or 58-70, and possesses amino acid modification(s), e.g., substitution(s), that are not present in a reference (e.g., unmodified or wild-type) TACI. Also provided herein are nucleic acids comprising a sequence exhibiting at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOs: 49-55 or 58-70, or any one of SEQ ID NOs: 549-55 or 58-70.

[0250] [Table 1] TIFF2025535041000003.tif232165TIFF2025535041000004.tif141165

[0251] Also provided herein, in some embodiments, are TACI ECD fusion sequences in which any of the above TACI ECD sequences are linked or fused to a multimerization domain, such as any of those described herein.

[0252] The interaction of two or more polypeptides of immunomodulatory protein can be promoted by any part that can interact with itself to form a stable structure or by direct or indirect binding to other polypeptides.For example, separate coded polypeptide chains can be linked by multimerization, whereby the multimerization of polypeptides is brought about by multimerization domain.Generally, multimerization domain provides the formation of stable protein-protein interaction between the first polypeptide and the second polypeptide.

[0253] In some embodiments, two or more individual polypeptides of an immunomodulatory protein can be linked by multimerization, for example, as 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 another example, a tetramer molecule can be formed from four copies of the same individual polypeptide. In a further example, a pentamer molecule can be formed from five copies of the same individual polypeptide. The multimerization domain can promote the dimerization, trimerization, tetramerization, or pentamerization of polypeptide chains.

[0254] In some embodiments, the immunomodulatory protein forms a multimer, for example, a dimer. In some embodiments, the dimer is a homodimer in which the two polypeptides of the immunomodulatory protein are the same. In some embodiments, the dimer is a heterodimer in which the two polypeptides of the immunomodulatory protein are different.

[0255] In some embodiments, multimerization domains include any that are capable of forming stable protein-protein interactions. The multimerization domain can interact through immunoglobulin sequences (e.g., Fc domains; see, e.g., International Patent Publication Nos. WO93 / 10151 and WO2005 / 063816US, U.S. Publication No. 2006 / 0024298, and U.S. 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 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 thiols that form intermolecular disulfide bonds between homo- or heteromultimeric chimeric molecules. Furthermore, the multimerization domain may contain an amino acid sequence containing a protrusion complementary to an amino acid sequence containing a hole, such as those described in, for example, U.S. Pat. No. 5,731,168, International Patent Publication Nos. WO 98 / 50431 and WO 2005 / 063816, and Ridgway et al. (1996) Protein Engineering, 9:617-621. Such multimerization regions may be engineered so that steric interactions not only promote stable interactions but also favor the formation of heterodimers rather than homodimers from a mixture of chimeric monomers. Generally, protrusions are constructed by replacing small amino acid side chains on the interface of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a "cavity" of the same or similar size as the protrusion is optionally created on the interface of a second polypeptide. Exemplary multimerization domains are described below.

[0256] A TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) can be attached anywhere, but typically via its N- or C-terminus, to the N- or C-terminus of a multimerization domain to form a chimeric polypeptide. The linkage can be direct or indirect via a linker. The chimeric polypeptide can also be a fusion protein or formed by chemical conjugation, such as through covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerization domain, a nucleic acid encoding all or a portion of a TACI polypeptide sequence, such as any of the described TACI ECDs, including a variant TACI polypeptide sequence, can be operably linked, directly or indirectly, optionally via a linker domain, to a nucleic acid encoding a multimerization domain sequence. In some cases, the construct encodes a chimeric protein in which the C-terminus of the TACI polypeptide sequence is linked to the N-terminus of the multimerization domain. In some cases, the construct can encode a chimeric protein in which the N-terminus of the TACI polypeptide sequence is linked to the N- or C-terminus of the multimerization domain.

[0257] Polypeptide multimers include two chimeric proteins created by directly or indirectly linking two identical or different TACI polypeptide sequences (e.g., two identical or different variant TACI polypeptide sequences) to a multimerization domain. In some examples, when the multimerization domain is a polypeptide, a gene fusion encoding the TACI polypeptide sequence (e.g., a 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 multimers, where the multimerization domains interact to form a multivalent polypeptide. Chemical linkage of the multimerization domain to the TACI polypeptide (e.g., a variant TACI polypeptide) can be achieved using a heterobifunctional linker.

[0258] The resulting chimeric polypeptide, e.g., a fusion protein, and multimers formed therefrom can be purified by any suitable method, for example, affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, homodimers and heterodimers are formed. Expression conditions can be adjusted so that heterodimer formation is preferred over homodimer formation.

[0259] In some embodiments, the multimerization domain is an Fc region of an immunoglobulin.

[0260] In some embodiments, the multimerization domain is an immunoglobulin (e.g., IgG1) Fc region, and the fusion protein is a TACI-Fc that comprises (1) a TACI sequence that comprises or consists of any of the provided TACI ECD sequences, and (2) an immunoglobulin Fc region. Thus, among the provided embodiments are TACI-Fc fusion proteins that comprise (1) a TACI sequence that comprises or consists of any of the above-described TACI ECD polypeptide sequences, e.g., variant TACI polypeptides, and (2) an immunoglobulin Fc region.

[0261] In some embodiments, provided herein are TACI-Fc fusion sequences comprising: (1) a TACI ECD sequence comprising the sequence set forth in SEQ ID NO: 13; and (2) an immunoglobulin Fc region. In some embodiments, provided herein are TACI-Fc fusion sequences comprising: (1) a TACI ECD sequence consisting of, or consisting essentially of, the sequence set forth in SEQ ID NO: 13; and (2) an immunoglobulin Fc region.

[0262] In some embodiments, the TACI-Fc fusion is a variant TACI-Fc fusion comprising or consisting of any of the variant TACI polypeptides described above and an immunoglobulin Fc region.

[0263] In some embodiments, provided herein are (1) a TACI ECD sequence comprising a CRD1 and a CRD2, e.g., a TACI sequence comprising a sequence set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and (2) a variant TACI-Fc fusion sequence comprising an immunoglobulin Fc region. In some embodiments, provided herein are (1) a TACI ECD sequence comprising a CRD1 and a CRD2, e.g., a TACI sequence consisting of, or consisting essentially of, a sequence set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120, and (2) a variant TACI-Fc fusion sequence comprising an immunoglobulin Fc region.

[0264] In some embodiments, provided herein are (1) a TACI ECD sequence that includes a CRD2 domain but lacks a CRD1 domain, e.g., a TACI sequence that includes a sequence set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, and 177-192, and (2) a variant TACI-Fc fusion sequence that includes an immunoglobulin Fc region. In some embodiments, provided herein are (1) a TACI ECD sequence that includes a CRD2 domain but lacks a CRD1 domain, e.g., a TACI sequence that consists of or consists essentially of a sequence set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, and 177-192, and (2) a variant TACI-Fc fusion sequence that includes an immunoglobulin Fc region.

[0265] In provided embodiments of TACI-Fc, the immunoglobulin Fc region can be a wild-type Fc of an immunoglobulin, such as an IgG1 Fc. Optionally, the Fc region can be a variant Fc that lacks effector function (also referred to as an "effector-less Fc"). Exemplary Fc regions and variants thereof in provided TACI-Fc fusion proteins are shown below.

[0266] In some embodiments, the Fc is a mouse or human Fc, hi some embodiments, the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0267] In some embodiments, the Fc region is or comprises a sequence set forth in any one of SEQ ID NOs: 71, 73, 75, 81, 82, 83, 134, 135, 136, 137, 138, 139, 140, 173, 174, 175, 176, 193, 218, 219, 220, or 221. In some embodiments, the Fc region is or is derived from an IgG1, such as that set forth in any one of SEQ ID NOs: 71, 73, 75, 81, 82, 83, 134, 135, 136, 137, 139, 140, 173, 174, 175, 176, 193, 218, 220, or 221. In some embodiments, the Fc region is or is derived from an IgG2, such as any set forth in SEQ ID NOs: 138 or 219. In some embodiments, the Fc region is or is derived from an IgG4, such as any set forth in SEQ ID NOs: 139, 140, or 220. In some embodiments, the Fc region of the Fc fusion proteins provided herein may also comprise an Fc region that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with any of the above Fc regions.

[0268] In some embodiments, the Fc is derived from an IgG1, such as human IgG1. In some embodiments, the Fc is an IgG1 Fc set forth in SEQ ID NO: 71, which has an allotype containing residues Glu (E) and Met (M) at positions 356 and 358 according to EU numbering. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 71, or a sequence of amino acids exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 71. In other embodiments, the Fc is an IgG1 Fc that contains amino acids of the human G1m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358 set forth in SEQ ID NO: 81. Thus, in some cases, the Fc provided herein may contain the amino acid substitutions E356D and M358L to reconstitute residues of the G1 m1 allotype. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:81, or a sequence of amino acids exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:81.

[0269] In some embodiments, the Fc region has the amino acid sequence set forth in SEQ ID NO:81. EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 81)

[0270] In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the Fc region consists of the amino acid sequence set forth in SEQ ID NO:81.

[0271] In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO: 173. In some embodiments, the variant Fc comprises the sequence set forth 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.

[0272] In some embodiments, the Fc is derived from an IgG2, such as human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 138, or a sequence of amino acids exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 138. In some embodiments, the Fc region is an IgG2 Fc having the sequence set forth in SEQ ID NO: 138. In some embodiments, the Fc region is an IgG2 Fc having the sequence set forth in SEQ ID NO: 219.

[0273] In some embodiments, the Fc is derived from IgG4, such as human IgG4. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 139, or a sequence of amino acids exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 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 inhibition of 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, respectively, or an antibody in which arginine at position 409 of human IgG4 as defined by the EU index proposed by Kabat et al. is replaced with lysine and exhibits inhibition of aggregate formation (see, e.g., U.S. Patent No. 8,911,726). In some embodiments, the Fc is an IgG4 comprising the S228P mutation, which has been shown to prevent recombination between therapeutic antibodies and endogenous IgG4 by Fab arm exchange (see, e.g., Labrijin et al. (2009) Nat. Biotechnol., 27(8):767-71). In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 140, or a sequence of amino acids exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 140. In some embodiments, the Fc region is an IgG4 Fc region set forth in SEQ ID NO: 140. In some embodiments, the Fc region is an IgG4 Fc region set forth in SEQ ID NO: 220.

[0274] In some embodiments, the Fc region is a variant Fc region in which the wild-type Fc has been modified with one or more amino acid substitutions that reduce effector activity or render the Fc inactive with respect to Fc effector function. Exemplary effectorless or inactive mutations include those described herein.

[0275] In some embodiments, the Fc region contains another modification that alters (e.g., reduces) one or more of its normal functions. Generally, the Fc region is involved in effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen-binding ability, which is the primary function of immunoglobulins. Furthermore, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by increasing in vivo half-life through conjugation with the in vivo FcRn receptor. In some embodiments, such functions may be reduced or modified in an Fc for use with the provided Fc fusion proteins.

[0276] In some embodiments, one or more amino acid modifications can be introduced into the Fc region to generate an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of Fc sequence changes or mutations that can alter effector function. For example, WO00 / 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 these publications are expressly incorporated herein by reference.

[0277] In some embodiments, the provided immunomodulatory proteins comprise an Fc region that exhibits reduced effector function, making them desirable candidates 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 deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the immunomodulatory protein lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.) Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the immunomodulatory protein is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0278] Immunomodulatory proteins with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with 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 variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0279] In some embodiments, the Fc region of the immunomodulatory protein has any one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as indicated by EU numbering) substituted with a different amino acid compared to a native Fc region. Such modifications of the Fc region include, for example, those listed in Current Opinion in Deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S ... -S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E modifications; modifications such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (indicated by EU numbering); and modifications at sites described in WO2000 / 042072.

[0280] Certain Fc variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO2004 / 056312, WO2006019447, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0281] In some embodiments, immunomodulatory proteins are provided that comprise a variant Fc region comprising one or more amino acid substitutions that increase half-life and / or improve binding to fetal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) and WO2015107026. These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 according to EU numbering, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0282] In some embodiments, the Fc region of the immunomodulatory protein comprises one or more amino acid substitutions C220S, C226S, and / or C229S according to EU numbering. In some embodiments, the Fc region of the immunomodulatory protein comprises one or more amino acid substitutions R292C and V302C. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0283] In some embodiments, modifications are made in the Fc region that result in reduced C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0284] In some embodiments, the variant Fc region comprising one or more amino acid modifications (e.g., amino acid substitutions) is derived from wild-type IgG1, such as wild-type human IgG1. In some embodiments, the wild-type IgG1 Fc can be the Fc set forth in SEQ ID NO: 71, which has an allotype comprising 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 set forth in SEQ ID NO: 71. In other embodiments, the wild-type IgG1 Fc comprises amino acids of the human G1m1 allotype, e.g., residues comprising Asp (D) and Leu (L) at positions 356 and 358 according to SEQ ID NO: 81. Thus, in some cases, the variant Fc is derived from the amino acid sequence set forth in SEQ ID NO: 81.

[0285] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 71 or 81 (corresponding to K447del according to EU numbering).

[0286] In some embodiments, the variant Fc region comprises the C5S amino acid modification of a wild-type or unmodified Fc region according to the numbering of SEQ ID NO: 71 (corresponding to C220S according to EU numbering).

[0287] In some embodiments, the Fc region is a variant Fc comprising at least one amino acid substitution that is N82G according to the numbering of SEQ ID NO: 71 (corresponding to N297G according to EU numbering). In some embodiments, the Fc further comprises at least one amino acid substitution that is 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 further comprises a C5S amino acid modification according to the numbering of SEQ ID NO: 71 (corresponding to C220S according to EU numbering). For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: N297G according to EU numbering and one or more of the following amino acid modifications C220S, R292C, or V302C (corresponding to N82G and one or more of the following amino acid modifications C5S, R77C, or V87C with reference to SEQ ID NO: 71), e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 82.

[0288] In some embodiments, the variant Fc comprises the amino acid substitutions L234A / L235E / G237A according to EU numbering. In some embodiments, the variant Fc comprises the amino acid substitutions A330S / P331S according to EU numbering. In some embodiments, the variant Fc comprises the amino acid substitutions L234A / L235E / G237A / A330S / P331S (Gross et al. (2001) Immunity 15:289). In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO: 175. In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO: 176. In some embodiments, the Fc region used in the constructs provided herein may further lack a C-terminal lysine residue.

[0289] In some embodiments, the Fc region is a variant Fc comprising the mutations L234A, L235E, and G237A according to EU numbering. In some embodiments, the wild-type Fc is further modified by removal of one or more cysteine ​​residues, such as by substitution of the cysteine ​​residue at position 220 according to EU numbering with a serine residue (C220S). Exemplary inactive Fc regions with reduced effector function are set forth in SEQ ID NO:83 and SEQ ID NO:75, which are based on the allotypes set forth in SEQ ID NO:71 or SEQ ID NO:81, respectively. In some embodiments, the Fc region may further lack a C-terminal lysine residue. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L234A, L235E, or G237A, e.g., the Fc region comprises the sequence set forth in SEQ ID NO:73, 75, 83, or 136. In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO:73. In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO:75. In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO: 83. In some embodiments, the variant Fc comprises the sequence set forth in SEQ ID NO: 136.

[0290] In some embodiments, the Fc region is a variant Fc having the sequence set forth in SEQ ID NO:73. EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73)

[0291] In some embodiments, the Fc region is an IgG1 Fc but does not include a hinge sequence. In some embodiments, the IgG1 Fc region does not include the hinge sequence EPKSC (SEQ ID NO: 239). In some embodiments, the IgG1 Fc region does not include the hinge sequence EPKSS (SEQ ID NO: 238).

[0292] In some embodiments, the Fc region is a variant Fc having the sequence set forth in SEQ ID NO:221. DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 221)

[0293] In some embodiments, the Fc region is a variant Fc region that comprises one or more of the amino acid modifications C220S, E233P, L234V, L235A, G236del, or S267K, e.g., the Fc region comprises the sequence set forth in SEQ ID NO: 134. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). In some embodiments, the Fc region comprises the sequence set forth in SEQ ID NO: 137.

[0294] In some embodiments, the Fc region is a variant Fc region comprising one or more of the amino acid modifications C220S, R292C, N297G, V302C. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). An exemplary variant Fc region is set forth in SEQ ID NO: 135.

[0295] In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S / E233P / L234V / L235A / G236del / S267K. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). An exemplary variant Fc region is set forth in SEQ ID NO: 137.

[0296] Examples of such Fc regions for inclusion in immunomodulatory polypeptides are shown in Table 2.

[0297] [Table 2]

[0298] In some embodiments, the Fc region is a variant Fc region comprising any combination of Fc mutations in Table 2. In some embodiments, the Fc region is a variant Fc region having a sequence set forth in any one of the SEQ ID NOs in Table 2.

[0299] For example, the variant Fc region can be an effector-less Fc that exhibits reduced effector activity compared to the wild-type IgG1 set forth in SEQ ID NO: 71 or SEQ ID NO: 81. In some embodiments, the variant Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 75, 82, 83, 134, 73, 135, 136, or 137, or a sequence of amino acids that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 75, 82, 83, 134, 73, 135, 136, or 137. In some embodiments, the variant Fc has the sequence set forth in SEQ ID NO: 73. In embodiments, when produced and expressed from a cell, the provided immunomodulatory protein (e.g., a TACI-Fc fusion) is a homodimer comprising two identical polypeptide chains.

[0300] 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 may be a region that promotes or facilitates heterodimer formation.

[0301] In some embodiments, one or both Fc domains of the first and second immunomodulatory Fc fusion polypeptides contain modifications (e.g., substitutions) that alter the interface of the Fc molecule and facilitate and / or promote heterodimerization. Methods for promoting heterodimerization of Fc chains include mutagenesis of the Fc region, for example, by including a set of "knobs-into-holes" mutations or mutations that result in electrostatic steering of the Fc, favoring attractive interactions between different polypeptide chains. In some embodiments, the Fc region of the heterodimeric molecule may further include one or more other Fc mutations, such as any of those described above. In some embodiments, the heterodimeric molecule includes an Fc region with a mutation that reduces effector function. In some embodiments, such an Fc region includes the mutations C220S, L234A, L235E, and / or G237A according to EU numbering. In some embodiments, any of the above mutations in the Fc backbone may be made in an allotype comprising 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 may be made in an allotype comprising residues Asp (D) and Leu (L) at positions 356 and 358 according to EU numbering.

[0302] In some embodiments, modifications include the introduction of a protrusion (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide, such that the protrusion is positioned within the cavity to facilitate complexation of the first and second Fc-containing polypeptides. The amino acids that are subject to substitution and / or modification to create a protrusion or cavity in a polypeptide are generally interface amino acids that interact or contact one or more amino acids at the interface of a second polypeptide.

[0303] In some embodiments, a first polypeptide modified to include a knob amino acid comprises a replacement of a natural or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide, thereby positioning within a counterbalanced cavity (hole) in the adjacent interface of the second polypeptide. In most cases, the replacement amino acid has a larger side chain volume than the original amino acid residue. Those skilled in the art will know how to determine and / or evaluate the properties of amino acid residues to identify amino acid residues that are ideal replacement amino acids for creating knobs. In some embodiments, the replacement residue for forming a knob is a naturally occurring amino acid residue, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residue identified for substitution is an amino acid residue with a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0304] In some embodiments, the second polypeptide modified to contain a cavity (hole) comprises a substitution of a natural 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 accommodate a corresponding protrusion from the interface of the first polypeptide. In most cases, the substituted 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 amino acid residues that are ideal replacement residues for cavity formation. Generally, replacement residues for cavity formation are naturally occurring amino acids, such as alanine (A), serine (S), threonine (T), and valine (V). In some examples, the original amino acid identified for substitution is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0305] For example, the CH3 interface of human IgG1 contains 16 residues in each domain located on four antiparallel β-strands buried 1090 Å from each surface (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). Modification of the CH3 domain to create a protrusion or cavity is described, for example, in U.S. Patent No. 5,731,168, International Patent Application Nos. WO98 / 50431 and WO2005 / 063816, and Ridgway et al., (1996) Prot.Engin., 9:617-621. In some examples, modification of the CH3 domain to create a protrusion or cavity generally targets residues located on the two central antiparallel β-strands. The purpose is to minimize the risk that the created protrusion may be accommodated by protruding into the surrounding solvent rather than being accommodated in the offset cavity of the partner CH3 domain.

[0306] In some embodiments, the heterodimeric molecule comprises a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain." In some cases, an additional interchain disulfide bridge between the CH3 domains can be used, for example, by introducing a Y349C mutation in the CH3 domain of the "knob" or "hole" chain and an E356C or S354C mutation in the CH3 domain of the other chain (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681). In some embodiments, the heterodimeric molecule comprises an S354C, T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. For example, the knob Fc may comprise the sequence set forth in SEQ ID NO: 89, comprising S354C and T366W, and the hole Fc may comprise the sequence set forth in SEQ ID NO: 90, comprising the mutations Y349C, T366S, L368A, and Y407V. In some embodiments, the heterodimeric molecule comprises E356C, T366W mutations in one of the two CH3 domains and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. Other examples of knobs-in-holes technology are known in the art, for example, as described in EP1870459A1.

[0307] In some embodiments, an Fc variant comprising a CH3 knob or hole modification may be attached to a multi-domain immunomodulatory polypeptide anywhere, but typically via its N- or C-terminus, to the N- or C-terminus of one or more TACI polypeptide sequences (e.g., variant TACI polypeptide sequences), e.g., to form a fusion polypeptide. The attachment may be direct or indirect via a linker. Typically, knob and hole molecules are produced by coexpression of a first immunomodulatory polypeptide linked to an Fc variant comprising a CH3 knob modification(s) and a second immunomodulatory polypeptide linked to an Fc variant comprising a CH3 cavity modification(s).

[0308] Exemplary sequences of knob and hole Fc polypeptides are set forth in SEQ ID NOs: 128 and 129, respectively. In some embodiments, the knob or hold Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 71 (corresponding to K447del according to EU numbering). Exemplary sequences of knob and hole Fc polypeptides are set forth in SEQ ID NOs: 89 and 90, respectively.

[0309] In some embodiments, individual polypeptides of a multi-domain polypeptide or individual polypeptides of a single-domain polypeptide are linked to a multimerization domain that forms an immunomodulatory protein that is a trimer, tetramer, or pentamer. In some embodiments, the individual polypeptides of such molecules are the same. 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.

[0310] In some embodiments, the multimerization domain is part of the cartilage oligomeric matrix protein (COMP) assembly domain (Voulgaraki et al., Immunology (2005) 115(3):337-346). In some examples, COMP is or comprises the amino acid sequence set forth in SEQ ID NO: 146 (e.g., amino acids 29-72 of full-length COMP, Uniprot Accession No. P49747), or a sequence having about 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 146.

[0311] 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, the VASP is or comprises the amino acid sequence set forth in SEQ ID NO: 147 (e.g., amino acids 343-375 of full-length VASP, Uniprot Accession No. P50552), or a sequence having about 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 147.

[0312] In some embodiments, a TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) is attached to a multimerization domain (e.g., an Fc region) via a linker, such as a peptide linker. In some embodiments, the peptide linker may be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length.

[0313] In some embodiments, the linker is (in single-letter amino acid code): GGGGS ("4GS"; SEQ ID NO: 77), or a multimer of 4GS linkers, e.g., 2, 3, 4, or 5 repeats of the 4GS linker. In some embodiments, the peptide linker is (GGGGS) (SEQ ID NO: 78), (GGGGS) (SEQ ID NO: 79), (GGGGS) (SEQ ID NO: 84), or (GGGGS) (SEQ ID NO: 91). In some embodiments, the linker may also include a series of alanine residues, alone or in addition to another peptide linker (such as a 4GS linker or a multimer thereof). In some embodiments, the linker (in single-letter amino acid code) is GSGGGGS (SEQ ID NO: 74) or GGGGSSA (SEQ ID NO: 80). In some examples, the linker is 2xGGGGS followed by 3 alanines (GGGGSGGGGSAAA; SEQ ID NO: 133). In some examples, the linker is set forth in SEQ ID NO: 194 or 195.

[0314] In some embodiments, a TACI polypeptide, such as a variant TACI polypeptide, is directly linked to an Fc sequence. In some embodiments, a TACI polypeptide, such as a variant TACI polypeptide, is indirectly linked to an Fc sequence, for example, via a linker. In some embodiments, one or more "peptide linkers" link a TACI polypeptide (e.g., a variant TACI polypeptide) to an Fc region. In some embodiments, the peptide linker may be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue, but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. Exemplary linkers include any of the linkers described herein.

[0315] 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, interaction between the Fc regions of two identical polypeptide fusions forms a covalent disulfide bond, resulting in a dimeric molecule comprising two TACI polypeptides (e.g., two variant TACI polypeptides).

[0316] In some embodiments, a TACI-Fc fusion protein is provided that includes, in order, for example, any of the TACI polypeptides described above, a linker, and an Fc region. In some embodiments, each TACI polypeptide of the TACI Fc fusion is any of the truncated wild-type TACI polypeptides described. In some embodiments, the TACI polypeptide of the TACI Fc fusion is set forth in SEQ ID NO: 13. The linker can be any of those described. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). In some embodiments, the linker is GS(GS)2 (SEQ ID NO: 194). The Fc region can be any of the Fc regions described. In some embodiments, the Fc region is a wild-type IgG1 Fc set forth in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc set forth in SEQ ID NO: 73.

[0317] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO: 171. In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO: 197. In some embodiments, the TACI-Fc fusion is encoded by the sequence set forth in SEQ ID NO: 208. SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 171)

[0318] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:172. SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 172)

[0319] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:196, which encoded the sequence set forth in SEQ ID NO:207.

[0320] In some embodiments, the TACI polypeptide is a variant TACI polypeptide. In some embodiments, a variant TACI-Fc fusion protein is provided comprising, in order, for example, any of the variant TACI polypeptides described above, a linker, and an Fc region. In some embodiments, the TACI polypeptide of the TACI Fc fusion is any of the variant TACI polypeptides described above. In some embodiments, the variant TACI of the variant TACI Fc fusion is set forth in any one of SEQ ID NOS: 2-12, 21, 22, or 101-120. In some embodiments, the variant TACI of the variant TACI Fc fusion is set forth in any one of SEQ ID NOS: 14-20, 23-35, 92-100, or 177-192. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). In some embodiments, the linker is GS(G4S)2 (SEQ ID NO: 194). In some embodiments, the Fc region is a wild-type IgG1 Fc set forth in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc set forth in SEQ ID NO: 73.

[0321] In some embodiments, the TACI-Fc fusion protein has the sequence of amino acids set forth in any one of SEQ ID NOs: 167-170, 200, or 222-237.

[0322] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:167. SLSCRKEQGEYYDHLLRDCISCASICGQHPKQCADFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 167)

[0323] In some embodiments, the TACI-Fc fusion is encoded by the sequence set forth in SEQ ID NO:211.

[0324] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:168. SLSCRKEQGEYYDHLLRDCISCASICGQHPKQCADFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 168)

[0325] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:169. SLSCRKEEGKFYDHLLQDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 169)

[0326] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:170. SLSCRKEEGKFYDHLLQDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 170)

[0327] In some embodiments, a TACI-Fc fusion protein comprises multiple copies of a TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence), e.g., two, three, or four TACI polypeptide sequences. In some embodiments, a TACI-Fc fusion protein comprises two TACI polypeptide sequences (e.g., two variant TACI polypeptide sequences). In some cases, the TACI polypeptide sequences may be directly linked or indirectly linked via a linker, such as a peptide linker, including any of those described. In such an example, one of the TACI polypeptide sequences is attached or linked to an Fc region, e.g., either the N-terminus or C-terminus of the Fc region. In other cases, the TACI polypeptide sequences may be separated from one another by an Fc region, each individually linked to the N-terminus or C-terminus of the Fc region. Linkage to the Fc region may be direct or indirect via a linker, such as a peptide linker, including any of those described.

[0328] In some embodiments, TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) can be arranged in tandem order in the fusion protein (hereinafter referred to as a "tandem" Fc fusion construct). In some embodiments, the TACI-Fc fusion protein has the structure: (TACI)-linker(TACI)-linker-Fc region. In some embodiments, the immunomodulatory protein is a tetravalent molecule that is a homodimer of two identical copies of the TACI-Fc fusion protein. For example, interaction between the Fc regions of two identical polypeptide fusions forms covalent disulfide bonds, resulting in a dimeric molecule comprising four TACI polypeptides (e.g., four variant TACI polypeptides).

[0329] In some embodiments, a TACI-Fc fusion protein is provided that includes, in order, e.g., any of the TACI polypeptides described above, a linker, e.g., another TACI polypeptide described, and an Fc region. In some embodiments, each TACI polypeptide of the TACI Fc fusion is any of the truncated wild-type TACI polypeptides described. In some embodiments, each TACI polypeptide of the TACI Fc fusion is set forth in SEQ ID NO: 13. In some embodiments, each TACI polypeptide of the TACI Fc fusion is any of the variant TACI polypeptides described. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a variant TACI polypeptide described in any one of SEQ ID NOs: 2-12, 21, 22, or 101-120. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a variant TACI polypeptide described in any one of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linker can be any of those described. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). The Fc region can be any Fc region described. In some embodiments, the Fc region is a wild-type IgG1 Fc set forth in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc set forth in SEQ ID NO: 73. In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO: 198 and is encoded by the sequence set forth in SEQ ID NO: 209.

[0330] In some embodiments, TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) may be separated in the fusion protein by an Fc region, with the Fc region positioned between two TACI polypeptide sequences (hereinafter referred to as a "barbell" Fc fusion construct). In some embodiments, the TACI-Fc fusion protein has the structure: (TACI)-linker-Fc region-linker-(TACI). In some embodiments, the linkers may 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, interaction between the Fc regions of the two identical polypeptide fusions forms a covalent disulfide bond, resulting in a dimeric molecule comprising four TACI polypeptides (e.g., four variant TACI polypeptides).

[0331] In some embodiments, a TACI-Fc fusion protein is provided that includes, 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., as described. In some embodiments, each TACI polypeptide of the TACI Fc fusion is any of the truncated wild-type TACI polypeptides described. In some embodiments, each TACI polypeptide of the TACI Fc fusion is set forth in SEQ ID NO: 13. In some embodiments, each TACI polypeptide of the TACI Fc fusion is any of the variant TACI polypeptides described. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a variant TACI polypeptide set forth in any one of SEQ ID NOs: 2-12, 21, 22, or 101-120. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a variant TACI polypeptide set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linker may be any of those described and may be the same or different. In some embodiments, the first linker is GSGGGGS (SEQ ID NO:74) and the second linker is (GGGGS)4 (SEQ ID NO:84). The Fc region may be any of the Fc regions described. In some embodiments, the Fc region is a wild-type IgG1 Fc set forth in SEQ ID NO:81. In some embodiments, the Fc region is a variant Fc set forth in SEQ ID NO:73. In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:201 and is encoded by the sequence set forth in SEQ ID NO:212. In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:202 and is encoded by the sequence set forth in SEQ ID NO:213.

[0332] In some embodiments, TACI-Fc fusion proteins are provided that are dimers formed by two identical TACI polypeptides (e.g., variant TACI polypeptides) as described linked to Fc domains. In some embodiments, identical species (also called copies) of any of the provided TACI-Fc fusion polypeptides, e.g., variant TACI-Fc fusions, are dimerized to create a homodimer. In some embodiments, the dimer is a homodimer, and the two TACI-Fc polypeptides, e.g., variant TACI-Fc polypeptides, are identical. When generating homodimeric Fc molecules, the Fc regions are capable of forming homodimers with matching Fc regions by coexpression of the individual Fc regions in a cell. In some embodiments, dimerization is mediated by covalent disulfide bond(s) formed between the Fc regions of the polypeptide fusions.

[0333] Nucleic acid molecules encoding immunomodulatory proteins are also provided. In some embodiments, for production of immunomodulatory proteins, the nucleic ac...

Claims

1. 1. A method of treating an autoantibody-associated 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 contains one or more amino acid substitutions selected from the group consisting of 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 once every four weeks (Q4W) at a dose of from at or about 80 mg to at or about 480 mg.

2. 1. A method of treating an autoantibody-associated 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 contains one or more amino acid substitutions selected from the group consisting of 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 once every four weeks (Q4W), once every eight weeks (Q8W), or once every twelve weeks (Q12W) at a dose of from at or about 24 mg to at or about 480 mg.

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

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

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

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

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

8. (i) the dose is at or about 24 mg Q4W; (ii) the dose is 24 mg or about 24 mg Q8W; (iii) the dose is 24 mg or about 24 mg Q12W; (iv) the dose is 80 mg or about 80 mg Q8W; (v) the dose is at or about 80 mg Q12W; (vi) the dose is at or about 240 mg Q8W; (vii) the dose is 240 mg or about 240 mg Q12W; The method according to claim 2 or claim 3.

9. 9. The method of any of claims 1 to 8, wherein the autoantibody-associated disease or disorder is selected from the group consisting of a rheumatic disease or disorder, a renal (kidney) disease or disorder, a hematological disease or disorder, a skin disease or disorder, or a neurological disease or disorder.

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

11. The method of any one of claims 1 to 10, wherein the autoantibody-associated disease or disorder is Sjogren's disease.

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

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

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

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

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

17. 17. The method of claim 15 or claim 16, wherein the 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 of any of claims 1 to 12 and 14, wherein administration of the TACI-Fc fusion protein does not reduce circulating IgG to <1.5 g / L in the subject.

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

20. 1. 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. Including, 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 set forth in SEQ ID NO: 13; and The TACI-Fc fusion protein is administered subcutaneously at a dose of at or about 80 mg to at or about 480 mg once every four weeks. The method.

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

22. 22. The method of claim 20 or claim 21, wherein the dose is at or about 80 mg Q4W.

23. 22. The method of claim 20 or claim 21, wherein the dose is at or about 240 mg Q4W.

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

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

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

0.

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

28. The method of claims 12-27, wherein 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 of care regimen characterized by stable use of standard of care to treat the SLE, optionally wherein the stable use is stable use of the standard of care for at least 30 days.

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

30. 30. The method of any of claims 27 to 29, wherein the standard of care comprises one or more of a corticosteroid, an antimalarial (e.g., hydroxychloroquine), a nonsteroidal anti-inflammatory drug (NSAID), or an immunosuppressant or immunomodulatory agent, or any combination thereof, optionally wherein the immunosuppressant or immunomodulatory agent is selected from the group comprising azathioprine, mycophenolic acid (e.g., mycophenolate mofetil or mycophenolate sodium), cyclophosphamide, methotrexate, leflunomide, tacrolimus, cyclosporine, and any combination of the foregoing.

31. The method of any of claims 27 to 30, wherein the standard of care comprises corticosteroids, and administration of the corticosteroids is tapered after administration of the TACI-Fc fusion protein.

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

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

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

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

36. 1. A method of treating glomerulonephritis, said 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. Including, 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 set forth in SEQ ID NO: 13; and The TACI-Fc fusion protein is administered subcutaneously at a dose of at or about 80 mg to at or about 480 mg once every four weeks. The method.

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

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

39. 38. The method of claim 36 or claim 37, wherein the dose is at or about 240 mg Q4W.

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

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

42. 42. The method of any of claims 36 to 41, wherein the glomerulonephritis is IgA nephropathy and the subject is selected for treatment if, at screening, the subject is characterized by one or both of the following: (i) the subject has been diagnosed with IgA nephropathy within 5 years prior to the screening; and (ii) ≥ 0.75 g / g urinary total protein to creatinine (proteinuria).

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

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

45. 45. The method of claim 44, wherein Gd-IgA1 is reduced by more than 50%.

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

47. 47. The method of any of claims 36 to 41 and 46, wherein the glomerulonephritis is lupus nephritis, and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject has been diagnosed with lupus nephritis class II-V within three years prior to the screening; (ii) ≥ 1 g / g urinary total protein to creatinine ratio (proteinuria); (iii) active urinary sediment; (iv) anti-dsDNA antibody and / or antinuclear antibody (ANA) positive, optionally anti-dsDNA positive with a titer of ≧30 IU / mL and ANA positive with a titer of ≧1:80; (v) a stable standard of care regimen characterized by stable use of standard of care for treating said SLE, optionally, said stable use is stable use of said standard of care for at least 30 days; and (vi) receiving stable basal immunosuppression, optionally said stable basal immunosuppression being a stable dose of ≧1 g / day of MMF, with or without corticosteroids, for at least 8 weeks prior to said time of screening or said time of administration of the TACI-Fc fusion protein.

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

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

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

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

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

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

54. 1. A method of treating an autoimmune cytopenia, the method comprising: a) selecting a subject diagnosed with an autoimmune cytopenia for administration of a TACI-Fc fusion protein; and b) administering the TACI-Fc fusion protein to the selected subject. Including, 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 set forth in SEQ ID NO: 13; and The TACI-Fc fusion protein is administered subcutaneously at a dose of at or about 80 mg to at or about 480 mg once every four weeks. The method.

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

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

57. 56. The method of claim 54 or claim 55, wherein the dose is at or about 240 mg Q4W.

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

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

60. 60. The method of any of claims 53 to 59, wherein the autoimmune cytopenia is ITP and the subject is selected for treatment if, at screening, the subject is characterized by one or more of the following: (i) the subject was diagnosed with ITP more than 3 months prior to the screening; (ii) platelet count <30,000 / μL; and (iii) had received two or more prior treatments to treat said ITP, optionally, four or more prior treatments to treat said ITP.

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

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

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

64. 63. The method of claim 62, wherein the autoimmune cytopenia is CAD.

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

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

67. 67. The method of any of claims 53-66, wherein the subject is not characterized by having a secondary cytopenia (e.g., a systemic autoimmune disease or malignancy) or Evans syndrome.

68. 67. The method of any of claims 53-66, wherein the subject is characterized by having a secondary cytopenia (e.g., a systemic autoimmune disease or malignancy) or Evans syndrome.

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

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

71. 1. A method of treating an autoimmune bullous (blistering) skin disease, said method comprising: a) selecting a subject diagnosed with an autoimmune bullous (blistering) skin disease for administration of a TACI-Fc fusion protein; and b) administering the TACI-Fc fusion protein to the selected subject. Including, 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 set forth in SEQ ID NO: 13; and The TACI-Fc fusion protein is administered subcutaneously at a dose of at or about 80 mg to at or about 480 mg once every four weeks. The method.

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

73. 73. The method of claim 71 or claim 72, wherein the dose is at or about 80 mg Q4W.

74. 73. The method of claim 71 or claim 72, wherein the dose is at or about 240 mg Q4W.

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

76. 76. The method of any of claims 70 to 75, wherein the autoimmune bullous (blistering) dermatosis is selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, or bullous pemphigoid.

77. 77. The method of any 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, at screening, the subject is characterized by one or both of the following: (i) Pemphigus severity international criteria (PDAI) ≧15; and (ii) Anti-Dsg1 antibody positive or anti-Dsg3 antibody positive.

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

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

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

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

82. 82. The method of claim 81, wherein the TACI-Fc fusion protein is administered to the subject in combination with the simultaneous administration of stable immunosuppression.

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

84. 84. The method of any 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 to 9, wherein the autoantibody-associated disease or disorder is a neurological disease or disorder.

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

87. 1. A method of treating encephalitis, said 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. Including, 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 set forth in SEQ ID NO: 13; and The TACI-Fc fusion protein is administered subcutaneously at a dose of at or about 80 mg to at or about 480 mg once every four weeks. The method.

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

89. 89. The method of claim 87 or claim 88, wherein the dose is at or about 80 mg Q4W.

90. 89. The method of claim 87 or claim 88, wherein the dose is at or about 240 mg Q4W.

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

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

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

94. 94. The method of any 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 more.

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

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

97. 95. The method of any preceding embodiment, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 24 weeks.

98. 95. The method of any preceding embodiment, wherein the TACI-Fc fusion protein is administered to the subject Q4W for 48 weeks.

99. The method of any preceding claim, wherein the variant TACI polypeptide is set forth in SEQ ID NO:

26.

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

101. 101. The method of any 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 (2xGGGGGS; SEQ ID NO:78), GGGGSGGGGSGGGGGS (3xGGGGGS; SEQ ID NO:79), GGGGSGGGGSGGGGGSGGGGGS (4xGGGGGS, SEQ ID NO:84), GGGGSGGGGSGGGGGSGGGGGS (5xGGGGGS, SEQ ID NO:91), GGGGSSA (SEQ ID NO:80), or GSGGGGSGGGGS (SEQ ID NO:194), or a combination thereof.

102. The method of any one of claims 1 to 101, wherein the linker is set forth in SEQ ID NO:

74.

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

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

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

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

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

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

109. 109. The method of any of claims 103-108, wherein the Fc region comprises K447del, said residues being numbered according to the EU index of Kabat.

110. The method of claims 1 to 107 and 109, wherein the Fc comprises the amino acid sequence set forth in SEQ ID NO:

73.

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

167.

112. The method of claims 1 to 103, 107 to 110, wherein the Fc comprises the amino acid sequence set forth in SEQ ID NO:

81.

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

168.

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

115. 115. The method of claim 114, wherein the formulation has a pH of about 5.

2.

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

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

118. 118. The method of any of claims 114 to 117, wherein the proline is at a concentration of about 2% to about 5%.

119. 118. The method of any of claims 114 to 117, wherein the proline is at a concentration of 3% or about 3%.

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

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

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

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

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

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

126. The method of any of claims 1 to 125, wherein B cell immune response or activity is reduced in said subject.

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

128. 128. The method of any of claims 1-127, wherein circulating serum immunoglobulins are reduced in said subject.

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

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

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

132. 132. The method of 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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