April and BAFF inhibitory immunomodulatory proteins and methods of use thereof

Abstract

To provide immunomodulatory proteins that exhibit neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers).SOLUTION: The immunomodulatory proteins provided include variant domains of Transmembrane Activator and CAML Interactor (TACI). Among the provided immunomodulatory proteins are TACI-Fc fusion proteins. Also provided are nucleic acid molecules encoding the immunomodulatory proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological diseases, disorders, or conditions. Also provided are compositions and methods for making and using such proteins.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Application No. 63 / 022,373, filed May 8, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF," U.S. Provisional Application No. 63 / 034,361, filed June 3, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF," and U.S. Provisional Application No. 63 / 034,361, filed September 18, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF," each of which is incorporated by reference in its entirety for all purposes. This application claims priority to U.S. Provisional Application No. 63 / 080,643, entitled "METHODS OF USE THEREOF."

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with an electronic Sequence Listing, which is provided as a 278,660 byte file entitled 761612003840SeqList.TXT, created on May 4, 2021. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides immunomodulatory proteins that exhibit neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers). The immunomodulatory proteins comprise a variant domain of Transmembrane Activator and CAML Interactor (TACI). Among the immunomodulatory proteins provided are TACI-Fc fusion proteins. The present disclosure also provides nucleic acid molecules encoding the immunomodulatory proteins. The immunomodulatory proteins provide therapeutic utility for various immunological diseases, disorders, or conditions. Compositions and methods for making and using such proteins are provided. [Background technology]

[0004] background There has been growing medical interest in modulating immune responses by intervening in processes involving the interaction between soluble ligands and their receptors. Currently, biologics used to enhance or suppress immune responses have generally been limited to antibodies (e.g., anti-PD-1 antibodies) or soluble receptors directed against a single type of cell surface molecule (e.g., Fc-CTLA-4). Improved therapeutic agents capable of modulating immune responses, particularly B cell immune responses, are needed. Embodiments that meet this need are provided. Summary of the Invention

[0005] overview Provided herein is an immunomodulatory protein containing at least one TACI polypeptide that is a truncated wild-type TACI extracellular domain or a variant thereof, wherein the truncated wild-type TACI extracellular domain contains cysteine-rich domain 2 (CRD2) but lacks the entire cysteine-rich domain 1 (CRD1), and the variant TACI polypeptide contains one or more amino acid substitutions in the truncated wild-type TACI extracellular domain.

[0006] Provided herein are immunomodulatory proteins containing at least one TACI polypeptide that is a truncated wild-type TACI extracellular domain or a variant thereof, wherein the truncated wild-type TACI extracellular domain consists of the contiguous sequence contained within amino acid residues 67-118, consisting of amino acid residues 71-104, relative to the positions set forth in SEQ ID NO: 122, and the variant TACI polypeptide contains one or more amino acid substitutions in the truncated wild-type TACI extracellular domain. In some of the embodiments, the truncated wild-type TACI extracellular domain is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51 amino acids in length. In some of the embodiments, the truncated wild-type TACI extracellular domain consists of amino acid residues 68-110 set forth in SEQ ID NO: 122. In some optional embodiments, the TACI polypeptide consists of the amino acid sequence set forth in SEQ ID NO:13; or is a variant thereof containing one or more amino acid substitutions in the sequence set forth in SEQ ID NO:13.

[0007] Provided herein are immunomodulatory proteins containing at least one TACI polypeptide that is a truncated TACI polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:13, or a variant thereof containing one or more amino acid substitutions in the sequence set forth in SEQ ID NO:13. In some of any of the embodiments, the truncated TACI polypeptide or variant thereof binds to APRIL, BAFF, or the BAFF / APRIL heterotrimer. In some of any of the embodiments, the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence set forth in SEQ ID NO:1. In some of any of the embodiments, the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence set forth in SEQ ID NO:13.

[0008] Provided herein is an immunomodulatory protein containing a truncated TACI polypeptide consisting of the sequence set forth in SEQ ID NO: 13. In some of the embodiments, the TACI polypeptide is a variant TACI polypeptide, and the variant TACI polypeptide has increased binding affinity for one or both of APRIL and BAFF compared to the truncated TACI polypeptide. In some of the embodiments, the variant TACI polypeptide contains one or more amino acid substitutions at positions selected from among 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the numbering set forth in SEQ ID NO: 122.

[0009] In some of the optional embodiments, the one or more amino acid substitutions are TIFF2025134866000001.tif19157, or a conservative amino acid substitution thereof. In some of the embodiments, the one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. In some of the embodiments, the one or more amino acid substitutions are selected from: TIFF2025134866000002.tif26132. In some embodiments, the one or more amino acid substitutions are K77E / F78Y / Y102D. In some embodiments, the one or more amino acid substitutions are Q75E / R84Q. In some embodiments, the variant TACI polypeptide is set forth in SEQ ID NO:26. In some embodiments, the variant TACI polypeptide is set forth in SEQ ID NO:27.

[0010] In some of the optional embodiments, the TACI polypeptide is a variant TACI polypeptide that includes one or more amino acid substitutions at positions selected from among 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 in the extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof, corresponding to the numbering of the positions set forth in SEQ ID NO:122.

[0011] Provided herein is an immunomodulatory protein containing at least one variant TACI polypeptide, wherein the at least one variant TACI polypeptide comprises one or more amino acid substitutions at positions selected from among 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 in the extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof, corresponding to the numbering of the positions set forth in SEQ ID NO:122.

[0012] Provided herein is an immunomodulatory protein that is a variant TACI Fc fusion protein containing a variant TACI polypeptide, an Fc region, and a linker between the TACI polypeptide and the Fc region, wherein the variant TACI polypeptide contains one or more amino acid substitutions at positions selected from among 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 in the extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof, corresponding to the numbering of the positions shown in SEQ ID NO:122.

[0013] In some of any of the embodiments, the reference TACI polypeptide is a truncated polypeptide consisting of the TACI extracellular domain, or a specific binding portion of the TACI extracellular domain, that binds to APRIL, BAFF, or the BAFF / APRIL heterotrimer.

[0014] In some of the optional embodiments, the reference TACI polypeptide comprises (i) the amino acid sequence set forth in SEQ ID NO:122; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:122; or (iii) a portion of (i) or (ii) that includes one or both of the CRD1 domain and the CRD2 domain that binds to APRIL, BAFF, or the BAFF / APRIL heterotrimer.

[0015] In some of the optional embodiments, the reference TACI polypeptide lacks an N-terminal methionine.

[0016] In some of the optional embodiments, the reference TACI polypeptide comprises a CRD1 domain and a CRD2 domain.

[0017] In some of any of the embodiments, the reference TACI polypeptide comprises the sequence set forth in SEQ ID NO: 1. In some of any of the embodiments, the reference TACI polypeptide consists of the sequence set forth in SEQ ID NO: 1.

[0018] In some of any of the embodiments, the reference TACI polypeptide consists essentially of the CRD2 domain.

[0019] In some of any of the embodiments, the reference TACI polypeptide comprises the sequence set forth in SEQ ID NO: 13. In some of any of the embodiments, the reference TACI polypeptide consists of the sequence set forth in SEQ ID NO:13.

[0020] In some of the optional embodiments, the one or more amino acid substitutions are TIFF2025134866000003.tif19163, or a conservative amino acid substitution thereof.

[0021] In some of the optional embodiments, the one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y.

[0022] In some of the optional embodiments, the one or more amino acid substitutions comprise an amino acid substitution selected from the group consisting of Q75E, K77E, F78Y, R84G, R84Q, A101D, and Y102D, or any combination thereof.

[0023] In some of the embodiments, the one or more amino acid substitutions include at least the amino acid substitution Q75E. In some of the embodiments, the one or more amino acid substitutions include at least the amino acid substitution K77E. In some of the embodiments, the one or more amino acid substitutions include at least the amino acid substitution F78Y. In some of the embodiments, the one or more amino acid substitutions include at least the amino acid substitution R84G. In some of the embodiments, the one or more amino acid substitutions include at least the amino acid substitution R84Q. In some of the embodiments, the one or more amino acid substitutions include at least the amino acid substitution A101D.

[0024] In some of the embodiments, the one or more amino acid substitutions include Q75E / R84Q. In some of the embodiments, the one or more amino acid substitutions include Q75E / K77E. In some of the embodiments, the one or more amino acid substitutions include Q75E / F78Y. In some of the embodiments, the one or more amino acid substitutions include Q75E / A101D. In some of the embodiments, the one or more amino acid substitutions include Q75E / Y102D. In some of the embodiments, the one or more amino acid substitutions include F77E / F78Y. In some of the embodiments, the one or more amino acid substitutions include K77E / R84Q. In some of the embodiments, the one or more amino acid substitutions include K77E / A101D. In some of the embodiments, the one or more amino acid substitutions include K77E / Y102D. In some of the embodiments, the one or more amino acid substitutions include F78Y / R84Q. In some of the embodiments, the one or more amino acid substitutions include F78Y / A101D. In some of the embodiments, the one or more amino acid substitutions include F78Y / Y102D. In some of the embodiments, the one or more amino acid substitutions include R84Q / A101D. In some of the embodiments, the one or more amino acid substitutions include R84Q / Y102D. In some of the embodiments, the one or more amino acid substitutions include A101D / Y102D.

[0025] In some of the optional embodiments, the one or more amino acid substitutions are TIFF2025134866000004.tif33146.

[0026] In some of the optional embodiments, the one or more amino acid substitutions are TIFF2025134866000005.tif19158.

[0027] In some of any of the embodiments, the one or more amino acid substitutions are K77E / F78Y / Y102D.

[0028] In some of any of the embodiments, the one or more amino acid substitutions are Q75E / R84Q.

[0029] In some of any of the embodiments, the one or more amino acid substitutions are K77E / A101D / Y102D.

[0030] In some of the embodiments, the variant TACI polypeptide has up to 10 amino acid modifications compared to the reference TACI polypeptide. In some of the embodiments, the variant TACI polypeptide has up to 5 amino acid modifications compared to the reference TACI polypeptide.

[0031] In some of the optional embodiments, the variant TACI polypeptide has at least 90% sequence identity to SEQ ID NO: 122; or a specific binding fragment thereof comprising the CRD1 domain and / or the CRD2 domain. In some embodiments, the variant TACI polypeptide has at least 95% sequence identity to SEQ ID NO: 122; or a specific binding fragment thereof comprising the CRD1 domain and / or the CRD2 domain. In some embodiments, the specific binding fragment is set forth in SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 130, or SEQ ID NO: 131.

[0032] In some of any of the embodiments, the variant TACI polypeptide has at least 90% sequence identity to SEQ ID NO: 13. In some of any of the embodiments, the variant TACI polypeptide has at least 95% sequence identity to SEQ ID NO: 13.

[0033] In some of the embodiments, the variant TACI polypeptide has increased binding affinity for one or both of APRIL and BAFF compared to a reference TACI polypeptide. In some of the embodiments, the variant TACI polypeptide has increased binding affinity for APRIL. In some of the embodiments, the variant TACI polypeptide has increased binding affinity for BAFF. In some of the embodiments, the variant TACI polypeptide has increased binding affinity for APRIL and BAFF.

[0034] In some of any of the embodiments, the increased binding affinity to BAFF or APRIL is, independently, 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, about 50-fold, or about 60-fold increase.

[0035] In some optional embodiments, the variant TACI polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 2-12, 21, 22, 101-120, or the variant TACI polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192.

[0036] In some optional embodiments, the variant TACI polypeptide consists of or essentially consists of the sequence set forth in any one of SEQ ID NOs:2-12, 21, 22, 101-120, or the variant TACI polypeptide consists of or essentially consists of the sequence set forth in any one of SEQ ID NOs:14-20, 23-35, 92-100, or 177-192.

[0037] In some of any of the embodiments, the variant TACI polypeptide consists of or consists essentially of the sequence set forth in SEQ ID NO: 26. In some of any of the embodiments, the variant TACI polypeptide consists of or consists essentially of the sequence set forth in SEQ ID NO: 27. In some of any of the embodiments, the variant TACI polypeptide consists of or consists essentially of the sequence set forth in SEQ ID NO: 107. In some of any of the embodiments, the variant TACI polypeptide consists of or consists essentially of the sequence set forth in SEQ ID NO: 20.

[0038] In some of the optional embodiments, the linker comprises a peptide linker, the peptide linker comprising: TIFF2025134866000006.tif33164 or a combination thereof.

[0039] In some embodiments, the immunomodulatory protein contains a heterologous moiety linked to at least one TACI polypeptide. In some embodiments, the heterologous moiety is a half-life extending moiety, a multimerization domain, a targeting moiety that binds to a molecule on a cell surface, or a detectable label. In some embodiments, the half-life extending moiety comprises a multimerization domain, albumin, an albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, or a combination thereof. In some embodiments, at least one TACI polypeptide is linked to the Fc region of an immunoglobulin. In some embodiments, the immunomodulatory protein of any of the embodiments provided herein that is a TACI-Fc fusion protein comprises at least one TACI polypeptide linked to the Fc region of an immunoglobulin.

[0040] In some embodiments, the immunomodulatory proteins provided herein do not comprise a TACI polypeptide linked to another targeting moiety that binds to a molecule on a cell surface. In some embodiments, the immunomodulatory proteins provided herein do not comprise a TACI polypeptide linked to a targeting moiety that is a binding partner of a T cell stimulatory receptor or a ligand of a T cell stimulatory receptor. In some embodiments, the immunomodulatory proteins provided herein do not comprise a TACI polypeptide linked to a targeting moiety that is a binding partner of CD28 or a ligand of CD28 (e.g., CD80 or CD86). In some embodiments, the immunomodulatory proteins provided herein do not comprise a TACI polypeptide linked to a CTLA-4 polypeptide or the extracellular domain or binding portion of CTLA-4, or a variant thereof. For example, in provided aspects, the immunomodulatory proteins provided herein do not comprise a TACI polypeptide linked to a wild-type CTLA-4 polypeptide or the extracellular domain or binding portion thereof. In provided aspects, the immunomodulatory proteins provided herein do not include a TACI polypeptide linked to a variant CTLA-4 polypeptide or its extracellular domain or binding portion, such as a variant CTLA-4 or binding portion thereof that contains one or more amino acid modifications (e.g., substitutions) in the CTLA-4 extracellular domain to increase binding affinity to one or more cognate binding partners.

[0041] In some of any of the embodiments, the immunoglobulin Fc is an IgG4 Fc domain or a variant thereof. In some embodiments, the IgG4 Fc domain has the amino acid sequence set forth in SEQ ID NO:139. In some embodiments, the IgG4 Fc domain is a variant thereof containing the mutation S228P. In some embodiments, the IgG4 Fc domain has the amino acid sequence set forth in SEQ ID NO:140 or SEQ ID NO:220.

[0042] In some of any of the embodiments, the TACI-Fc Fc fusion protein is a dimer. In some of any of the embodiments, the immunoglobulin Fc region is a homodimeric Fc region.

[0043] In some of any of the embodiments, the immunoglobulin Fc is an IgG1 Fc domain; or a variant Fc that optionally exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a wild-type IgG1 Fc domain. In some of any of the embodiments, the immunoglobulin Fc is set forth in SEQ ID NO:71. In some embodiments, the immunoglobulin Fc is an IgG1 Fc domain, and the Fc comprises the amino acid sequence set forth in SEQ ID NO:81. In some of any of the embodiments, the immunoglobulin Fc is a variant IgG1 Fc domain containing one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to EU numbering. In some of the optional embodiments, the immunoglobulin Fc region contains amino acid substitutions L234A, L235E, G237A, according to EU numbering, or amino acid substitutions R292C, N297G, and V302C, according to EU numbering. In some embodiments, the Fc region includes amino acid substitutions L234A, L235E, G237A, according to EU numbering. In some embodiments, the Fc region is set forth in SEQ ID NO:73, 75, 83, 136, or 221. In some embodiments, the immunoglobulin Fc region further includes amino acid substitutions A330S and P331S. In some embodiments, the immunoglobulin Fc region is set forth in SEQ ID NO:175 or SEQ ID NO:176.

[0044] In some embodiments, the Fc is a variant Fc comprising the amino acid sequence set forth in SEQ ID NO:73.

[0045] In some embodiments, the immunomodulatory protein is a heterodimer, and each polypeptide of the dimer is individually linked to an immunoglobulin Fc domain containing one or more amino acid modifications in the wild-type Fc domain to form a heterodimer between the polypeptides. In some embodiments, the wild-type immunoglobulin Fc is an IgG1 Fc domain. In some embodiments, the one or more amino acid modifications are selected from knob-into-hole modifications and charge mutations that reduce or prevent self-association due to charge repulsion.

[0046] In some of the embodiments, the immunomodulatory protein optionally contains one or more amino acid substitutions for reduced binding affinity to an Fc receptor and / or reduced effector function compared to a wild-type IgG1 Fc domain. In some of the embodiments, the one or more amino acid substitutions are selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to EU numbering. In some of the embodiments, the immunoglobulin Fc region contains amino acid substitutions L234A, L235E, G237A according to EU numbering, or R292C, N297G, and V302C according to EU numbering.

[0047] In some of the optional embodiments, the TACI-Fc fusion protein has the structure: TACI polypeptide (TACI)-linker-Fc region In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 168. In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 170. In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 167. In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 169. In some embodiments, the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein.

[0048] Provided herein is an immunomodulatory TACI Fc fusion protein that is a homodimer, comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:167, linked by a covalent disulfide bond.

[0049] Provided herein is an immunomodulatory TACI Fc fusion protein that is a homodimer, comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:168, linked by a covalent disulfide bond.

[0050] Provided herein is an immunomodulatory TACI Fc fusion protein that is a homodimer, comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:169, linked by a covalent disulfide bond.

[0051] Provided herein is an immunomodulatory TACI Fc fusion protein that is a homodimer, comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:170, linked by a covalent disulfide bond.

[0052] In some of the optional embodiments, the TACI-Fc fusion protein has the structure: (TACI)-linker-Fc region-linker-(TACI) In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 201. In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 202. In some embodiments, the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein.

[0053] In some of the optional embodiments, the TACI-Fc fusion protein has the structure: (TACI)-linker-(TACI)-linker-Fc region In some embodiments, the TACI-Fc fusion protein is set forth in SEQ ID NO: 198. In some embodiments, the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein.

[0054] In some of the embodiments, the immunomodulatory protein (e.g., an Fc fusion protein) blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI. The immunomodulatory protein reduces the level of circulating APRIL, BAFF, or APRIL / BAFF in the blood after administration to a subject. In some of the embodiments, the immunomodulatory protein (e.g., an Fc fusion protein) blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI. In some of the embodiments, the immunomodulatory protein (e.g., an Fc fusion protein) reduces the level of circulating APRIL, BAFF, or APRIL / BAFF in the blood after administration to a subject.

[0055] In some of the embodiments, the immunomodulatory protein (e.g., Fc fusion protein) reduces or inhibits B cell maturation, differentiation, and proliferation. In some of the embodiments, the immunomodulatory protein reduces or inhibits B cell maturation, differentiation, or proliferation.

[0056] In some embodiments, the Fc-fusion protein neutralizes APRIL and BAFF. In some embodiments, the IC50 for APRIL neutralization is less than 100 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, less than 10 pM, less than 5 pM, or less than 1 pM, or any value between any of the aforementioned values, and / or the IC50 for BAFF neutralization is less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 75 pM, less than 50 pM, less than 25 pM, or less than 10 pM, or any value between any of the aforementioned values.

[0057] Provided herein is a nucleic acid molecule encoding any of the immunomodulatory proteins (e.g., Fc fusion proteins) of the embodiments described herein. In some of any of the embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some of any of the embodiments, the nucleic acid molecule is a cDNA.

[0058] Provided herein is a vector containing the nucleic acid molecule of any of the embodiments described herein. In some of any of the embodiments, the vector is an expression vector. In some of any of the embodiments, the vector is a mammalian expression vector or a viral vector.

[0059] Provided herein is a cell containing the nucleic acid of any of the embodiments described herein or the vector of any of the embodiments described herein. In some of any of the embodiments, the cell is a mammalian cell. In some of any of the embodiments, the cell is a human cell.

[0060] Provided herein is a method for producing an immunomodulatory protein, the method comprising introducing into a host cell a nucleic acid molecule of any of the embodiments described herein or a vector of any of the embodiments described herein under conditions that allow the protein to be expressed in the cell. In some of any of the embodiments, the method comprises isolating or purifying the immunomodulatory protein (e.g., an Fc fusion protein) from the cell. Provided herein is a method for producing an Fc fusion protein, the method comprising introducing into a host cell a nucleic acid molecule of any of the embodiments provided herein or a vector of any of the embodiments provided herein under conditions that allow the protein to be expressed in the cell.

[0061] Provided herein is an immunomodulatory protein (e.g., an Fc fusion protein) produced by the method of any of the embodiments described herein. Provided herein is an Fc fusion protein produced by the method of any of the embodiments described herein.

[0062] Provided herein is a pharmaceutical composition containing any of the immunomodulatory proteins (e.g., Fc fusion proteins) of the embodiments described herein. In some of the embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some of the embodiments, the pharmaceutical composition is sterile.

[0063] Provided herein is an article of manufacture comprising a pharmaceutical composition of any of the embodiments described herein in a vial or container. In some of any of the embodiments, the vial or container is sealed.

[0064] Provided herein are kits comprising the pharmaceutical composition of any of the aspects provided herein and instructions for use. In some of any of the aspects, the kit comprises an article of manufacture of any of the aspects described herein and instructions for use.

[0065] Provided herein is a method of reducing an immune response in a subject, the method comprising administering to a subject in need thereof an immunomodulatory protein of any of the aspects described herein.

[0066] Provided herein is a method of reducing an immune response in a subject, the method comprising administering to a subject in need thereof an Fc-fusion protein of any of the aspects described herein.

[0067] Provided herein is a method for reducing an immune response in a subject, comprising administering to a subject in need thereof a pharmaceutical composition according to any of the embodiments described herein. In some of any of the embodiments, the B cell immune response is reduced in the subject, thereby reducing or inhibiting B cell maturation, differentiation, and / or proliferation. In some of any of the embodiments, the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer are reduced in a control.

[0068] Provided herein is a method for reducing the circulating level of APRIL, BAFF or APRIL / BAFF heterotrimer in a subject, comprising administering to the subject any pharmaceutical composition of any of the embodiments described herein.In some of any embodiments, the T cell immune response is reduced in the subject, thereby reducing or inhibiting T cell costimulation.In some of any embodiments, reducing the immune response treats the disease or condition in the subject.

[0069] Provided herein are methods of treating a disease, disorder, or condition in a subject, comprising administering to a subject in need thereof an immunomodulatory protein of any of the aspects described herein.

[0070] Provided herein are methods of treating a disease, disorder, or condition in a subject, comprising administering to a subject in need thereof an Fc-fusion protein of any of the embodiments described herein.

[0071] Provided herein are methods for treating a disease, disorder, or condition in a subject, comprising administering to a subject in need thereof a pharmaceutical composition according to any of the embodiments described herein. In some of any of the embodiments, the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, a kidney disease, a transplant rejection, a graft-versus-host disease, or a viral infection. In some of any of the embodiments, the disease, disorder, or condition is selected from the group consisting of systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), autoimmune polyglandular syndrome type II (APSII), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris. In some of any of the embodiments, the disease, disorder, or condition is a B-cell cancer and the cancer is a myeloma.

[0072] Also provided herein are pharmaceutical compositions for use in reducing an immune response in a subject.

[0073] Also provided herein is the use of any of the provided immunomodulatory proteins (e.g., Fc fusion proteins) or any of the provided pharmaceutical compositions in the manufacture of a medicament for reducing an immune response in a subject.

[0074] In some embodiments of the pharmaceutical compositions for use or uses provided herein, the immune response is a B cell immune response, and reducing the immune response reduces or inhibits B cell maturation, differentiation, and / or proliferation. In some embodiments, reducing the immune response reduces the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer in the subject. In some embodiments, reducing the immune response treats a disease, disorder, or condition in the subject.

[0075] Also provided herein are pharmaceutical compositions for use in treating a disease, disorder, or condition in a subject.

[0076] Also provided herein is the use of any of the provided immunomodulatory proteins or pharmaceutical compositions in the manufacture of a medicament for treating a disease, disorder, or condition in a subject.

[0077] In some embodiments of any of the pharmaceutical compositions for use or uses provided herein, the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, a kidney disease, a transplant rejection, a graft-versus-host disease, or a viral infection. In some embodiments, the disease, disorder, or condition is selected from the group consisting of systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), autoimmune polyglandular syndrome type II (APSII), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris. In some embodiments, the disease, disorder, or condition is a B-cell cancer, and the cancer is myeloma. In some of the embodiments, the type of myeloma comprises multiple myeloma, plasmacytoma, multiple plasmacytoma, and / or extramedullary myeloma. In some of the embodiments, the type of myeloma comprises light chain myeloma, non-secretory myeloma, and / or IgD or IgE myeloma. [Brief explanation of the drawings]

[0078] [Figure 1] Figure 1 shows a schematic diagram of a functional inhibition assay involving recombinant APRIL and BAFF by TACI. In this assay, Jurkat cells were transduced to stably express mouse or human TACI on the cell surface along with a luciferase-based NF-κB reporter. After activation by recombinant APRIL or BAFF, the endogenous NK-κB transcription factor binds to a DNA response element that controls 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 for blocking signaling mediated by human APRIL (top panel) and BAFF (bottom panel) are shown. 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 3] The function of an exemplary TACI TD Fc fusion molecule to block APRIL (top panel of this figure) or BAFF (bottom panel of this figure) is shown. [Figure 4] Human TACI TD Fc fusion molecules are shown to block signaling mediated by mouse APRIL (left panel) and BAFF (right panel). [Figure 5] Human TACI TD Fc fusion molecules are shown for blocking signaling mediated by human APRIL (upper panel) and BAFF (lower panel) compared to TACI 13-118-Fc, TACI 30-110-Fc, and belimumab. [Figure 6A] Figures 6A-6I show the analysis of parameters evaluated in the NZB / NZW mouse model of human SLE. Proteinuria score (Figure 6A), mean percent body weight change (Figure 6B), and percent survival (Figure 6C) were assessed from 20 weeks of age. Serum anti-double-stranded DNA IgG titers (Figure 6D) and blood urea nitrogen (BUN) (Figure 6E) were analyzed (for anti-dsDNA IgG, **** vs. Fc, p<0.0001 by Student's t-test; for BUN-4, *** vs. Fc, p=0.0008 by Student's t-test). Kidneys were processed and analyzed by histology on duplicate periodic acid-Schiff (PAS)-stained sections. Individual components and overall histology scores are illustrated in Figure 6F. Frozen kidneys were also sectioned and stained for immunohistochemical analysis of mouse IgG as shown in Figure 6G and for immunohistochemical analysis of complement C3 glomerular deposits as shown in Figure 6H. Figure 6I shows the histological scores ± SEM. [Figure 6B] See legend to Figure 6A. [Figure 6C]See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. [Figure 6I] See legend to Figure 6A. [Figure 7] Figure 1 shows the ability of TACI mutations (K77E / F78Y / Y102D) to inhibit signaling mediated by APRIL (left panel) and BAFF (right panel), as quantified by luciferase production in Jurkat / NF-κB / TACI cells. [Figure 8] Figures 8A and 8B illustrate schematic diagrams of exemplary TACI-Fc fusion proteins. Figure 8A illustrates an exemplary TACI-Fc fusion protein containing two cysteine-rich pseudo-repeats (CRDs). Figure 8B illustrates an exemplary TACI-Fc fusion protein containing one cysteine-rich pseudo-repeat (CRD, e.g., CRD2). [Figure 9]An exemplary sequence alignment is shown to identify corresponding residues in 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 with identical residues in common, one skilled in the art can identify "corresponding" positions in a sequence by comparing them to the reference sequence using the conserved identical amino acid residues as a guide. Figure 9 shows an exemplary alignment of the reference TACI extracellular domain sequence shown in SEQ ID NO: 122 (containing the complete extracellular domain with CRD1 and CRD2 and an initiating methionine residue) with the TACI extracellular domain sequence shown in SEQ ID NO: 13 (containing only one CRD, CRD2). Alignment of identical residues demonstrates, for example, that amino acid residue E7 in SEQ ID NO:13 corresponds to residue E74 in SEQ ID NO:122, amino acid residue K10 in SEQ ID NO:13 corresponds to residue K77 in SEQ ID NO:122, amino acid residue Y12 in SEQ ID NO:13 corresponds to Y79 in SEQ ID NO:122, amino acid residue L15 in SEQ ID NO:13 corresponds to L82 in SEQ ID NO:122, amino acid residue R17 in SEQ ID NO:13 corresponds to R84 in SEQ ID NO:122, and amino acid residue D16 in SEQ ID NO:13 corresponds to D85 in SEQ ID NO:122. 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 identifying corresponding residues based on the examples and explanations herein. [Figure 10]Figures 10A-10D show the analysis of parameters evaluated from the mouse keyhole limpet hemocyanin (KLH) model. Serum KLH IgM OD levels were evaluated as the primary response (Figure 10A) and secondary response (Figure 10B). Similarly, serum anti-KLH IgG1 OD levels were evaluated as the primary response (Figure 10C) and secondary response (Figure 10D). [Figure 11] 11A-11B show an analysis of harvested spleens evaluated from a mouse keyhole limpet hemocyanin (KLH) immunization model. Spleens were processed and analyzed by weight (FIG. 11A) as well as total cell count (FIG. 11B). [Figure 12] Illustrated is a spleen analysis assessing cell subtype population composition from the mouse keyhole limpet hemocyanin (KLH) model, showing results for B cell subset numbers compared to group means. [Figure 13] Illustrated is a spleen analysis assessed for cell subtype phenotype composition from the mouse keyhole limpet hemocyanin (KLH) model, showing results for germinal center B cell and plasma cell counts (FIG. 13). [Figure 14] Figures 14A-D illustrate T cell numbers in the mouse keyhole limpet hemocyanin (KLH) model. Splenic CD3+, CD8+, CD4+, and follicular helper T cells are depicted in Figures 14A, 14B, 14C, and 14D, respectively. [Figure 15] Illustrates Tcm and Tem cell populations in the mouse keyhole limpet hemocyanin (KLH) model. [Figure 16] 16A-16B illustrate the overall incidence and severity of sialadenitis (FIGS. 16A-16B) in diabetes-prone mice after treatment with test molecules. [Figure 17] 17A-17B illustrate the overall incidence and extent of insulitis (FIGS. 17A-17B) in diabetes-prone mice after treatment with test molecules. DETAILED DESCRIPTION OF THE INVENTION

[0079] Detailed Description Provided herein are immunomodulating proteins that bind to one or more ligands, e.g., one or more ligands produced as soluble factors, to suppress or reduce B cell responses or activity. Among the provided immunomodulating proteins are proteins that bind to BAFF or APRIL ligands to neutralize their activity and block or antagonize the activity of B cell stimulatory receptors, e.g., TACI or BCMA. The provided immunomodulating proteins may also be fusion proteins of the TACI extracellular domain or a binding portion thereof (hereinafter, TACI ECD) with 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 a dysregulated immune response, e.g., diseases, disorders, or conditions associated with inflammatory or autoimmune conditions, including inflammatory or autoimmune diseases.

[0080] 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 attack against pathogen infection.However, in some cases, the immune system may become dysregulated, and abnormal immune response may be initiated against normal body parts or tissues, resulting in autoimmune disease or condition or autoimmune symptoms.In other cases, undesirable immune response may be initiated against foreign tissues such as transplants, resulting in transplant rejection.

[0081] In some aspects, certain diseases, disorders, and conditions in which the immune response is dysregulated can be treated by immunotherapy, which alters immune cell activity, such as B cell activity. 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 graft rejection. However, therapeutic approaches that attempt to modulate the interaction between a ligand and its receptor, which mediates the immune response, are not entirely satisfactory. In some cases, therapies that intervene in the activation of immune cells, such as B cells, and alter their immunomodulatory effects are constrained by the spatial and size constraints imposed by the extent of the immune synapse. In some aspects, existing therapeutic drugs, including antibody drugs, may not be able to simultaneously interact with multiple target proteins involved in modulating these interactions. For example, soluble receptors and antibodies generally competitively bind (e.g., do not bind to multiple target species at once) and therefore lack the ability to simultaneously bind to multiple targets. Furthermore, pharmacokinetic differences between drugs that independently target one of these receptors can create difficulties in adequately maintaining desirable blood concentrations of a drug combination targeting two different targets throughout the course of treatment.

[0082] BAFF and APRIL are TNF superfamily members that bind to both TACI and BCMA on B cells. BAFF also binds to a third receptor, BAFF-R. BAFF and APRIL cooperate to support the development, differentiation, and survival of B cells, particularly plasmablasts and plasma cells, and play a role in the pathogenesis of B cell-related autoimmune diseases. Co-neutralization of these two receptors dramatically reduces B cell function, including antibody production, whereas inhibition of either BAFF or APRIL alone mediates relatively modest effects. Fc fusions of wild-type (WT) TACI (e.g., atacicept and telitacicept) target both BAFF and APRIL and have demonstrated promising clinical potential in, for example, systemic lupus erythematosus (SLE) and IgA nephropathy, but have yet to demonstrate long-term and / or complete disease remission. While B cell-targeted therapies have demonstrated promising therapeutic potential, they remain unsatisfactory. For example, soluble recombinant TACI shows great promise as a therapeutic agent, but its usefulness appears to be hindered by its low to moderate affinity for APRIL.

[0083] Some provided embodiments result in improved neutralizing activity and suppression or reduction of B cell responses. In some embodiments, the improved activity is mediated by 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., BAFF or APRIL homotrimers, BAFF / APRIL heterotrimers, or 60-mer BAFF, with 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, the provided immunomodulatory proteins (TACI-Fc fusion proteins) are candidate therapeutic agents for treating multiple autoimmune and inflammatory diseases, particularly B-cell-related diseases, such as SLE, SjS, and other connective tissue diseases.

[0084] Provided embodiments relate to the identification of variant TACI polypeptides engineered to have improved affinity for APRIL and / or BAFF following random mutagenesis and directed evolution of the second cysteine-rich domain (CRD2) of TACI spanning residues 68-110. As shown herein, affinity maturation involved five rounds of selection alternating between APRIL and BAFF while simultaneously decreasing the concentration of the selection reagent to maintain selective pressure. Results demonstrated variant TACI polypeptides that exhibit substantially enhanced affinity for BAFF and APRIL when compared to wild-type TACI. For example, provided herein are variant TACI polypeptides containing one or more amino acid substitutions (exchanges or mutations) that confer improved binding affinity of the protein to BAFF and / or APRIL. Notably, some provided embodiments result in improved combined BAFF and APRIL inhibition. Accordingly, provided immunomodulatory proteins 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.

[0085] For example, provided embodiments are based on the discovery that directed evolution through 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). Fusing such molecules to immunoglobulin Fc results in immunomodulatory proteins that suppress B cell activity and responses. For example, when affinity-matured TACI variant outputs are reformatted as soluble Fc fusion proteins, they inhibit APRIL and BAFF with lower IC than wild-type TACI-Fc and belimumab controls, as shown herein in TACI-dependent reporter assays. 50Values ​​are shown. Furthermore, results in the animal models evaluated demonstrate rapid and significant reduction of important lymphocyte subsets, including plasma cells, germinal center B cells, and follicular T helper cells. Furthermore, the tested variant molecules demonstrated improved activity in mouse models, including significantly reduced autoantibodies and sialadenitis in a spontaneous SjS model, inhibited glomerular IgG deposition in a bm12-induced lupus model, and potently suppressed anti-dsDNA autoAb, blood urea nitrogen levels, proteinuria, sialadenitis, renal lesions, and renal immune complex deposition in a NZB / W lupus model. Furthermore, when compared with wild-type TACI-Fc, the tested TACI Fc fusions significantly and sustainably reduced serum IgM, IgG, and IgA antibody titers in mice. The findings herein demonstrate that these immunomodulatory proteins consistently exhibit potent immunosuppressive activity and efficacy in vitro and in vivo, and appear to be superior to existing and / or approved immunomodulatory drugs 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 diseases, such as SLE, Sjögren's syndrome, and other connective tissue diseases.

[0086] All publications referenced in this application, including patent documents, scientific papers, and databases, are incorporated by reference in their entirety for all purposes, as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0087] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0088] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0089] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0090] The term "about" as used herein refers to the normal error range of each value, which is readily known to those skilled in the art. Reference to "about" any value or parameter herein includes (and describes) aspects that are directed to the value or parameter itself. For example, a statement that refers to "about X" includes the statement "X".

[0091] The term "affinity modified" as used in reference to a domain of a protein refers to a mammalian protein having an amino acid sequence altered in its extracellular domain or specific binding portion (compared to the corresponding wild-type parent domain or unmodified domain) such that it has increased or decreased binding activity, such as binding affinity for at least one of its binding partners (or "counter-structure"), compared to the parent wild-type protein or the 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. See also Larsen et al., American Journal of Transplantation, Vol. 5:443-453 (2005). Linsley et al., Immunity, 1:7930801 (1994). The increase in binding activity, e.g., affinity, of the protein for its binding partners is at least 10% greater than that of the wild-type control, and in some embodiments, by at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10,000% greater than that of the wild-type control. The decrease in binding activity, e.g., affinity, of the protein for at least one of its binding partners is 90% or less of the control but 10% or more of the wild-type control, and in some embodiments, by 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type control 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" is not to be construed as imposing any requirement 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 transformed 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 to obtain affinity-modified domain compositions of matter, then modeled in silico to bind to their binding partners, and finally recombinantly or chemically synthesized. However, in an alternative example, affinity-modified domains can be generated by site-directed mutagenesis of wild-type domains. Thus, affinity-modified TD domains refer to a product, not necessarily a product produced by any given process. A variety of techniques can be used, including recombinant methods, chemical synthesis, or a combination thereof.

[0092] 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 the TNFR domain or TNF domain, respectively. 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 the corresponding wild-type parent domain or unmodified domain), such that it has increased or decreased binding activity, such as binding affinity, for at least one of its binding partners (or "partner structures"), compared to a parent wild-type or unmodified protein containing a non-affinity-modified TD domain or unmodified TD domain.

[0093] "Affinity-modified TACI (also referred to as variant TACI)" refers to a TACI protein molecule that antagonizes or blocks the activity of a B cell stimulatory receptor. For example, TACI binds to APRIL and / or BAFF, which are ligands for the B cell stimulatory receptor B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), and 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 heterotrimers of APRIL and BAFF. Affinity-modified variants of the extracellular domain of TACI, or portions thereof, can include another amino acid modification (e.g., amino acid substitution) in the TD that increases the binding affinity for the cognate ligand (e.g., APRIL and / or BAFF, and heterotrimers of APRIL and BAFF).

[0094] As used herein, "B cell stimulating receptor" refers to one or more of the related tumor necrosis factor receptor (TNFR) superfamily receptors expressed on B cells: B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), and transmembrane activator and calcium-regulated and cyclophilin ligand-interactor (TACI). Engagement 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, and 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), to mediate B cell signaling pathways that regulate B cell homeostasis.

[0095] As used herein, "binding," "bound," or grammatical variations thereof, refers to any attractive interaction between a molecule and 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 covalent bonds and irreversible covalent bonds), and includes interactions between molecules such as compounds including, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, e.g., drugs.

[0096] As used herein, avidity refers to the characteristics of a molecule, such as a polypeptide, regarding whether or not it binds to one or more binding partners and how it binds.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, the affinity (e.g., high affinity) of binding to a binding partner, the avidity of binding to a binding partner, the strength of binding to a binding partner, and / or the specificity or selectivity of binding to a binding partner.

[0097] The term "binding affinity" as used herein refers to the specific binding affinity of a protein to its binding partner (i.e., its partner structure) under specific binding conditions. Binding affinity refers to the strength of the interaction between two or more molecules, such as binding partners, typically the strength of the non-covalent interaction between two binding partners. The increase or decrease in the binding affinity of an affinity-modifying domain or an immunomodulating protein comprising an affinity-modifying domain to a binding partner is determined by comparing it with the binding affinity of an unmodified domain (e.g., a natural or wild-type TD domain). Methods for determining binding affinity or relative binding affinity are known in the art, and include solid-phase ELISA immunoassay, ForteBio Octet, Biacore measurement, or flow cytometry. For example, see Larsen et al., American Journal of Transplantation, vol.5:443-453 (2005); Linsley et al., Immunity, Vol 1(9):793-801 (1994). In some embodiments, binding affinity can be measured by flow cytometry, for example, based on mean fluorescence intensity (MFI) in a flow binding assay.

[0098] As used herein, the term "binding avidity" refers to the specific binding avidity of a protein to its binding partner (i.e., its counterpart structure) under specific binding conditions.In biochemical dynamics, avidity refers to the cumulative strength of individual non-covalent binding interactions, for example, the multiple affinities between proteins to their binding partners (i.e., their counterpart structures).Therefore, avidity is different from affinity, which represents the strength of a single interaction.

[0099] 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 a particular organ or tissue of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to 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).

[0100] As used herein, the term "cell surface counterpart structure" (or "cell surface binding partner") refers to a counterpart structure (or binding partner) expressed on a mammalian cell. Typically, a cell surface binding partner is a transmembrane protein. In some embodiments, a cell surface binding partner is a receptor.

[0101] With respect to a protein such as a receptor, a soluble ligand, or an extracellular domain or portion thereof, or an affinity-modified variant thereof, the term "binding partner" or "partner structure" refers to at least one molecule (typically 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 decreased affinity. A "cell surface binding partner" is a binding partner expressed on a mammalian cell. Typically, a cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor or a receptor ligand expressed on and by cells, such as mammalian cells that form an immune synapse, e.g., immune cells.

[0102] With respect to binding to cell surface molecules, the term "cis" 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 exclusively on one or exclusively on the other (but not both) of the two mammalian cells that form the IS.

[0103] As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which an 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 groups of amino acids 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 acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0104] With respect to protein positions, such as when 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, the term "corresponding to" refers to a nucleotide or amino acid position identified when aligned with a disclosed sequence based on structural sequence alignment or using a standard alignment algorithm such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Figure 9 illustrates identifying corresponding residues by aligning two sequences.

[0105] As used herein, a "domain" (typically a sequence of three or more, generally five or seven or more amino acids, e.g., 10-200 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 from and identifiable with the rest 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 that is recognized by a functional activity, such as binding activity. A protein can 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 or non-linear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For illustrative purposes herein, definitions are provided, but it is understood that it is well within the skill of one in the art to recognize specific domains by name. If necessary, appropriate software can be used to identify domains. It is understood that reference to amino acids comprising a particular sequence, as set forth as a SEQ ID NO, used to describe a domain configuration (e.g., 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. In some cases, the adjacent N- and / or C-terminal amino acids of a given domain (e.g., a TD) can also be included in the sequence, for example, to ensure proper folding of the domain when expressed.Thus, the exact locus may vary and is not necessarily the same for each protein. For example, a particular TD domain, e.g., a particular CRD domain, may be several amino acids (1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) longer or shorter.

[0106] The terms "ectodomain," "extracellular domain," or "ECD," as 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 space outside the cell) when the full-length form of the membrane protein is expressed from a cell. For purposes herein, reference to an ECD is understood to refer to the sequences and domains that make up this region and do not require that the protein containing the ECD be a membrane protein or that the domain reside outside the cell. 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, for example, 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 herein to an ECD includes the full-length sequence of the ECD of a membrane protein, as well as specific binding fragments thereof that include the CRD that binds to a ligand or cognate binding partner.

[0107] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition containing an immunomodulatory protein or Fc-fusion protein that, when administered ex vivo (by contact with cells from a patient) or in vivo (by administration to a patient), e.g., 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 cause of the disease. An effective amount for treating a disease, condition, or disorder, e.g., an immune system disease, immune system condition, or immune system disorder, can be an amount that relieves, reduces, or alleviates at least one symptom or biological response or effect associated with the disease, condition, or disorder, prevents the progression of the disease, condition, or disorder, or improves the patient's physical function. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to the patient. In some embodiments, the patient is a human patient.

[0108] 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 typically less than 20, 15, 10, 9, 8, 7, or 6 amino acids. The protein product encoded by the fusion construct is referred to as a fusion polypeptide. An example of a fusion protein according to the provided embodiments is an Fc-fusion protein containing an affinity-modifying domain (e.g., a variant or portion of the extracellular domain of TACI containing a CRD) linked to an immunoglobulin Fc domain.

[0109] 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 conjugated to the moiety. In some embodiments, the half-life is extended by more than 1.2-fold, or about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, or about 6.0-fold. In some embodiments, 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, by 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).

[0110] The Fc (Fragment Crystallizable) region or Fc (Fragment Crystallizable) domain (also referred to as an Fc polypeptide) of an immunoglobulin molecule primarily corresponds to the constant region of an immunoglobulin heavy chain and is responsible for various functions, including, in some cases, antibody effector functions. The Fc domain comprises part or all of the hinge domain, CH2 domain, and CH3 domain of an immunoglobulin molecule. In some cases, all or part of the Fc hinge sequence may be deleted 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 reduced activity (e.g., greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) to promote effector function. In some embodiments, references to amino acid substitutions within the Fc region are in accordance with the EU numbering system, unless described with reference to a specific SEQ ID NO. EU numbering is known and follows the EU index reported in the latest updated IMGT Scientific Chart (IMGT®, international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013) and Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0111] 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 can 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 provided. The immunoglobulin Fc region can 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. Fc fusions of the same species can be dimerized to form Fc fusion homodimers. Fc fusions of non-identical species (e.g., knob-into-hole engineering) can also be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a murine Fc or a human Fc.

[0112] The term "host cell" refers to any cell that can be used to express a protein encoded by a recombinant expression vector. Host cells can be prokaryotes, such as Escherichia coli (E. coli), or eukaryotes, such as unicellular eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridomas. Examples of host cells include Chinese hamster ovary (CHO) cells or derivatives thereof grown in serum-free medium, such as the Veggie CHO cell line and related cell lines, or the DHFR-deficient CHO line DX-B11.

[0113] As used herein, the term "immunological synapse" or "immune synapse" (abbreviated as "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.

[0114] 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, whether wholly or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, including any fragment thereof comprising 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 linked VH and VL in one chain, and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc and Fd polypeptide fragments. 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 the term. Antibodies include polyclonal or monoclonal antibodies. Antibodies also include synthetic or recombinantly produced antibodies. For the structure and properties of various classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0115] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to antibodies in a substantially intact form, as opposed to antibody fragments. Full-length antibodies typically have two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4), two full-length light chains (VL-CL), and a hinge region, such as antibodies produced by antibody-secreting B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.), as well as synthetically produced antibodies with the same domains. Specifically, whole antibodies include those having heavy and light chains comprising an Fc region. The constant domains can be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies can have one or more effector functions.

[0116] "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 Fvs (dsFvs), 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 Fvs (scFvs) or single-chain Fabs (scFabs); antigen-binding fragments of any of the above, and multispecific antibodies derived from antibody fragments.

[0117] An "Fv" consists of one heavy-chain variable domain 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 in each of the heavy and light chains) that contribute 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 an antigen, although in some cases with lower affinity than the entire binding site.

[0118] "dsFv" is V H -V L It refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the pair.

[0119] An "Fd fragment" is a fragment of the variable domain (V H ) and one constant region domain (C H 1) is a fragment of an antibody.

[0120] A "Fab fragment" is an antibody fragment resulting from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure produced synthetically, for example 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

[0121] 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, for example, by recombinant methods. An F(ab')2 fragment essentially contains two Fab fragments, each heavy chain of which contains an additional few amino acids, including a cysteine ​​residue that forms a disulfide bond connecting the two fragments.

[0122] A "Fab' fragment" is a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain).

[0123] An "Fd' fragment" is a fragment of an antibody that contains one heavy chain portion of the F(ab')2 fragment.

[0124] "Fv' fragment" refers to the V fragment of an antibody molecule. H Domains and V L A fragment containing only the domain.

[0125] An "scFv fragment" is a fragment of variable light chains (VLC) covalently linked by a polypeptide linker in any order. L ) and variable heavy chain (V H ) The linker is of a length that allows for substantial uninterrupted bridging of the two variable domains. Exemplary linkers include those with several Glu or Lys residues dispersed throughout to enhance solubility (Gly-Ser). n There are residues.

[0126] "Diabodies" are dimeric scFvs. Diabodies typically have shorter peptide linkers than scFvs and preferentially dimerize.

[0127] As used herein, the term "immunological activity" refers to one or more activities of immune cells, such as T cells or B cells, such as 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 lead to activation of transcription factors in immune cells. Assays to assess the immunological activity of immunomodulatory proteins can be compared to control proteins with known activity.

[0128] An "immunomodulating protein" or "immunomodulating polypeptide" is a protein that modulates immunological activity. "Modulation" of an immune response or "modulating" an immune response means that immunological activity is enhanced or suppressed. Such modulation includes any induction of immunological activity of immune cells such as B cells or T cells, or a change in the degree or extent of immunological activity of immune cells such as B cells or T cells, or suppression of immunological activity of immune cells such as B cells or T cells. For example, the soluble Fc fusion protein herein can suppress the immunological 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 proteins, dimeric proteins, and higher order multimeric proteins are within the scope of this defined term. A multimeric protein can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains).

[0129] As used herein, modification refers to modification of the amino acid sequence of a polypeptide or the nucleotide sequence in a nucleic acid molecule, including changes in the amino acids or nucleotides of the sequence, respectively. Amino acid modifications or changes can be deletions, insertions, or replacements (substitutions) of amino acids or nucleotides, respectively. Methods for modifying polypeptides are familiar to those skilled in the art, for example, by using recombinant DNA techniques.

[0130] The term "multimerization domain" refers to an amino acid sequence that promotes the formation of multimers of two or more polypeptides. The multimerization domain comprises a sequence that promotes stable interaction between a polypeptide molecule and one or more additional polypeptide molecules, each of which contains complementary multimerization domains (e.g., a first multimerization domain and a second multimerization domain), which may be the same or different. The interaction between the complementary multimerization domains, for example, the interaction between the first multimerization domain and the second multimerization domain, forms a stable protein-protein interaction to generate a multimer of the polypeptide molecule and the additional polypeptide molecule. In some cases, the multimerization domains are the same and interact with themselves to form a stable protein-protein interaction between the two polypeptide chains. Generally, the polypeptide is directly or indirectly linked to the 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 may be, for example, an immunoglobulin constant region or domain, such as an Fc domain or portion thereof from IgG, including IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM, and modified forms thereof.

[0131] The terms "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless specifically limited, the terms encompass nucleic acids that contain known analogs of natural nucleotides, have similar binding properties to natural nucleotides, and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0132] As used herein, the terms "in functional combination," "in functional order," and "operably linked" refer to the linking of nucleic acid sequences in a manner or orientation such that the segments are arranged to function in concert for their intended purpose.In some embodiments, the terms refer to the linking of nucleic acids to produce a nucleic acid molecule that can direct the transcription of a given gene and / or to produce a functional desired protein molecule.For example, segments of DNA sequences, such as coding sequences and regulatory sequences, are linked to allow gene expression when appropriate molecules (such as transcriptional activator proteins) are bound to the regulatory sequences.

[0133] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0134] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked via peptide bonds. The term does not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The term includes post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, and the like. The term also includes molecules containing one or more amino acid analogs, or non-canonical or non-natural amino acids, which may be synthesized or recombinantly expressed using known protein engineering techniques. Furthermore, proteins may be derivatized as described herein by well-known organic chemistry techniques.

[0135] For example, the term "purified," as applied to a nucleic acid encoding an immunomodulatory protein, or 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 forms a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that 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).

[0136] 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. The alteration can be made to the material in its natural environment or state, or can be removed from its natural environment or state. 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 that has been genetically altered, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein, such as the immunomodulatory protein provided herein. In eukaryotes, transcriptional control signals include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, 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 which cell type will be used to express the protein of interest.

[0137] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing a desired coding sequence (e.g., encoding an immunomodulatory protein) and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular cell. In prokaryotes, nucleic acid sequences necessary for expression include a promoter, optionally an operator sequence, a ribosome binding site, and optionally other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence may also be optionally encoded by the recombinant expression vector and operably linked to the coding sequence so that the expressed protein can be secreted by the recombinant host cell, for example, for its expression as a secretable protein, or for easier isolation or purification of the immunomodulatory protein from the cell, if desired. The term also includes vectors as autonomously replicating nucleic acid constructs, as well as vectors that integrate into the genome of the host cell into which they are introduced. Among vectors are viral vectors, such as lentiviral vectors.

[0138] As used herein, the term "sequence identity" 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 comparing the amino acid sequence at a given position within each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position within each sequence when the sequences are aligned. 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 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 the amino acid residues (or nucleotide residues) in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Reference to sequence identity includes sequence identity over the entire length of each of the sequences being compared. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0139] The term "soluble" used herein in relation 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, and not including the transmembrane domain.In some cases, the solubility of a protein can be improved by linking or binding, 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 cases.In some aspects, the soluble protein is an Fc fusion protein.

[0140] As used herein, the term "specifically binds" refers to the ability of a protein to bind to a target protein under specific 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 population of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not exclusively bind to a single target molecule but may specifically bind to multiple target molecules. In some cases, a specifically binding protein may bind to a protein (e.g., a paralog or ortholog) that has a similar structural conformation to the target protein. Those skilled in the art will recognize that specific binding to molecules with the same function (i.e., orthologs) in different animal species or to molecules with substantially similar epitopes to the target molecule (e.g., a paralog) is possible without compromising the specificity of binding as determined by comparison with a statistically valid population of unique non-targets (e.g., random polypeptides). Thus, the immunomodulatory proteins of the present invention may specifically bind to target molecules of multiple different species due to cross-reactivity. Specific binding between two proteins can be determined using solid-phase ELISA immunoassays, ForteBio Octet, or Biacore measurements. Generally, the interaction between two binding proteins is approximately 1 x 10 -5 Less than M, often about 1 × 10-12 In certain aspects of the present disclosure, the interaction between two binding proteins has a dissociation constant (Kd) as low as about 1×10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 It has a dissociation constant below M or less.

[0141] The term "specific binding fragment" or "fragment" as used herein with respect to a protein refers to a polypeptide that is shorter than the full-length protein or a specific domain or region thereof and specifically binds to a binding partner of the full-length protein or specific domain or region in vitro and / or in vivo. A specific binding fragment relates to a fragment of the full-length extracellular domain of a polypeptide or a binding domain of a polypeptide, but still binds to a binding partner of the binding domain. For example, a specific binding fragment relates to a fragment of the extracellular domain of a full-length TNFR family member or a full-length TNFR domain (TD) (e.g., CRD) thereof, but still binds to a binding partner of the 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 can have an amino acid length of at least 50 amino acids, e.g., 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.

[0142] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human. The subject may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

[0143] As used herein, "synthetic," for example with respect to a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or a polypeptide molecule that is produced by recombinant and / or chemical synthesis methods.

[0144] As used herein, the term "TNF receptor superfamily" or "TNFRSF" refers to a group of cell surface cytokine receptors that are all type I (N-terminal extracellular) transmembrane glycoproteins containing one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on shared structural features, including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular regions, which often play a role in protein binding of cognate binding partners or cognate ligands. TNFRSF proteins can have only one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or ectodomain of TNFRSF members contains one to six pseudorepeats of the CRDs. For example, the BAFF receptor and BCMA each contain one CRD, whereas TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are usually trimeric or multimeric complexes stabilized by their cysteine-cysteine ​​disulfide bonds. Binding of TNFRSF proteins to their ligands promotes various biological activities within the cell, such as the induction of apoptotic cell death or cell survival and proliferation.

[0145] The term "TD" refers to one or more structural domains of a TNFRSF protein or a TNF family ligand. For example, the TD of a TNFRSF protein is a cysteine-rich domain (CRD) module of approximately 40 amino acids containing six conserved cysteines. Therefore, reference to a CRD can also be used interchangeably with the term TD when referring to the TD of a TNFRSF protein. The six cysteines are involved in the formation of intrachain disulfide bonds. The extracellular domain (ECD) of a TNFRSF member contains one or more CRD domains. Therefore, the term TD is also used 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.

[0146] With respect to binding to cell surface molecules, the term "trans" refers to binding to two different cell surface molecules, each present on the surface of a different cell. In some embodiments, trans means, with respect to two different cell surface molecules, that the first one is present exclusively on one of the two mammalian cells that form the IS and the second one is present exclusively on the other of the two mammalian cells that form the IS.

[0147] 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 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 makes this integration thermodynamically stable under physiological conditions. A transmembrane domain can generally be predicted from its amino acid sequence through any number of commercially available bioinformatics software applications based on its increased hydrophobicity compared to the region of the protein that interacts with the aqueous environment (e.g., cytosol, extracellular fluid). A transmembrane domain is often a hydrophobic α-helix that spans the membrane. A transmembrane protein can span both layers of a lipid bilayer one or more times.

[0148] As used herein, the terms "treating" a disease, condition, or disorder, "treatment," or "therapy" of a disease, condition, or disorder means slowing, halting, or reversing the progression of the disease or disorder by administering an immunomodulatory protein, or an engineered cell of the invention, alone or in combination with another compound described herein, as evidenced by the reduction, halting, or elimination of any clinical or diagnostic symptoms. "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, for example, in the case of 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 this invention, "preventing" a disease, condition or disorder, "prophylaxis" or "prevention" of a disease, condition or disorder refers to the administration of an immunomodulatory protein of the invention, alone or in combination with another compound, to prevent the onset or development of a disease, condition or disorder, or some or all of the symptoms of a disease, condition or disorder, or to reduce the likelihood of developing a disease, condition or disorder.

[0149] The term "variant" (which may also be used interchangeably with "modified" or "mutant") as used with respect to a variant protein or variant polypeptide refers to a protein, such as a mammalian (e.g., human or murine) protein, that has been created by human intervention. A variant is a polypeptide that has an altered or modified amino acid sequence, such as by one or more amino acid substitutions, deletions, additions, or a combination thereof, compared to the unmodified or wild-type protein or domain thereof. A variant polypeptide can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences, e.g., amino acid substitutions. Variant polypeptides 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 corresponding form of the wild-type or unmodified protein, e.g., its mature sequence (lacking the signal sequence) or a portion thereof, including the extracellular domain or its binding domain. 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 production method, 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, the modified amino acid sequence results in altered (i.e., increased or decreased) binding activity, e.g., binding affinity or binding avidity, for one or more binding partners. Thus, the variant proteins may be "affinity-modified" proteins as described herein.

[0150] As used herein, the terms "wild-type" or "natural" or "native," used interchangeably, are used in reference to biological material, such as, for example, nucleic acid molecules, proteins, host cells, etc., that are found in nature and have not been modified by human intervention.

[0151] II. TACI Immunomodulatory Proteins and Variant TACI Polypeptides Provided herein is a TACI immunomodulatory protein, comprising a portion of the extracellular domain (ECD) of a TACI receptor or a variant thereof, which binds to at least one TACI cognate binding partner.Also provided herein is a variant TACI polypeptide that exhibits altered (e.g., increased) binding activity or binding affinity to one or more of the TACI cognate binding partners.In some embodiments, the TACI cognate binding partner is one or more of BAFF or APRIL, or is a BAFF / APRIL heterotrimer.The provided TACI immunomodulatory proteins and TACI immunomodulatory polypeptides include soluble fusion proteins thereof, in which the TACI portion of the extracellular domain or its variant is linked to another moiety, such as 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, a TACI-Fc fusion protein is provided, comprising: (1) a TACI polypeptide or variant TACI polypeptide consisting of the extracellular domain of a TACI receptor or a portion thereof, which binds to at least one TACI cognate binding partner; and (2) an Fc domain. The TACI polypeptide or variant TACI polypeptide can be linked to the Fc domain directly or indirectly (e.g., via a peptide linker).

[0152] 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 B cells and a subset of 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), as well as variously called 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)). The other ligand is called APRIL, and 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.

[0153] The amino acid sequence of full-length TACI is set forth in SEQ ID NO:88. The protein is a type III membrane protein and lacks a signal peptide. After expression in eukaryotic cells, the N-terminal methionine is removed. In some embodiments, the mature TACI protein does not contain the N-terminal methionine 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), which exhibit affinity for binding to BAFF and APRIL, respectively. 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 a stalk region of approximately 60 amino acids following the second cysteine ​​repeat in the extracellular domain, corresponding to amino acid residues 105-165 of the sequence set forth in SEQ ID NO:122.

[0154] In some embodiments, the variant TACI polypeptides provided herein comprise one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "replacements"), deletions, or additions, in the extracellular domain of a reference TACI polypeptide, e.g., a wild-type or unmodified TACI polypeptide comprising a CRD (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 in the CRD. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "replacements"), deletions, or additions, are in the CRD1 region. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or alternatively, "mutations" or "replacements"), deletions, or additions, are in the CRD2 region. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions or additions, are in amino acids within the CRD1 and CRD2 regions.

[0155] 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 essentially consists of CRD1 or a specific binding fragment thereof. In some embodiments, a variant TACI polypeptide comprises or essentially consists 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.

[0156] 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.

[0157] In some embodiments, the reference (e.g., unmodified) TACI sequence has (i) the amino acid sequence set forth in SEQ ID NO:122, or a sequence thereof lacking the N-terminal methionine; (ii) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: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 TIFF2025134866000007.tif27155

[0158] In some embodiments, the reference (e.g., unmodified) TACI sequence is the extracellular domain sequence of TACI, a portion of the ECD that includes an N-terminal deletion compared to the amino acid sequence set forth in SEQ ID NO:122. In some embodiments, the N-terminal deletion is a deletion of N-terminal amino acid residues 1-28, which 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.

[0159] In some of 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 stalk portion of the TACI extracellular domain. In some embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion that lacks one or more 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 of the ECD sequence are deleted.

[0160] In some embodiments, a reference (e.g., unmodified) TACI sequence includes an ECD portion having a contiguous amino acid sequence that includes CRD1 and / or CRD2 (e.g., CRD1 and CRD2, or only CRD2) and only a segment or portion of the stalk sequence. A suitable stalk segment includes one or more amino acids from amino acid residues 105-154 of SEQ ID NO:122. For example, the stalk segment may include, based on SEQ ID NO:122, amino acid residues 105, amino acid residues 105-106, amino acid residues 105-107, amino acid residues 105-108, amino acid residues 105-109, amino acid residues 105-110, amino acid residues 105-111, amino acid residues 105-112, amino acid residues 105-113, amino acid residues 105-114, amino acid residues 105-115, amino acid residues 105-116, amino acid residues 105-117, amino acid residues 105-118, amino acid residues 105-119, amino acid residues 105-120, amino acid residues 105-121, amino acid residues 105-122, amino acid residues 105-123, amino acid residues 105-124, amino acid residues 105-125, amino acid residues 105-126, amino acid residues 105-127, amino acid residues 105-128, amino acid residues 105-129, amino acid residues 105-130, amino acid residues 105-131, amino acid residues 105-132, amino acid residues 105-133, amino acid residue 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 105-130, amino acid residues 105-131, amino acid residues 105-132, amino acid residues 105-133, amino acid residues 105-134, amino acid residues 105-135, amino acid residues 105-136, amino acid residues 105-137, amino acid residues 105-138, amino acid residues 105-139, amino acid residues 105-140, amino acid residues 105-141, amino acid residues 105-142 42, amino acid residues 105-143, amino acid residues 105-144, amino acid residues 105-145, amino acid residues 105-146, amino acid residues 105-147, amino acid residues 105-148, amino acid residues 105-149, amino acid residues 105-150, amino acid residues 105-151, amino acid residues 105-152, amino acid residues 105-153, and amino acid residues 105-154.

[0161] In some embodiments, the reference (e.g., unmodified) TACI sequence lacks one or more potential furin cleavage sites or has one or more potential furin cleavage sites mutated. In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or portion in which the arginine residue at position 119 has been 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 has been 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 has been mutated (e.g., R122Q).

[0162] 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.

[0163] 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 TIFF2025134866000008.tif19141

[0164] 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 TIFF2025134866000009.tif12153

[0165] 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 nucleotide sequence set forth in SEQ ID NO:36). TACI ECD(CRD1 / CRD2):SEQ ID NO:1 TIFF2025134866000010.tif12159

[0166] In some embodiments, the reference TACI sequence is a region of the extracellular domain of TACI that consists essentially of only the CRD2 sequence, with the entire CRD1 sequence and substantially all of the stalk region deleted or absent. Previous studies have shown that residues within the stalk region may contain protease cleavage sites, but at least CRD1 and CRD2 are believed to be necessary for sufficient expression and / or binding activity of TACI to its cognate ligand. For example, International PCT Publication No. WO2002 / 094852 demonstrated that a TACI molecule containing CRD1 and CRD2 but lacking the entire amino-terminal region and a partial sequence of the stalk region exhibited reduced proteolysis when expressed. Other studies have shown that at least a portion of the N-terminal region preceding CRD1 was 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 region were determined to be necessary for biological activity while minimizing degradation of TACI during expression. Surprisingly, it is found herein (e.g., Example 3) that a TACI extracellular region consisting essentially of only CRD2 with a small portion of the stalk region exhibits substantially improved cognate binding activity compared to longer TACI molecules containing both CRD1 and CRD2.

[0167] For example, 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, lacking the N-terminal region and CRD1, and lacking one or more residues in the stalk portion of the TACI extracellular domain, as compared 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 immunomodulatory protein TACI polypeptide includes 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 immunomodulatory protein TACI polypeptide includes a deletion of N-terminal amino acid residues 1-70, which correspond to the residues set forth in SEQ ID NO: 122. In some of any such embodiments, the immunomodulatory protein TACI polypeptide 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 of the ECD sequence are deleted.

[0168] In some aspects, the immunomodulatory proteins (e.g., TACI-Fc fusion proteins) provided herein have a TACI polypeptide having a sequence that includes an ECD portion having the continuous amino acid sequence of the TACI ECD, e.g., compared to the amino acid sequence set forth in SEQ ID NO:122, including CRD2 (e.g., residues 71-104, based on SEQ ID NO:122), but lacking the N-terminal region and CRD1, and lacking one or more residues in the stalk portion of the TACI extracellular domain. For example, the TACI ECD portion may consist of amino acid residues 67-118, amino acid residues 67-117, amino acid residues 67-116, amino acid residues 67-115, amino acid residues 67-114, amino acid residues 67-113, amino acid residues 67-112, amino acid residues 67-111, amino acid residues 67-110, amino acid residues 67-109, amino acid residues 67-108, amino acid residues 67-107, amino acid residues 67-106, amino acid residues 67-105, or amino acid residues 67-104, based on the amino acid residues set forth in SEQ ID NO:122. In some examples, the TACI ECD portion can consist of amino acid residues 68-118, amino acid residues 68-117, amino acid residues 68-116, amino acid residues 68-115, amino acid residues 68-114, amino acid residues 68-113, amino acid residues 68-112, amino acid residues 68-111, amino acid residues 68-110, amino acid residues 68-109, amino acid residues 68-108, amino acid residues 68-107, amino acid residues 68-106, amino acid residues 68-105, or amino acid residues 68-104, based on the residues set forth in SEQ ID NO:122. In some examples, the TACI ECD portion can consist of amino acid residues 69-118, amino acid residues 69-117, amino acid residues 69-116, amino acid residues 69-115, amino acid residues 69-114, amino acid residues 69-113, amino acid residues 69-112, amino acid residues 69-111, amino acid residues 69-110, amino acid residues 69-109, amino acid residues 69-108, amino acid residues 69-107, amino acid residues 69-106, amino acid residues 69-105, or amino acid residues 69-104, based on the residues set forth in SEQ ID NO:122.In some examples, the TACI ECD portion can consist of amino acid residues 70-118, amino acid residues 70-117, amino acid residues 70-116, amino acid residues 70-115, amino acid residues 70-114, amino acid residues 70-113, amino acid residues 70-112, amino acid residues 70-111, amino acid residues 70-110, amino acid residues 70-109, amino acid residues 70-108, amino acid residues 70-107, amino acid residues 70-106, amino acid residues 70-105, or amino acid residues 70-104, based on the residues set forth in SEQ ID NO:122. In some examples, the TACI ECD portion can consist of amino acid residues 71-118, amino acid residues 71-117, amino acid residues 71-116, amino acid residues 71-115, amino acid residues 71-114, amino acid residues 71-113, amino acid residues 71-112, amino acid residues 71-111, amino acid residues 71-110, amino acid residues 71-109, amino acid residues 71-108, amino acid residues 71-107, amino acid residues 71-106, amino acid residues 71-105, or amino acid residues 71-104, relative to the residues set forth in SEQ ID NO: 122. Any of the above TACI ECD sequences can also be a TACI reference sequence according to the immunomodulatory proteins provided herein, including variant TACI polypeptides modified by one or more amino acid modifications (e.g., substitutions) described herein compared to such a TACI reference sequence.

[0169] 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 nucleotide sequence 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 TIFF2025134866000011.tif5128

[0170] Among the provided TACI polypeptides are variant TACI polypeptides. Also provided are immunomodulatory proteins, e.g., TACI-Fc fusion proteins, comprising the provided TACI polypeptides. In some of any of the provided embodiments, the variant TACI sequence has the sequence of a reference (e.g., unmodified) TACI sequence, e.g., any of those described above, but further comprises another amino acid modification, e.g., one or more amino acid substitutions. In particular, provided herein are variant TACI polypeptides comprising at least one affinity-modified TD domain (e.g., CRD1 and / or CRD2) or a specific-binding fragment thereof, comprising one or more amino acid substitutions in the TD domain of a reference (e.g., unmodified or wild-type) TACI polypeptide, such that the variant TACI polypeptide exhibits altered (e.g., increased) binding activity or affinity for one or both of APRIL 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 the binding affinity 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. That is, 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.

[0171] 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, e.g., human or mouse proteins. In some embodiments, BAFF and / or APRIL are human. In some embodiments, a variant TACI polypeptide having increased or enhanced binding affinity for APRIL and / or BAFF has an increase in 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).

[0172] In some embodiments, the equilibrium dissociation constant (K) of any of the foregoing 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 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 M, or 1 x 10 -12 Less than M or about 1 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 M, or 1 x 10 -12 In some embodiments, the K of any of the foregoing embodiments for BAFF is less than M. d is 1 x 10 -9 From M, 1 x 10 -12 M or approximately 1 x 10 -12 In some embodiments, the K of any of the foregoing embodiments for BAFF is 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 -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 -12 In some embodiments, provided embodiments comprise the variant TACI polypeptides described above, which have a K d is decreased by more than or more than about 1.5 fold, e.g., by more than or more than about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more (an increase in binding affinity).

[0173] 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 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 M, or 1 x 10 -12 Less than M or approximately 1 x 10 -9 M, 1 x 10 -10 M or 1 x 10 -11 M, or 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 From M, 1 x 10 -12 m or approximately 1 x 10 -12 In some embodiments, K of any of the foregoing embodiments for APRIL is 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 -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 -12 In some embodiments, provided embodiments comprise the variant TACI polypeptides described above, having a K d is decreased by more than or more than about 1.5 fold, e.g., by more than or more than about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more (an increase in binding affinity).

[0174] A reference (e.g., unmodified or wild-type) TACI sequence need not necessarily be used as the starting material for generating the variant TACI polypeptides described herein. Thus, the use of the term "modification," e.g., "substitution," does not imply that the present embodiment is limited to a particular method for producing a variant TACI polypeptide or an immunomodulatory protein comprising the same. Variant TACI polypeptides can be produced, for example, by de novo peptide synthesis and therefore do not necessarily require a modification, such as a "substitution," in the sense of changing a codon to encode 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 for designing or producing a variant TACI polypeptide are not limited to a particular method. However, in some embodiments, a reference (e.g., unmodified or wild-type) TACI-encoding nucleic acid is mutagenized from a reference (e.g., unmodified or wild-type) TACI genetic material and screened for a desired specific binding affinity or other functional activity. In some embodiments, variant TACI polypeptides are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases, and then screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, such as the UniProtKB database mentioned above.

[0175] Unless otherwise indicated, as set forth throughout this disclosure, amino acid modifications in variant TACI polypeptides are designated by amino acid position numbers that correspond 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, within 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; if the modification is an insertion at that position, "ins" is indicated. In some cases, insertions are listed with the amino acid position indicated in the center, the corresponding reference amino acid is listed before and after the number, and the identified variant amino acid insertion is listed after the unaltered (e.g., wild-type) amino acid.

[0176] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, e.g., substitutions, within a reference (e.g., unmodified or wild-type) TACI sequence, such as any described. The one or more amino acid modifications, e.g., substitutions, can be present within 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 within the CRD1 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 within the CRD1 domain or a specific binding fragment thereof, and some of the one or more amino acid modifications, e.g., substitutions, are within the CRD2 domain or a specific binding fragment thereof.

[0177] In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., substitutions, in the reference TACI sequence. The modifications, e.g., substitutions, can be in the CRD1 domain or the CRD2 domain. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in the CRD1 domain of the reference TACI sequence, or a specific-binding fragment thereof. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions within the CRD2 domain of a reference TACI sequence or a specific binding fragment thereof.

[0178] In some embodiments, a variant TACI polypeptide containing one or more 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, containing the CRD1 and / or CRD2 domain. In some embodiments, the specific binding fragment contains the CRD1 domain, e.g., the specific binding fragment contains the sequence set forth in amino acids 34-66 of SEQ ID NO:122. In some cases, the CRD1 domain is the only complete CRD domain present in the specific binding fragment. In some embodiments, the specific binding fragment is or contains the CRD2 domain, e.g., the specific binding fragment contains the sequence set forth in amino acids 71-104 of SEQ ID NO:122. In some embodiments, the CRD2 domain is the only complete CRD domain present in the specific binding fragment. In some embodiments, the specific binding fragment is or contains the CRD1 and CRD2 domains, e.g., the specific binding fragment contains amino acids 34-104 of SEQ ID NO:122. In some embodiments, the specific binding fragment contains a continuous portion of the stalk domain, e.g., the specific binding fragment contains a continuous portion of amino acids 105-165 of SEQ ID NO:122. In some of the optional embodiments, the specific binding fragment of SEQ ID NO:122 is shorter 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.

[0179] 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, such as having the amino acid sequence of SEQ ID NO:1, 13 or 122.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions, in a reference TACI polypeptide or a specific binding fragment thereof corresponding to 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, BIM, has one or more amino acid modifications, such as 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.

[0186] In some embodiments, the amino acid substitutions are in the CRD2 domain only. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as 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:709. In some embodiments, the variant TACI polypeptide, BIM, has one or more amino acid modifications, such as 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 but 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.

[0187] A conservative amino acid modification, e.g., substitution, is any amino acid, other than the reference (e.g., unmodified) or wild-type amino acid, that is within the same class of amino acids as the substituting 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 (aspartate, glutamate, asparagine, and glutamine).

[0188] In some embodiments, the variant TACI polypeptide, BIM, comprises at least one amino acid substitution at position 75 relative to the numbering of SEQ ID NO:709. In some embodiments, the amino acid substitution at position 75 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid is an acidic amino acid or amide, e.g., a different acidic amino acid or amide compared to the 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).

[0189] In some embodiments, the variant TACI polypeptide, BIM, 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 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 77 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 77 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 77 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 77 is glutamine (Gln, Q).

[0190] In some embodiments, the variant TACI polypeptide, BIM, 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 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 78 is an aromatic amino acid, e.g., 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).

[0191] In some embodiments, the variant TACI polypeptide, BIM, 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 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 84 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 84 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 84 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 84 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 84 is glutamine (Gln, Q).

[0192] 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 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 101 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 101 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 101 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 101 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 101 is glutamine (Gln, Q).

[0193] 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 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 102 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 102 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 102 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 102 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 102 is glutamine (Gln, Q).

[0194] 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 contains any two or more of the amino acid substitutions described above. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions that are any of the conservative amino acid substitutions described above. 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 in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the at least one amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the 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 shown in SEQ ID NO:131.

[0195] 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 contains two or more amino acid substitutions of any two or more of the foregoing. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions that are any of the foregoing conservative amino acid substitutions. In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution that is found in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:131.

[0196] 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 found in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO: 13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO: 130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO: 131.

[0197] In some of the optional embodiments, 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 contains one of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains two of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains three of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains four of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains five of the above amino acid substitutions. In some embodiments, the variant TACI polypeptide contains six of the above amino acid substitutions.

[0198] In some of the embodiments, the one or more amino acid substitutions include Q75E / R84Q. In some of the embodiments, the one or more amino acid substitutions include Q75E / K77E. In some of the embodiments, the one or more amino acid substitutions include Q75E / F78Y. In some of the embodiments, the one or more amino acid substitutions include Q75E / A101D. In some of the embodiments, the one or more amino acid substitutions include Q75E / Y102D. In some of the embodiments, the one or more amino acid substitutions include F77E / F78Y. In some of the embodiments, the one or more amino acid substitutions include K77E / R84Q. In some of the embodiments, the one or more amino acid substitutions include K77E / A101D. In some of the embodiments, the one or more amino acid substitutions include K77E / Y102D. In some of the embodiments, the one or more amino acid substitutions include F78Y / R84Q. In some of any of the embodiments, the one or more amino acid substitutions include F78Y / A101D. In some of any of the embodiments, the one or more amino acid substitutions include F78Y / Y102D. In some of any of the embodiments, the one or more amino acid substitutions include R84Q / A101D. In some of any of the embodiments, the one or more amino acid substitutions include R84Q / Y102D. In some of any of the embodiments, the one or more amino acid substitutions include A101D / Y102D. In provided embodiments, the variant TACI polypeptide comprises an amino acid substitution found in any of the reference TACI polypeptide sequences set forth, 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.

[0199] In some embodiments, the variant TACI polypeptide comprises an amino acid substitution TIFF2025134866000012.tif19158. In provided embodiments, the variant TACI polypeptide contains an amino acid substitution found 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.

[0200] 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 found in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:131.

[0201] 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 found in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:131.

[0202] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions F78Y and Y102D (F78Y / Y012D). In provided embodiments, the variant TACI polypeptide includes an amino acid substitution that is in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:131.

[0203] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions K77E, F78Y, and Y102D (K77E / F78Y / Y102D). In provided embodiments, the variant TACI polypeptide includes an amino acid substitution that is in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:131.

[0204] In some embodiments, the variant TACI polypeptide contains the amino acid substitution Q75E / R84Q. In provided embodiments, the variant TACI polypeptide includes an amino acid substitution found in any of the reference TACI polypeptide sequences described. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:13. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:130. In some embodiments, the amino acid substitution is in the reference TACI sequence set forth in SEQ ID NO:131.

[0205] 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 particular domain may vary, depending, for example, on the method used to identify or classify the domain. In addition, in some cases, the adjacent N- and / or C-terminal amino acids of a particular domain (e.g., a CRD) may also be included in the sequence of the variant TACI polypeptide, for example, to ensure proper folding of the domain upon expression. Thus, it is understood that the exemplary SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain of a variant TACI polypeptide, e.g., an ECD domain or portion thereof containing 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 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0206] 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 containing the amino acid sequence set forth in SEQ ID NO: 122. In some examples, the mutations (amino acid substitutions) are made in a reference TACI containing the CRD1 and CRD2 domains of TACI, e.g., as set forth in SEQ ID NO: 1. In some examples, the mutations (amino acid substitutions) are made in a reference TACI that has been further truncated by deleting the N- and C-terminal amino acid residues to retain CRD2, e.g., as set forth in SEQ ID NO: 13.

[0207] 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 a modification, such as a "substitution" in the sense of altering a codon to encode a 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 created are not limited to any particular method. However, in some embodiments, nucleic acids encoding wild-type or unmodified TDs are mutagenized from wild-type or unmodified TD genetic material and screened for alterations in the desired specific binding activity, e.g., binding affinity, and / or NF-κB modulation or other functional activity. In some embodiments, vTDs are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases, and then subsequently screened. The National Center for Biotechnology Information provides such information, and its website is publicly available via the Internet, such as the UniProtKB database.

[0208] In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence containing CRD1 and CRD2, e.g., 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 that retains an amino acid modification, e.g., a substitution, that is 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 contains a contiguous sequence containing an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI.

[0209] In some embodiments, the 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, the variant TACI polypeptide consists of, or consists essentially of, a polypeptide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, 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 that retains an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or essentially consists 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 contains a contiguous sequence containing an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI.

[0210] In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence containing CRD2 but lacking CRD1 of a reference TACI polypeptide, e.g., 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 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 comprises a polypeptide sequence that retains an amino acid modification, e.g., a substitution, that is 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 contains a contiguous sequence containing an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI.

[0211] 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 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 an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or essentially consists 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 contains a contiguous sequence containing an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence set forth in any of SEQ ID NOs: 37-47, 56, or 57. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, 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 that retains an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI. Also provided herein are nucleic acids containing a sequence set forth in any 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.

[0217] In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence set forth in any of SEQ ID NOs: 49-55 or 58-70. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, 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 that retains an amino acid modification, e.g., a substitution, that is not present in a reference (e.g., unmodified or wild-type) TACI. Also provided herein are nucleic acids containing a sequence set forth in any of SEQ ID NOs:49-55 or 58-70, 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:549-55 or 58-70.

[0218] Table 1. Exemplary variants of TACI TIFF2025134866000013.tif116165TIFF2025134866000014.tif230165TIFF2025134866000015.tif207165

[0219] In some embodiments, TACI ECD fusion sequences are also provided herein in which any of the above-described TACI ECD sequences are linked or fused to a multimerization domain, e.g., any of the multimerization domains described herein.

[0220] The interaction of two or more polypeptides of immunomodulatory protein can be facilitated by directly or indirectly linking them to any part or other polypeptide that can interact with itself to form a stable structure.For example, different encoded polypeptide chains can be linked by multimerization, and the multimerization of polypeptides is mediated by multimerization domain.Typically, the multimerization domain forms a stable protein-protein interaction between the first polypeptide and the second polypeptide.

[0221] In some embodiments, two or more individual polypeptides of an immunomodulatory protein may be linked by multimerization, for example, as a dimeric molecule, a trimeric molecule, a tetrameric molecule, or a pentameric molecule. In some cases, the individual polypeptides are the same. For example, a trimeric molecule may be formed from three copies of the same individual polypeptide. In another example, a tetrameric molecule is formed from four copies of the same individual polypeptide. In a further example, a pentameric molecule is formed from five copies of the same individual polypeptide. The multimerization domain may be a domain that facilitates the dimerization, trimerization, tetramerization, or pentamerization of polypeptide chains.

[0222] 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.

[0223] In some embodiments, multimerization domains include any that are capable of forming stable protein-protein interactions. The multimerization domain can be an immunoglobulin sequence (e.g., an Fc domain; see, e.g., International Patent Publication Nos. WO93 / 10151 and WO2005 / 063816 US; U.S. Patent Application Publication No. 2006 / 0024298; U.S. Patent No. 5,457,035); a leucine zipper (e.g., derived from the nuclear transforming proteins fos and jun, or derived from the proto-oncogene c-myc or General Control of Nitrogen (GCN4)) (see, e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz ​​et al., (1989) Science, 243:1695-1699); a hydrophobic region; a hydrophilic region; or a free thiol that forms an intermolecular disulfide bond between homo- or heteromultimeric chimeric molecules. Furthermore, the multimerization domain may contain an amino acid sequence containing a protrusion complementary to the amino acid sequence containing the hole, as described, for example, in U.S. Patent No. 5,731,168; International Patent Publication Nos. WO 98 / 50431 and WO 2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such multimerization regions can be engineered so that steric interactions not only promote stable interactions but also further promote the formation of heterodimers over homodimers from a mixture of chimeric monomers. Generally, the protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Optionally, a compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0224] A TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) can be linked anywhere, but typically via its N- or C-terminus, to the N- or C-terminus of a multimerization domain to form a chimeric polypeptide. Linkage can be direct or indirect via a linker. Chimeric polypeptides can also be fusion proteins, formed by chemical linkage, e.g., via 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, or 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 encodes 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.

[0225] The polypeptide multimer contains two chimeric proteins created by directly or indirectly linking two of the same or different TACI polypeptide sequences (e.g., two of the same or different variant TACI polypeptide sequences) directly or indirectly to a multimerization domain. In some cases where 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 a recombinant expression vector and assembled to form a multimer. In this case, the multimerization domains interact to form a multivalent polypeptide. The multimerization domain and the TACI polypeptide (e.g., a variant TACI polypeptide) can be chemically linked using a heterobifunctional linker.

[0226] The resulting chimeric polypeptide, e.g., a fusion protein, and the resulting multimers can be purified by any suitable method, for example, by affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are introduced into a cell by transformation, homodimers and heterodimers are formed. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

[0227] In some embodiments, the multimerization domain is an Fc region of an immunoglobulin.

[0228] In some embodiments, the multimerization domain is an immunoglobulin (e.g., IgG1) Fc region, and the fusion protein is a TACI-Fc that contains (1) a TACI sequence that contains 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 contain (1) a TACI sequence, e.g., a variant TACI polypeptide, that contains or consists of any of the above-described TACI ECD polypeptide sequences, and (2) an immunoglobulin Fc region.

[0229] In some embodiments, provided herein are TACI Fc fusion proteins containing (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 proteins containing (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.

[0230] In some embodiments, the TACI-Fc fusion is a variant TACI-Fc fusion that contains or consists of any of the variant TACI polypeptides described above and an immunoglobulin Fc region.

[0231] In some embodiments, provided herein are variant TACI-Fc fusion sequences comprising: (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) an immunoglobulin Fc region. In some embodiments, provided herein are variant TACI-Fc fusion sequences comprising: (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) an immunoglobulin Fc region.

[0232] In some embodiments, provided herein are variant TACI-Fc fusion sequences comprising: (1) a TACI ECD sequence containing a CRD2 domain but lacking a CRD1 domain, e.g., a TACI sequence containing a sequence set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192; and (2) an immunoglobulin Fc region. In some embodiments, provided herein are variant TACI-Fc fusion sequences comprising: (1) a TACI ECD sequence containing a CRD2 domain but lacking a CRD1 domain, e.g., a TACI sequence consisting of or consisting essentially of a sequence set forth in any one of SEQ ID NOs: 14-20, 23-35, 92-100, 177-192; and (2) an immunoglobulin Fc region.

[0233] In provided embodiments of TACI-Fc, the immunoglobulin Fc region can be a wild-type Fc of an immunoglobulin, e.g., an IgG1 Fc. Optionally, the Fc region can be a variant Fc that lacks effector function (also referred to as an "effectorless Fc"). Exemplary Fc regions and variants thereof found in the provided TACI-Fc fusion proteins are described below.

[0234] In some embodiments, the Fc is a murine Fc or a human Fc. In some embodiments, the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0235] 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, e.g., an IgG1 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, e.g., any of the IgG2 set forth in SEQ ID NO: 138, or 219. In some embodiments, the Fc region is or is derived from an IgG4, e.g., any of the IgG4 set forth in SEQ ID NO: 139, 140, or 220. In some embodiments, the Fc region in the Fc fusion proteins provided herein may also include an Fc region exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the above Fc regions.

[0236] In some embodiments, the Fc is derived from IgG1, e.g., human IgG1. In some embodiments, the Fc is an IgG1 Fc set forth in SEQ ID NO:71, with 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 an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:71. In other embodiments, the Fc is an IgG1 Fc containing amino acids of the human G1m1 allotype, e.g., residues containing Asp (D) and Leu (L) at positions 356 and 358, e.g., the residues 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 allotype G1m1. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:81 or an amino acid sequence 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.

[0237] In some embodiments, the Fc region has the amino acid sequence set forth in SEQ ID NO:81. TIFF2025134866000016.tif33160

[0238] 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.

[0239] 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 an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:138. In some embodiments, the Fc region is an IgG2 Fc region having the sequence set forth in SEQ ID NO:138. In some embodiments, the Fc region is an IgG2 Fc region having the sequence set forth in SEQ ID NO:219.

[0240] In some embodiments, the Fc is derived from IgG4, e.g., human IgG4. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:139, or an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 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 inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which the arginine at position 409 of human IgG4 as defined in the EU index proposed by Kabat et al. is replaced with a lysine and exhibits inhibited aggregate formation (see, e.g., U.S. Patent No. 8,911,726). In some embodiments, the Fc is an IgG4 containing the S228P mutation, which has been shown to prevent recombination between 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 an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 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.

[0241] In some embodiments, the Fc region is a variant Fc region in which the wild-type Fc has been modified by one or more amino acid substitutions to reduce effector activity or render the Fc inactive with respect to Fc effector function. Exemplary effectorless or inactive mutations include those described herein.

[0242] In some embodiments, the Fc region contains one or more modifications that alter (e.g., reduce) one or more of its normal functions. In addition to antigen-binding ability, which is generally the primary function of immunoglobulins, the Fc region is responsible for effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Furthermore, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by extending in vivo half-life through binding to the in vivo FcRn receptor. In some embodiments, such functions may be reduced or altered in the Fc for use with the provided Fc fusion proteins.

[0243] In some embodiments, one or more amino acid modifications may be introduced into the Fc region, thereby creating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of changes or mutations to the Fc sequence 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.

[0244] 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 / depleted 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 is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas 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-Radiotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox96™ Non-Radiotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in an animal model, for example, in the animal model 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 an immunomodulatory protein cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC assays can be used to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0245] Immunomodulating proteins with reduced effector function include those with one or more substitutions at 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, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0246] In some embodiments, the Fc region of the immunomodulatory protein has an Fc region in which any one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as indicated by EU numbering) have been substituted with a different amino acid compared to a native Fc region. Such Fc region modifications include, for example, modifications such as those described in Current Opinion in Biotechnology (2009) 20 (6), deglycosylated chains (N297A and N297Q) described in 685-691, IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, Includes IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E; alterations such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (as indicated by EU numbering), and alterations at the sites described in WO2000 / 042072.

[0247] Certain Fc variants have been described that have improved or reduced binding to FcRs (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)).

[0248] In some embodiments, immunomodulatory proteins are provided that comprise a variant Fc region comprising one or more amino acid substitutions that enhance half-life and / or improve binding to fetal Fc receptor (FcRn). Antibodies with enhanced half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.) or 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 Fc variants with a substitution at one or more of the following Fc region residues according to EU numbering: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0249] In some embodiments, the Fc region of the immunomodulatory protein comprises one or more amino acid substitutions C220S, C226S, and / or C229S according to EU numbering. In some embodiments, the Fc region of the immunomodulatory protein comprises one or more amino acid substitutions R292C and V302C. For other examples of Fc region variants, see 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.

[0250] In some embodiments, changes are made in the Fc region that reduce 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).

[0251] In some embodiments, the variant Fc region comprising one or more amino acid modifications (e.g., amino acid substitutions) is derived from a wild-type IgG1, e.g., a 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 containing residues Glu (E) and Met (M) at positions 356 and 358 according to EU numbering. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO:71. In other embodiments, the wild-type IgG1 Fc contains amino acids of the human G1m1 allotype, e.g., residues containing Asp (D) and Leu (L) at positions 356 and 358, e.g., as set forth in 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.

[0252] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO:71 or 81 (corresponding to K447del according to EU numbering).

[0253] 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).

[0254] In some embodiments, the Fc region is a variant Fc containing 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 contains 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 and one or more of the following amino acid modifications C220S, R292C, or V302C, according to EU numbering (which corresponds to N82G and one or more of the following amino acid modifications C5S, R77C, or V87C, based on SEQ ID NO:71), e.g., the Fc region comprises the sequence set forth in SEQ ID NO:82.

[0255] In some embodiments, the variant Fc contains the amino acid substitutions L234A / L235E / G237A, according to EU numbering. In some embodiments, the variant Fc contains the amino acid substitutions A330S / P331S, according to EU numbering. In some embodiments, the variant Fc contains the amino acid substitutions L234A / L235E / G237A / A330S / P331S (Gross et al. (2001) Immunity 15:289). In some embodiments, the variant Fc 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.

[0256] 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, e.g., by replacement 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.

[0257] In some embodiments, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:73. TIFF2025134866000017.tif33159

[0258] In some embodiments, the Fc region is an IgG1 Fc but does not contain the hinge sequence. In some embodiments, the IgG1 Fc region does not contain the hinge sequence EPKSC (SEQ ID NO:239). In some embodiments, the IgG1 Fc region does not contain the hinge sequence EPKSS (SEQ ID NO:238).

[0259] In some embodiments, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:221. TIFF2025134866000018.tif33160

[0260] In some embodiments, the Fc region is a variant Fc region that comprises one or more of the amino acid modifications C220S, L235P, 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).

[0261] 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.

[0262] 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.

[0263] Examples of such Fc regions for inclusion in immunomodulatory polypeptides are provided in Table 2.

[0264] Table 2. Exemplary IgG1 Fc regions, wild-type or variant (effectorless) TIFF2025134866000019.tif127160

[0265] In some embodiments, the Fc region is a variant Fc region containing 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.

[0266] For example, the variant Fc region may 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 the amino acid sequence set forth in any of SEQ ID NOs:75, 82, 83, 134, 73, 135, 136, or 137, or an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any 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, the immunomodulatory protein (eg, a TACI-Fc fusion) provided when produced and expressed from a cell is a homodimer containing two identical polypeptide chains.

[0267] 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.

[0268] 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 to facilitate and / or promote heterodimerization. Methods for promoting heterodimerization of Fc chains include mutagenesis of the Fc region, for example, by including a series of "knob-into-hole" mutations or by including mutations that result in electrostatic steering of the Fc and promote attractive interactions between different polypeptide chains. In some embodiments, the Fc region of the heterodimeric molecule can 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 that includes residues Glu (E) and Met (M) at positions 356 and 358 (EU numbering). In other embodiments, any of the above mutations in the Fc backbone may be made in an allotype that includes residues Asp (D) and Leu (L) at positions 356 and 358 (EU numbering).

[0269] In some embodiments, the modification comprises introducing a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide, such that the protrusion is positionable within the cavity to facilitate complexation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create the protrusion or cavity within the polypeptide are typically interface amino acids that interact or contact one or more amino acids at the interface of the second polypeptide.

[0270] In some embodiments, a first polypeptide modified to include a knob amino acid comprises replacing a natural or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide and can therefore be positioned in a complementary cavity (hole) in the adjacent interface of a 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 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 residues for forming knobs are naturally occurring amino acid residues, including, for example, 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.

[0271] In some embodiments, the second polypeptide modified to contain a cavity (hole) comprises substituting 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 therefore 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 forming a cavity. Generally, the replacement residues for forming a cavity are naturally occurring amino acids, including, for example, 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.

[0272] For example, the CH3 interface of human IgG1 contains 16 residues on 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). Modifications of the CH3 domain to create protrusions or cavities have been described, for example, in U.S. Patent No. 5,731,168; International Patent Applications WO98 / 50431 and WO2005 / 063816; and Ridgway et al., (1996) Prot. Engin., 9:617-621. In some instances, modifications of the CH3 domain to create protrusions or cavities are typically targeted to residues located on the two central antiparallel β-strands, with the goal being to minimize the risk that the created protrusions may be accommodated by protruding into the surrounding solvent rather than being accommodated by a compensatory cavity in the partner CH3 domain.

[0273] 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 also 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, which includes S354C and T366W, and the hole Fc may comprise the sequence set forth in SEQ ID NO:90, which includes mutations Y349C, T366S, L368A, and Y407V. In some embodiments, the heterodimeric molecule comprises an E356C, T366W mutation in one of the two CH3 domains and an Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises an Y349C, T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. Other examples of knob-in-hole technology are known in the art, for example, as described in EP1870459A1.

[0274] In some embodiments, an Fc variant comprising a CH3 knob or hole modification can be linked to a multi-domain immunomodulatory polypeptide, typically via its N- or C-terminus, at any location, 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. Linkage can 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 and a second immunomodulatory polypeptide linked to an Fc variant comprising a CH3 cavity modification.

[0275] Exemplary sequences of knob and hole Fc polypeptides are set forth in SEQ ID NOs:123 and 129, respectively. In some embodiments, the knob or hole Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc 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.

[0276] In some embodiments, the individual polypeptides of a multi-domain polypeptide or the individual polypeptides of a single-domain polypeptide are linked to a multimerization domain that forms a trimer, tetramer, or pentamer of the immunomodulatory protein. 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.

[0277] In some embodiments, the multimerization domain is a portion 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.

[0278] 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.

[0279] In some embodiments, a TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) is connected to a multimerization domain (e.g., an Fc region) via a linker, e.g., a peptide linker. In some embodiments, the peptide linker can be one or more amino acid residues in length. In some embodiments, the peptide linker has at least one amino acid residue, but is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue or less in length.

[0280] 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., two, three, four, or five repeats of the 4GS linker. In some embodiments, the peptide linker is (GGGGS)2 (SEQ ID NO:78), (GGGGS)3 (SEQ ID NO:79), (GGGGS)4 (SEQ ID NO:84), or (GGGGS)5 (SEQ ID NO:91). In some embodiments, the linker may also comprise a series of alanine residues alone or in addition to another peptide linker (e.g., a 4GS linker or multimer thereof). In some embodiments, the linker is (in single-letter amino acid code): GSGGGGS (SEQ ID NO:74) or GGGGSSA (SEQ ID NO:80). In some examples, the linker is 2xGGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 133). In some examples, the linker is set forth in SEQ ID NO: 194 or 195.

[0281] 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, e.g., 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 one or more amino acid residues in length. In some embodiments, the peptide linker has at least one amino acid residue, but is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue or less in length. Exemplary linkers include any of the linkers described herein.

[0282] 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 a 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 containing two TACI polypeptides (e.g., two variant TACI polypeptides).

[0283] In some embodiments, a TACI-Fc fusion protein is provided containing, in order, a TACI polypeptide, e.g., 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 a truncated wild-type TACI polypeptide, e.g., 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 the linkers 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.

[0284] 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. TIFF2025134866000020.tif41160

[0285] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:172. TIFF2025134866000021.tif41160

[0286] In some embodiments, the TACI-Fc fusion protein has the sequence set forth in SEQ ID NO:196 and is encoded by the sequence set forth in SEQ ID NO:207.

[0287] 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, a variant TACI polypeptide, e.g., 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 a variant TACI polypeptide, e.g., 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 as set forth in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc as set forth in SEQ ID NO: 73.

[0288] In some embodiments, the TACI-Fc fusion protein has the amino acid sequence set forth in any one of SEQ ID NOs: 167-170, 200, or 222-237.

[0289] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:167. TIFF2025134866000022.tif41154

[0290] In some embodiments, the TACI-Fc fusion is encoded by the sequence shown in SEQ ID NO:211.

[0291] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:168. TIFF2025134866000023.tif41154

[0292] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:169. TIFF2025134866000024.tif41159

[0293] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:170. TIFF2025134866000025.tif41154

[0294] In some embodiments, a TACI-Fc fusion protein contains 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 contains 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, e.g., a peptide linker, including any of the peptide linkers described. In such examples, one of the TACI polypeptide sequences is connected 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 connected to the N-terminus or C-terminus of the Fc region. Linkage to the Fc region may be direct or indirect via a linker, e.g., a peptide linker, including any of the peptide linkers described.

[0295] In some embodiments, TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) may be arranged in tandem in a fusion protein (hereinafter referred to as a "tandem" Fc fusion construct). In some embodiments, a 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 consisting of two identical copies of a TACI-Fc fusion protein. For example, interaction between the Fc regions of two identical polypeptide fusions results in the formation of covalent disulfide bonds, resulting in a dimeric molecule containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0296] In some embodiments, a TACI-Fc fusion protein is provided, comprising, in order: a TACI polypeptide, e.g., any of the TACI polypeptides described above; a linker; another TACI polypeptide, e.g., any of the TACI polypeptides described; and an Fc region. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a truncated wild-type TACI polypeptide, e.g., 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 a variant TACI polypeptide, e.g., 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 set forth in any one of SEQ ID NOs:14-20, 23-35, 92-100, or 177-192. The linker can be any of the linkers described. In some embodiments, the linker is GSGGGGS (SEQ ID NO:74). The Fc region can be any of the Fc regions described. In some embodiments, the Fc region is 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.

[0297] In some embodiments, the TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) may be separated in the fusion protein by an Fc region, with the Fc region being positioned between the 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 can be the same or different. In some embodiments, the immunomodulatory protein is a tetravalent molecule that is a homodimer of two identical copies of a 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 containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0298] In some embodiments, a TACI-Fc fusion protein is provided that contains, in this order: a TACI polypeptide, e.g., any of the TACI polypeptides described above; a linker; an Fc region; a linker; and another TACI polypeptide, e.g., any of the TACI polypeptides described. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a truncated wild-type TACI polypeptide, e.g., 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 a variant TACI polypeptide, e.g., 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:2-2, 21, 22, or 101-120. In some embodiments, each TACI polypeptide of the TACI Fc fusion is a variant TACI set forth in any one of SEQ ID NOs:14-20, 23-35, 92-100, or 177-192. The linkers can be any of the described linkers and can be the same or different. In some embodiments, the first linker is GSGGGGS (SEQ ID NO:74) and the second linker is (GGGGS)4 (SEQ ID NO:84). The Fc region can be any of the described Fc regions. 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.

[0299] In some embodiments, a TACI-Fc fusion protein is provided that is a dimer formed by two identical TACI polypeptides (e.g., variant TACI polypeptides) as described above linked to an Fc domain. In some embodiments, any of the provided TACI-Fc fusion polypeptides of the same species (also called copies), e.g., variant TACI-Fc fusions, are dimerized to produce a homodimer. In some embodiments, the dimer is a homodimer in which the two TACI-Fc polypeptides, e.g., variant TACI-Fc polypeptides, are identical. When generating a homodimeric Fc molecule, the Fc region is an Fc region that can form a homodimer with the corresponding Fc region by co-expressing the individual Fc regions in a cell. In some embodiments, dimerization is mediated by a covalent disulfide bond formed between the Fc regions of the polypeptide fusion.

[0300] Nucleic acid molecules encoding immunomodulatory proteins are also provided. In some embodiments, when producing immunomodulatory proteins, the nucleic acid molecules encoding the immunomodulatory proteins are inserted into an appropriate expression vector. The resulting immunomodulatory proteins can be expressed in host cells transformed with the vector, and in the host cells, interchain disulfide bonds formed between the Fc portions cause assembly between the Fc domains to form dimers, e.g., bivalent immunomodulatory proteins.

[0301] Nucleic acid molecules encoding TACI-Fc fusion proteins, e.g., variant TACI-Fc fusion proteins, are also provided. In some embodiments, to produce Fc fusion proteins, nucleic acid molecules encoding TACI-Fc fusion proteins, e.g., variant TACI-Fc fusion proteins, are inserted into an appropriate expression vector. The resulting TACI-Fc fusion protein, e.g., variant TACI-Fc fusion protein, can be expressed in a host cell transformed with the vector, where interchain disulfide bonds formed between the Fc moieties cause assembly between the Fc domains to form a dimer, e.g., a bivalent TACI-Fc fusion protein. The resulting Fc fusion protein can be easily purified by affinity chromatography on a Protein A or Protein G column. When heterodimers are produced, additional purification steps may be required. For example, when two nucleic acids encoding different immunomodulatory proteins are introduced into cells by transformation, the immunomodulatory protein bearing the Fc domain is also expressed as a disulfide-linked homodimer, and the heterodimer must be formed biochemically. Therefore, homodimers can be reduced under conditions that favor the disruption of interchain disulfides but not intrachain disulfides. In some cases, different immunomodulatory protein monomers are mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of this mixture are separated by chromatographic methods. Alternatively, the formation of this type of heterodimer can be biased by genetically engineering and expressing immunomodulatory proteins containing Fc fusion molecules containing one or more TACI variants using the knob-into-hole method described.

[0302] In embodiments, when produced and expressed from a cell, the provided immunomodulatory proteins, such as TACI-Fc (e.g., variant TACI-Fc), are homodimers containing two identical polypeptide chains. Figures 8A and 8B illustrate the structure of exemplary TACI-Fc fusion proteins provided herein.

[0303] Provided herein is a TACI(26)-Fc_73 homodimer consisting of two identical variant TACI-Fc fusion proteins containing the TACI cysteine-rich domain 2 (CRD2) variant shown in SEQ ID NO: 26, which is designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI(26)-Fc_73 homodimer is a dimer consisting of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, each containing a variant TACI CRD2 domain human Fc fusion shown in SEQ ID NO: 167.

[0304] Provided herein is a TACI(26)-Fc_81 homodimer consisting of two identical variant TACI-Fc fusion proteins containing the TACI cysteine-rich domain 2 (CRD2) variant shown in SEQ ID NO: 26, which is designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI(26)-Fc_81 homodimer is a dimer consisting of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, each containing a variant TACI CRD2 domain human Fc fusion shown in SEQ ID NO: 168.

[0305] Provided herein is a TACI(27)-Fc_73 homodimer consisting of two identical variant TACI-Fc fusion proteins containing a variant of the TACI cysteine-rich domain 2 (CRD2) shown in SEQ ID NO: 27, designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI(27)-Fc_73 homodimer is a dimer consisting of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, each bearing a variant TACI CRD2 domain human Fc fusion shown in SEQ ID NO: 169.

[0306] Provided herein is a TACI(27)-Fc_81 homodimer consisting of two identical variant TACI-Fc fusion proteins containing a variant of the TACI cysteine-rich domain 2 (CRD2) shown in SEQ ID NO: 27, designed to neutralize the B cell stimulatory activity of APRIL and BAFF. The TACI(27)-Fc_81 homodimer is a dimer consisting of two identical receptor Fc fusion protein chains linked by a covalent disulfide bond, each bearing a variant TACI CRD2 domain human Fc fusion shown in SEQ ID NO: 170.

[0307] In some embodiments, the provided TACI-Fc (e.g., variant TACI-Fc) fusion proteins, e.g., homodimers thereof, have an IC of less than 400 pM in BAFF neutralization. 50 In some embodiments, the IC50 for BAFF neutralization is 1 pM to 400 pM, e.g., 10 pM to 300 pM, 10 pM to 200 pM, 10 pM to 100 pM, 10 pM to 50 pM, 10 pM to 20 pM, 20 pM to 400 pM, 20 pM to 300 pM, 20 pM to 200 pM, 20 pM to 100 pM , 20 pM to 50 pM, 50 pM to 400 pM, 50 pM to 300 pM, 50 pM to 200 pM, 50 pM to 100 pM, 100 pM to 400 pM, 100 pM to 300 pM, 100 pM to 200 pM, 200 pM to 400 pM, 200 pM to 300 pM, or 300 pM to 400 pM. 50 is 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, or 100 pM, or any value between any of the aforementioned values, or about 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, or 100 pM, or any value between any of the aforementioned values.

[0308] In some embodiments, the provided TACI-Fc (e.g., variant TACI-Fc) fusion proteins, e.g., homodimers thereof, have an IC of less than 400 pM in neutralizing APRIL. 50 In some embodiments, the IC50 for neutralizing APRIL is 0.5 pM to 100 pM, e.g., 0.5 pM to 50 pM, 0.5 pM to 25 pM, 0.5 pM to 10 pM, 0.5 pM to 5 pM, 0.5 pM to 1 pM, 1 pM to 100 pM, 1 pM to 50 pM, 1 pM to 25 pM, 1 pM to 10 pM, 1 pM to 5 pM, 5 pM to 100 pM, 5 pM to 50 pM, 5 pM to 25 pM, 5 pM to 10 pM, 10 pM to 100 pM, 10 pM to 50 pM, 10 pM to 25 pM, or 25 pM to 100 pM, 25 pM to 50 pM, or 50 pM to 100 pM. 50 is 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 20 pM, or 25 pM, or any value between any of the aforementioned values, or about 0.5 pM, 0.75 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 20 pM, or 25 pM, or any value between any of the aforementioned values.

[0309] III. Nucleic Acids, Vectors, and Methods for Producing Polypeptides or Cells Provided herein are isolated or recombinant nucleic acids, collectively referred to as "nucleic acids," that encode any of the immunomodulatory proteins provided herein. In some embodiments, including all of those described below, the nucleic acids provided herein are useful for the recombinant production (e.g., expression) of the immunomodulatory proteins provided herein. In some embodiments, including all of those described below, the nucleic acids provided herein are useful for the expression of the immunomodulatory proteins provided herein, such as the TACI fusion proteins provided herein. The nucleic acids provided herein can be in the form of RNA or DNA, including mRNA, cRNA, recombinant or synthetic RNA, and recombinant or synthetic DNA, as well as cDNA. The nucleic acids provided herein are typically DNA molecules, usually double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA / RNA nucleic acids, or combinations thereof, comprising any of the nucleotide sequences of the present invention are also provided.

[0310] In some cases, a heterologous (non-native) signal peptide can be added to a nucleic acid encoding an immunomodulatory protein. This may be desirable, for example, when expressing a TACI fusion protein that does not contain an amino-terminal signal sequence. In some embodiments, the signal peptide is derived from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (e.g., interleukin-2 (IL-2) or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide that allows for efficient expression, and in some aspects, secretion, of the protein from the cell. Exemplary signal peptides include any of those listed in Table 3.

[0311] Table 3. Exemplary signal peptides TIFF2025134866000026.tif119166

[0312] In some embodiments, the immunomodulatory protein includes a signal peptide when expressed, and the signal peptide (or portion thereof) is cleaved from the immunomodulatory protein upon secretion.

[0313] Also provided herein are recombinant expression vectors and recombinant host cells useful for producing immunomodulatory proteins, such as the TACI fusion proteins provided herein.

[0314] In any of the above-provided aspects, the nucleic acids encoding the immunomodulatory polypeptides provided herein can be introduced into cells using recombinant DNA and cloning techniques. To this end, recombinant DNA molecules encoding the immunomodulatory polypeptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the peptide-encoding sequence can be excised from DNA using a suitable restriction enzyme. Alternatively, the DNA molecule can be synthesized using chemical synthesis techniques, such as the phosphoramidite method. A combination of these techniques can also be used. In some examples, recombinant or synthetic nucleic acids can be generated by polymerase chain reaction (PCR). The DNA insert encoding the immunomodulatory protein can be cloned into an appropriate transduction / transfection vector, as known to those skilled in the art. Expression vectors containing the nucleic acid molecules are also provided.

[0315] In some embodiments, the expression vector is capable of expressing the immunomodulatory protein in a suitable cell under conditions suitable for expression of the protein. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding the immunomodulatory protein operably linked to an appropriate expression control sequence. Methods for achieving this functional linkage, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in regulating transcription or translation.

[0316] In some aspects, expression of the immunomodulatory protein is controlled by a promoter or enhancer to control or regulate expression. The promoter is operably linked to a portion of the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein.

[0317] The resulting recombinant expression vector carrying the DNA molecule thereon is used to transform a suitable host. This transformation can be carried out using methods well known in the art. In some embodiments, the nucleic acid provided herein further comprises a nucleotide sequence encoding a secretory or signal peptide operably linked to the nucleic acid encoding the immunomodulatory polypeptide, so that the resulting soluble immunomodulatory polypeptide can be recovered from the culture medium, the host cell, or the host cell periplasm. In other embodiments, an appropriate expression control signal is selected to enable membrane expression of the immunomodulatory polypeptide. In addition, commercially available kits and contract manufacturers can be used to produce the engineered cells or recombinant host cells provided herein.

[0318] In some embodiments, the resulting expression vector carrying the DNA molecule thereon is used to transform, e.g., transduce, suitable cells. Introduction can be performed using methods well known in the art. Exemplary methods include methods for transferring receptor-encoding nucleic acids, including via viruses, e.g., retroviruses or lentiviruses, transduction, transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred into cells by lentivirus or retrovirus transduction.

[0319] For the preparation of polypeptides or engineered cells, any of a number of publicly available and well-known mammalian host cells, including mammalian T cells or APCs, can be used. The selection of cells depends on a number of factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of peptide recovery, expression characteristics, biological safety, and cost. The balance of these factors must be negotiated with the understanding that not all cells may be equally effective in expressing a particular DNA sequence.

[0320] In some embodiments, the host cell is a mammalian cell. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61, CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), and SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and mouse embryonic cells (NIH-3T3; ATCC CRL 1658).

[0321] In some embodiments, host cells can be various eukaryotic cells (e.g., yeast cells) or mammalian cells such as Chinese hamster ovary (CHO) cells or HEK293 cells. In some embodiments, the host cells are suspension cells, and the polypeptide is engineered or produced in culture suspension, e.g., culture suspension CHO cells, e.g., CHO-S cells. In some examples, the cell line is a DHFR-deficient (DHFR-) CHO cell line, such as DG44 and DUXB11. In some embodiments, the cells are glutamine synthase (GS)-deficient, e.g., CHO-S cells, CHOK1 SV cells, and CHOZN((R))GS- / - cells. In some embodiments, the CHO cells, such as suspension CHO cells, can be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

[0322] In some embodiments, the host cell may be a prokaryotic cell (e.g., using E. coli). The transformed recombinant host is cultured under polypeptide expression conditions to obtain soluble protein, which is then purified. The recombinant host cell may be cultured under conventional fermentation conditions to express the desired polypeptide. Such fermentation conditions are well known in the art. Finally, the polypeptides provided herein may be recovered and purified from the recombinant cell culture by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. If desired, a protein refolding step can be used to complete the construction of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be used in a final purification step.

[0323] In some embodiments, the recombinant vector is a viral vector.Exemplary recombinant viral vectors include lentivirus vector genome, poxvirus vector genome, vaccinia virus vector genome, adenovirus vector genome, adenovirus-associated virus vector genome, herpesvirus vector genome and alphavirus vector genome.The viral vector can be live, attenuated, replication-conditional or replication-defective, non-pathogenic (defective), replication-competent viral vector, and / or modified to express heterologous gene products, for example, the variant immunomodulatory polypeptides provided herein.The vector for generating the virus can also be modified to change the attenuation of the virus, including any method that increases or decreases the transcriptional or translational load.

[0324] Exemplary viral vectors that can be used include modified vaccinia virus vectors (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartaglia et al., AIDS Research and Human Retroviruses 8:1445-47 (1992); Gheradi et al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14 (1975); Hu et al., J. Virol. 75:10300-308 (2001); U.S. Pat. Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615, and 7,368,116); adenovirus or adenovirus-associated virus vectors (see, e.g., Molin ... al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93 (2004); see U.S. Patent Nos. 6,143,290; 6,596,535; 6,855,317; 6,936,257; 7,125,717; 7,378,087; and 7,550,296; retroviral vectors (e.g., Buchscher, et al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93 (2004); see U.S. Patent Nos. 6,143,290; 6,596,535; 6,855,317; 6,936,257; 7,125,717; 7,378,087; and 7,550,296), including those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations (e.g., Buchscher, et al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936- et al.,J.Virol.66:2731-39(1992);Johann et al.,J.Virol.66:1635-40(1992);Sommerfelt et al.,Virology 176:58-59(1990);Wilson et al.,J.Virol.63:2374-78(1989);Miller et al. al., J. Virol. 65:2220-24 (1991); Miller et al., Mol. Cell Biol.10:4239 (1990); Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215 (1991); lentiviral vectors, including those based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV), and Maedi-Visna virus (see, e.g., Pfeifer et al., Annu. Rev. Genomics Hum. Genet. 2:177-211 (2001); Zufferey et al., J. Virol. 72:9873, 1998; Miyoshi et al., J. Virol. 72:8150, 1998; Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al. al., J. Virol. 69:2729, 1995; Nightingale et al., Mol. Therapy, 13:1121, 2006; Brown et al., J. Virol. 73:9011 (1999); WO 2009 / 076524; WO 2012 / 141984; WO 2016 / 011083; McWilliams et al., J. Virol. 77:11150, 2003; Powell et al., J. Virol. 70:5288, 1996), or any variants thereof, and / or vectors that can be used to generate any of the above viruses. In some embodiments, the recombinant vector can include a regulatory sequence, such as a promoter or enhancer sequence, that can regulate expression of the viral genome in the packaging cell line, for example, in the case of an RNA virus (see, e.g., U.S. Pat. Nos. 5,385,839 and 5,168,062).

[0325] In some aspects, the nucleic acid or expression vector contains a nucleic acid sequence encoding an immunomodulatory protein operably linked to an appropriate expression control sequence. Methods for performing this functional linkage, either before or after the nucleic acid sequence encoding the immunomodulatory protein is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation. A promoter can be operably linked to a portion of the nucleic acid sequence encoding the immunomodulatory protein.

[0326] Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TK promoter of herpes virus (McKnight, Cell 31:355 (1982)), the SV40 early promoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor virus promoter (see generally Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 101-112). 163-181 (John Wiley & Sons, Inc. 1996). One useful combination of promoter and enhancer is provided by the myeloproliferative sarcoma virus promoter and the human cytomegalovirus enhancer.

[0327] Alternatively, when a prokaryotic promoter is regulated by a eukaryotic promoter, for example, the bacteriophage T3 RNA polymerase promoter, can be used to control the production of immunomodulatory proteins in mammalian cells (Zhou et al., Mol Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

[0328] Expression vectors can be introduced into host cells using a variety of standard techniques, including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, etc. Transfected cells can be selected and grown to provide recombinant host cells containing the expression vector stably integrated into the host cell genome. Techniques for introducing vectors into eukaryotic cells and for selecting such stable transformants using dominant selectable markers are described, for example, by Ausubel (1995) and Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).

[0329] For example, one suitable selectable marker is a gene that confers resistance to the antibiotic neomycin. In this case, selection is performed in the presence of a neomycin-type drug, such as G-418. A selection system can also be used to increase the expression level of a gene of interest, a process called "amplification." Amplification is achieved by culturing transfectants in the presence of low levels of the selection agent and then increasing the amount of the selection agent to select for cells that produce high levels of the introduced gene's product. A suitable amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used. Alternatively, markers that introduce an altered phenotype, such as green fluorescent protein or cell surface proteins such as CD4, CD8, class I MHC, or placental alkaline phosphatase, can be used to select transfected cells from untransfected cells by means such as FACS sorting or magnetic bead separation techniques.

[0330] In some embodiments, the polypeptides provided herein can also be produced by synthetic methods.Solid-phase synthesis is the most cost-effective method for producing small peptides, and therefore is the preferred technique for producing individual peptides.For example, well-known solid-phase synthesis techniques include the use of protecting groups, linkers and solid-phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid-phase peptide synthesis.The peptides can then be assembled into the polypeptides provided herein.

[0331] IV. Pharmaceutical Compositions Provided herein is a composition that contains any of the immunomodulatory proteins described herein.Pharmaceutical compositions can further comprise pharmaceutically acceptable excipients.For example, pharmaceutical compositions can contain one or more excipients to modify, maintain or preserve the pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, absorption or penetration of the composition.Such compositions can include buffer solutions, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives.

[0332] In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition may be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.

[0333] In some embodiments, the pharmaceutical composition is an immunomodulatory protein dissolved in a liquid, e.g., an aqueous solution (e.g., physiological saline or Ringer's solution). In some embodiments, the pH of the pharmaceutical composition is about 4.0 to about 8.5 (e.g., about 4.0 to about 5.0, about 4.5 to about 5.5, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, about 6.5 to about 7.5, about 7.0 to about 8.0, or about 7.5 to about 8.5).

[0334] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, binder, coating, preservative, lubricant, flavoring agent, sweetener, colorant, solvent, buffer, chelating agent, or stabilizer. Examples of pharmaceutically acceptable fillers include cellulose, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Examples of pharmaceutically acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, or sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate or stearic acid.Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid or sorbic acid.Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame or sorbitol.Examples of pharmaceutically acceptable buffers include carbonate, citrate, gluconate, acetate, phosphate or tartrate.

[0335] In some embodiments, the pharmaceutical composition further comprises an agent for controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes.

[0336] In some embodiments, the pharmaceutical composition is sterile. Sterilization can be achieved by filtration through sterile filtration membranes or by irradiation. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions are generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0337] Pharmaceutically acceptable carrier can be pharmaceutically acceptable material, composition or vehicle.For example, carrier can be liquid or solid filler, diluent, excipient, solvent or encapsulating material, or any combination thereof.Each component of carrier must be " pharmaceutically acceptable " in that it must be compatible with other components of the formulation.It must also be suitable for contact with any tissue, organ or body part that it may encounter, which means that it must not have the risk of toxicity, irritation, allergic reaction, immunogenicity or any other complications that excessively outweigh its therapeutic benefits.

[0338] In some embodiments, the pharmaceutical composition is administered to a subject. Generally, the dosage and route of administration of the pharmaceutical composition are determined according to the size and condition of the subject, according to standard pharmaceutical practice. For example, a therapeutically effective dose can be first estimated using cell culture assays or animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine the dosage and route useful for human administration. The exact dosage is determined by taking into account factors related to the subject requiring treatment. Dosage and administration are adjusted to provide a sufficient level of the active compound or to maintain the desired effect. Factors that can be considered include the severity of the disease state, the subject's general health, the subject's age, weight, and sex, the time and frequency of administration, drug combinations, reaction sensitivities, and response to treatment.

[0339] Long-acting pharmaceutical compositions may be administered every 3-4 days, weekly, or once every two weeks, depending on the half-life and clearance rate of the particular formulation. The frequency of administration depends on the pharmacokinetic parameters of the molecule in the formulation used. Typically, the composition is administered until a dosage that achieves the desired effect is reached. Thus, the composition may be administered as a single dose, as multiple doses (at the same or different concentrations / dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely performed. The appropriate dosage may be confirmed through the use of appropriate dose-response data.

[0340] In some embodiments, the pharmaceutical composition is administered to a subject via any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, by direct application to a wound site, by application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by spray, intrapleurally, intracerebroventricularly, intraarticularly, intraocularly, or intraspinally.

[0341] The pharmaceutical formulations provided can be, for example, in a form suitable for intravenous infusion.

[0342] In some embodiments, the dosage of the pharmaceutical composition is a single dose or multiple doses. In some embodiments, the dose is administered to the subject once a day, twice a day, three times a day, or four or more times a day. In some embodiments, about one or more doses (such as about two or more, about three or more, about four or more, about five or more, about six or more, or about seven or more) are administered per week. In some embodiments, multiple doses are administered over the course of several days, weeks, months, or years. In some embodiments, a course of treatment is about one or more doses (such as about two or more doses, about three or more doses, about four or more doses, about five or more doses, about seven or more doses, about ten or more doses, about fifteen or more doses, about twenty-five or more doses, about forty or more doses, about fifty or more doses, or about one hundred or more doses).

[0343] In some embodiments, the administered dosage of the pharmaceutical composition is about 1 μg or more of protein per kg of subject body weight (such as about 2 μg or more of protein per kg of subject body weight, about 5 μg or more of protein per kg of subject body weight, about 10 μg or more of protein per kg of subject body weight, about 25 μg or more of protein per kg of subject body weight, about 50 μg or more of protein per kg of subject body weight, about 100 μg or more of protein per kg of subject body weight, 250 μg or more of protein per kg of subject body weight, about 500 μg or more of protein per kg of subject body weight, about 1 mg or more of protein per kg of subject body weight, about 2 mg or more of protein per kg of subject body weight, or about 5 mg or more of protein per kg of subject body weight).

[0344] V. Methods for Assessing the Activity and Immunomodulation of Immunomodulatory Proteins In some embodiments, the provided immunomodulatory proteins, e.g., the TACI fusion proteins provided herein, exhibit immunomodulatory activity. The provided immunomodulatory proteins, e.g., the TACI fusion proteins, can regulate one or more of B cell activities, e.g., B cell proliferation, differentiation, or survival.

[0345] The function of immunomodulatory proteins can be examined using various methods to evaluate the ability of proteins to bind to their cognate binding partners.For example, TACI fusion proteins can be evaluated for binding to APRIL or BAFF.Various assays are known for evaluating binding affinity and / or determining whether a binding molecule (e.g., immunomodulatory protein) specifically binds to a specific binding partner.For example, it is within the level of a person skilled in the art to determine the binding affinity of a binding molecule (e.g., immunomodulatory protein) to a binding partner (e.g., APRIL or BAFF) using any of a number of binding assays well known in the art.Various binding assays are known, including, but not limited to, ELISA K, including those described herein. D, KinExA, flow cytometry, and / or surface plasmon resonance instruments). Such methods include, but are not limited to, methods involving BIAcore®, Octet®, or flow cytometry. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and binding constant of a complex between two proteins using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents). SPR measures the change in concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, thereby allowing for measurement of the binding kinetics between the two molecules. The dissociation constant of the complex can be determined by monitoring the change in refractive index versus time as a buffer solution passes over the chip. Other suitable assays for measuring the binding of one protein to another include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or determining binding by monitoring changes in the spectroscopic or optical properties of the proteins via fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting expressed polynucleotides or protein binding.

[0346] The provided immunomodulatory proteins can also be evaluated in a variety of assays to assess modulation of B cell activity. One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound (e.g., an immunomodulatory protein), and cell proliferation is detected, for example, by measuring the incorporation of tritiated thymidine or by a colorimetric assay based on the metabolic degradation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65:55-63, 1983). An alternative assay format uses cells further engineered to express a reporter gene. The reporter gene is linked to a promoter element responsive to a receptor-linked pathway, and the assay detects activation of transcription of the reporter gene. Numerous reporter genes that can be easily assayed in cell extracts are known in the art, such as E. coli lacZ, chloramphenicol acetyltransferase (CAT) and serum response element (SRE) (see, for example, Shaw et al., Cell 56:563-72, 1989). An exemplary reporter gene is the luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). The expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:29094-101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase activity assay kits are commercially available, for example, from Promega Corp., Madison, Wis.

[0347] The provided immunomodulatory proteins can be characterized by their ability to inhibit stimulation of human B cells by soluble APRIL or soluble BAFF, as described in Gross et al., International Publication No. WO 00 / 40716. Briefly, human B cells are isolated from peripheral blood mononuclear cells, for example, using CD19 magnetic bead separation (e.g., Miltenyi Biotec, Auburn, CA). Purified B cells can be incubated under stimulatory conditions, for example, in the presence of soluble APRIL and increasing concentrations of the immunomodulatory protein. B cells can be labeled with a proliferation dye to measure proliferation, or 1 μCi of soluble APRIL can be used to detect B cell proliferation. 3 The number of B cells can be determined over time.

[0348] Transcription factors, e.g., NF- K Reporter cell lines expressing reporter genes under the functional control of B, NFAT-1, and AP-1 can be created to express TACI or BCMA. For example, reporter cells can include Jurkat cell lines and other B lymphoma cell lines. Incubation of these cells with soluble BAFF or APRIL ligands transmits signals through the reporter genes in these constructs. The effect of the provided immunomodulatory proteins to regulate this signal transduction can be evaluated.

[0349] Well-established animal models are available for testing the in vivo efficacy of provided immunomodulatory proteins in certain disease states, including those involving autoimmune or inflammatory conditions. For example, animal models of autoimmune disease include the MRL-lpr / lpr or NZB x NZW F1 congenic mouse strains, which serve as models of systemic lupus erythematosus (SLE). Such animal models are known in the art; see, for example, Autoimmune Disease Models: A Guidebook, Cohen and Miller, eds., Academic Press. The offspring of crosses between New Zealand Black (NZB) and New Zealand White (NZW) mice develop a spontaneous form of SLE that closely resembles human SLE. The offspring, known as NZBW mice, begin to develop IgM autoantibodies against T cells at 1 month of age, and by 5–7 months of age, Ig anti-DNA autoantibodies become the predominant immunoglobulin. Polyclonal B cell hyperactivity leads to the overproduction of autoantibodies. The deposition of these autoantibodies, particularly those against single-stranded DNA, is associated with the development of glomerulonephritis, which manifests clinically as proteinuria, azotemia, and death due to renal failure. Renal failure is the primary cause of death in mice with spontaneous SLE, and in the NZBW strain, this process is chronic and obstructive. The disease is more rapid and severe in females than in males, with a median survival time of only 245 days compared with 406 days in males. While most female mice become symptomatic (proteinuric) by 7–9 months of age, some may develop symptoms at a much younger or older age.The fatal immune nephritis seen in NZBW mice closely resembles the glomerulonephritis seen in human SLE, making this spontaneous mouse model extremely attractive for testing potential SLE therapeutics (Putterman and Naparstek, Murine Models of Spontaneous Systemic Lupus Erythematosus, Autoimmune Disease Models: A Guidebook, chapter 14, pp. 217-34, 1994; Mohan et al., J. Immunol. 154:1470-80, 1995; and Daikh et al., J. Immunol. 159:3104-08, 1997). Administration of provided immunomodulatory proteins to these mice can be evaluated to assess their effect on ameliorating symptoms and altering the course of the disease.

[0350] Another mouse model of inflammation and lupus-like disease is the bm12-inducible mouse model of SLE (Klarquist and Janssen, 2015. J. Vis. Exp. (105), e53319). bm12 B6(C)-H2-Ab1 bm12 Splenocyte suspensions from H2-Ab1 / KhEgJ ("bm12") mice are adoptively transferred into female C57BL / 6NJ recipient mice. bm12 is H2-Ab1 bThe gene differs by three nucleotides from the gene encoding bm12, resulting in three amino acid changes in the β-chain of the MHC class II IA molecule. Alloactivation of donor bm12 CD4+ T cells by recipient antigen-presenting cells results in chronic GVHD with symptoms similar to SLE, including autoantibody production, changes in immune cell subsets, and mild renal disease. Glomerulonephritis with immune complex deposition develops late in this model and is primarily composed of autoantigens bound to IgG1, IgG2b, IgG2c, and IgG3 antibodies. Endpoints in this model may include anti-dsDNA antibody concentrations, selected IgG isotypes, blood urea nitrogen (BUN), and serum creatinine, immune cell subset composition in the spleen and cervical LN, and renal histology.

[0351] In some embodiments, a mouse model of Sjögren's syndrome (SjS) can be used. Based on a modified version of the protocol published by Zhou et al., 2016 Sci. Rep. 6, 39105, repeated administration of anti-mouse (m)PD-L1 antibodies can be used to induce SjS disease and accelerate the onset of diabetes in female, diabetes-prone non-obese diabetic (NOD) mice. Starting at 6 weeks of age, mice are intraperitoneally (IP) injected with 100 μg of anti-PD-L1 antibody on study days 0, 2, 4, and 6, and treated with the provided immunomodulatory protein on various days. Naive mice are included as controls for endpoint analysis. All mice are typically sacrificed on study day 10, and the submandibular gland (SMG) and pancreas from each mouse are harvested for histopathological evaluation to assess the signs and severity of sialadenitis and insulitis. Blood glucose levels can be measured on various days.

[0352] In some embodiments, mouse models of experimental allergic encephalomyelitis (EAE) can be used. These models resemble human multiple sclerosis, resulting in demyelination as a result of T cell activation against neuronal proteins such as myelin basic protein (MBP) or proteolipid protein (PLP). Antigen inoculation leads to the induction of CD4+, class II MHC-restricted T cells (Th1). Modifications to EAE protocols can be used to generate acute, chronic relapsing, or passive transfer variants of the model (Weinberg et al., J. Immunol. 162:1818-26, 1999; Mijaba et al., Cell. Immunol. 186:94-102, 1999; and Glabinski, Meth. Enzym. 288:182-90, 1997). Administration of provided immunomodulatory proteins can be evaluated to improve symptoms and alter the course of the disease.

[0353] In some embodiments, collagen-induced arthritis (CIA) model can be used, in which mice develop chronic inflammatory arthritis similar to human rheumatoid arthritis (RA).CIA shares similar immunological and pathological characteristics with RA, making it an ideal model for screening potential human anti-inflammatory compounds.Another advantage of using the CIA model is that the pathogenesis is known. The T cell epitope and B cell epitope on type II collagen have been identified, and various immunological parameters (delayed type hypersensitivity and anti-collagen antibody) and inflammatory parameters (cytokines, chemokines and matrix degrading enzymes) related to immune-mediated arthritis have been determined and can be used to evaluate the effectiveness of test compounds in this model (Wooley, Curr.Opin.Rheum.3:407-20,1999; Williams et al., Immunol.89:9784-788,1992; Myers et al., Life Sci.61:1861-78,1997; and Wang et al., Immunol.92:8955-959,1995).The administration of provided immunomodulatory protein can be evaluated to improve symptoms and change the course of disease.

[0354] In some embodiments, a model of bronchial infection, such as asthma, can be created by injecting mice with ovalbumin and restimulating them intranasally with an antigen, which produces an asthmatic response in the bronchi, similar to asthma. Administration of the provided immunomodulatory proteins to ameliorate symptoms and alter the course of the disease can be evaluated.

[0355] In some embodiments, myasthenia gravis (MG) is another autoimmune disease for which a mouse model is available. MG is a disorder of neuromuscular transmission associated with the production of autoantibodies against nicotinic acetylcholine receptors (AChRs). MG can be acquired or hereditary and is associated with clinical features including abnormal weakness and fatigue during exercise. A mouse model of MG has been established. (Christadoss et al., Establishment of a Mouse Model of Myasthenia Gravis Which Mimics Human Myasthenia Gravis Pathogenesis for Immune Intervention, in Immunobiology of Proteins and Peptides VIII, Atassi and Bixler, eds., 1995, pp. 195-99.) Experimental autoimmune myasthenia gravis (EAMG) is an antibody-mediated disease characterized by the presence of antibodies against AChRs. These antibodies destroy the receptors, resulting in defective neuromuscular electrical impulses and muscle weakness. In the EAMG model, mice are immunized with nicotinic acetylcholine receptors. Clinical signs of MG become evident several weeks after the second immunization. EAMG can be assessed by several methods, including measuring serum levels of AChR antibodies by radioimmunoassay (Christadoss and Dauphinee, J. Immunol. 136:2437-40, 1986; and Lindstrom et al., Methods Enzymol. 74:432-60, 1981), measuring muscle AChRs, or electromyography (Wu et al., Protocols in Immunology. Vol. 3, Eds. Coligen, Kruisbeak, Margulies, Shevach, and Strober. John Wiley and Sons, New York, p. 158.1, 1997).

[0356] Another use of in vivo models involves delivering antigen challenge to animals, followed by administration of immunomodulatory proteins, and measuring T cell and B cell responses.T cell-dependent and T cell-independent immune responses can be measured as described in Perez-Melgosa et al., J.Immunol.163:1123-7,1999.To measure the effect on B cell responses, the immune response can be measured in animals that are subjected to periodic antigen challenge (e.g., keyhole limpet hemocyanin (KLH), sheep red blood cells (SRBC), ovalbumin, or collagen), and then administered the immunomodulatory proteins provided.

[0357] Pharmacokinetic studies can be used in conjunction with radiolabeled immunomodulating proteins to determine the distribution and half-life of such polypeptides in vivo.

[0358] VI. Therapeutic applications The pharmaceutical compositions described herein (including pharmaceutical compositions comprising the immunomodulatory proteins described herein) can be used for various therapeutic applications, such as disease treatment. For example, in some embodiments, the pharmaceutical compositions are used to treat inflammatory or autoimmune disorders, cancer, organ transplants, viral infections, and / or bacterial infections in mammals. The pharmaceutical compositions can regulate (e.g., reduce) immune responses to treat diseases.

[0359] Such methods and uses include, for example, therapeutic methods and uses involving the administration of a molecule or a composition containing the same to a subject having a disease, condition, or disorder. In some cases as described, the disease, condition, or disorder is an autoimmune or inflammatory disease or disorder. In some embodiments, the molecule or engineered cell is administered in an amount effective to treat the disease or disorder. Uses include the use of molecules including immunomodulatory proteins and their use in preparing medicaments for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering the provided immunomodulatory protein or a composition containing the same to a subject having or suspected of having a disease or condition. In some embodiments, the method thereby treats the disease or disorder or condition or disorder in the subject.

[0360] Exemplary subjects include mammalian subjects, e.g., livestock, farm animals, and human patients. In certain embodiments, the subject is a human subject.

[0361] The pharmaceutical compositions described herein can be used for various therapeutic applications, such as disease treatment. For example, in some embodiments, the pharmaceutical compositions are used to treat inflammatory or autoimmune disorders, organ transplants, viral infections and / or bacterial infections in mammals. The pharmaceutical compositions can regulate immune responses to treat diseases. In some embodiments, the pharmaceutical compositions suppress immune responses, which can be useful in the treatment of inflammatory or autoimmune disorders or organ transplants.

[0362] The provided methods are believed to be useful for a variety of applications, including, but not limited to, prophylactic or therapeutic methods for treating various immune system diseases or conditions in mammals in which regulating or modulating the immune system and immune system responses is beneficial. For example, suppressing the immune response can be beneficial in prophylactic and / or therapeutic methods for suppressing the recipient's rejection of a tissue, cell, or organ transplant from a donor. In therapeutic settings, the mammalian subject typically has an immune system disease or condition, and administration is performed to prevent the disease or condition from further progression.

[0363] The provided immunomodulatory proteins, including TACI fusion proteins, can be used to treat autoimmune diseases, B cell cancers, immunomodulation, EBD and any antibody-mediated pathology (e.g., ITCP, myasthenia gravis, etc.), renal disease, indirect T cell immune response, graft rejection, and graft-versus-host disease. Administration of the immunomodulatory proteins can specifically regulate B cell responses during immune responses. Furthermore, administration of the provided immunomodulatory proteins (e.g., TACI-Fc) can be used to regulate B cell development, other cell development, antibody production, and cytokine production. Administration or use of the provided immunomodulatory proteins can also regulate B cell proliferation, for example, by neutralizing the proliferative effects of BAFF or APRIL alone.

[0364] In some embodiments, the pharmaceutical composition suppresses the immune response, which may be useful in treating inflammatory or autoimmune disorders or organ transplantation. In some embodiments, the pharmaceutical composition contains an immunomodulatory protein that exhibits antagonist activity of a B cell stimulatory receptor, thereby reducing or diminishing the immune response.

[0365] In some embodiments, the composition can be used to treat autoimmune diseases. In some embodiments, administering a therapeutic composition containing an immunomodulatory protein provided herein to a subject suffering from an immune system disease (e.g., an autoimmune disease) can suppress or inhibit such immune system attack or its associated biological response. By suppressing this immune system attack on healthy body tissues, the resulting physical symptoms (e.g., pain, joint inflammation, joint swelling, or tenderness) caused by or associated with such attack on healthy tissues can be reduced or alleviated, and the biological and physical damage caused by or associated with the immune system attack can be reduced, delayed, or stopped. In preventive situations, the subject may have an immune system disease, disorder, or condition, be susceptible to an immune system disease, disorder, or condition, or be thought to exhibit an immune system disease, disorder, or condition, and administration is typically carried out to prevent the progression of the disease, disorder, or condition, inhibit or alleviate the associated symptoms, signs, or biological responses, prevent the physical damage potentially resulting therefrom, and / or maintain or improve the subject's physical function.

[0366] In some embodiments, the disease or condition that can be treated by the pharmaceutical compositions described herein is any disease mediated by immune complex deposition (e.g., lupus nephritis, vasculitis); direct interference with a pathway (e.g., catastrophic antiphospholipid syndrome, myasthenia gravis crisis; anti-Jo-1 disease); opsonization or direct damage to cells (e.g., idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia); antibody-mediated rejection of allografts (e.g., highly sensitized kidney transplant patients); or anti-drug antibodies against biological replacement factors, vectors (e.g., anti-Factor 8).

[0367] In some embodiments, inflammatory and autoimmune disorders, conditions, or diseases that can be treated by the pharmaceutical compositions described herein are systemic lupus erythematosus (SLE), including glucocorticoid-free flare prevention; Sjogren's syndrome; primary biliary cirrhosis (PBC); systemic sclerosis; polymyositis; diabetes prevention; IgA nephropathy; IgA vasculitis; B-cell cancers, such as myeloma; multiple sclerosis, or optic neuritis.

[0368] In some embodiments, the provided immunomodulatory proteins can be used to treat pre-B cell or B cell leukemias, such as plasma cell leukemia, chronic or acute lymphocytic leukemia, myelomas, such as multiple myeloma, plasma cell myeloma, endothelial myeloma, and giant cell myeloma, and lymphomas, such as non-Hodgkin's lymphoma. In some of any of the embodiments, the type of myeloma includes multiple myeloma, plasmacytoma, multiple solitary plasmacytoma, and / or extramedullary myeloma. In some of any of the embodiments, the type of myeloma includes light chain myeloma, non-secretory myeloma, and / or IgD or IgE myeloma.

[0369] In some embodiments, the provided immunomodulatory proteins can be used as immunosuppressants that selectively block the action of B lymphocytes for use in disease treatment. For example, certain autoimmune diseases are characterized by the production of autoantibodies that contribute to tissue destruction and disease progression. Autoantibodies can also lead to the development of immune complex deposition complications, resulting in many symptoms of systemic lupus erythematosus, including renal failure, neuralgia, and death. Regulating antibody production independently of cellular responses is also beneficial in many disease states. B cells have also been shown to play a role in the secretion of arthritis-inducing immunoglobulins in rheumatoid arthritis. Methods and uses of the provided immunomodulatory proteins to inhibit, block, or neutralize the action of B cells, thereby suppressing antibody production, are beneficial for the treatment of autoimmune diseases such as myasthenia gravis, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis.

[0370] In some embodiments, the provided immunomodulatory proteins can be used to block or neutralize the action of B cells associated with end-stage renal disease, which may or may not be associated with an autoimmune disease. Such methods are also useful for treating immunological renal diseases. Such methods are useful for treating glomerulonephritis associated with diseases such as membranous nephropathy, IgA nephropathy or Berger's disease, IgM nephropathy, IgA vasculitis, Goodpasture's disease, post-infectious glomerulonephritis, mesangial proliferative disease, chronic lymphocytic leukemia, and minimal change nephrotic syndrome. Such methods also serve as therapeutic applications for treating secondary glomerulonephritis or vasculitis associated with diseases such as lupus, polyarteritis nodosa, Henoch-Schönlein purpura, scleroderma, HTV-associated disease, amyloidosis, or hemolytic uremic syndrome. The provided method is also useful as part of therapeutic applications for treating chronic pyelonephritis, analgesic abuse, nephrocalcinosis, nephropathy caused by other drugs, nephrolithiasis, or interstitial nephritis or pyelonephritis associated with chronic or acute interstitial nephritis.The provided method also includes the use of the provided immunomodulatory protein in the treatment of hypertensive disease or macrovascular disease, including renal artery stenosis or occlusion, and cholesterol embolism or renal embolism.The provided method and use can also be used to treat renal or urinary neoplasms, multiple myeloma, lymphoma, light chain neuropathy or amyloidosis.

[0371] In some embodiments, the provided immunomodulating proteins can also be used to treat asthma and other chronic airway diseases, such as bronchitis and emphysema. The provided immunomodulating proteins can also be used to treat Sjogren's syndrome.

[0372] In some embodiments, the methods and uses of the provided immunomodulatory proteins include immunosuppression, particularly for therapeutic uses such as graft-versus-host disease and transplant rejection. In some embodiments, the methods and uses of the provided immunomodulatory proteins include the treatment of autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM) and Crohn's disease. The methods provided herein have additional therapeutic value for treating chronic inflammatory diseases, particularly for reducing joint pain, swelling, anemia, and other associated symptoms, as well as for treating septic shock.

[0373] In some embodiments, inflammatory and autoimmune disorders that may be treated by pharmaceutical compositions containing the immunomodulatory proteins described herein include, but are not limited to, achalasia; Addison's disease; adult Still's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome; autoimmune adrenalitis (Addison's disease); autoimmune angioedema; autoimmune autonomic neuropathy; autoimmune encephalomyelitis; autoimmune hepatitis; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune orchitis; autoimmune pancreatitis; autoimmune polyglandular syndrome type II (APS). II); autoimmune retinopathy; autoimmune thyroid disease (AITD), i.e., Hashimoto's disease; autoimmune urticaria; axonal and neurogenic neuropathy (AMAN); Barrow's disease; Behçet's disease; benign mucous membrane pemphigoid; bullous pemphigoid; Castleman's disease (CD); celiac disease; Chagas' disease; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA); cicatricial pemphigoid; Cogan's syndrome; cold agglutinin disease; congenital heart block; Coxsackie myocarditis; CREST syndrome; Crohn's disease; dermatitis herpetiformis; dermatomyositis; Devic's disease (neuromyelitis optica); lupus circularis (Discoid) lupus); Dressler syndrome; endometriosis; eosinophilic esophagitis (EoE); eosinophilic fasciitis; erythema nodosum; essential mixed cryoglobulinemia; Evans syndrome; fibromyalgia; fibrosing alveolitis; giant cell arteritis (temporal arteritis); giant cell myocarditis; glomerulonephritis; Goodpasture syndrome; granulomatosis with polyangiitis; Graves' disease; Guillain-Barré syndrome; Hashimoto's thyroiditis; hemolytic anemia; Henoch-Schönlein purpura (HSP) ); herpes gestationis or pemphigoid of gestationis (PG); hidradenitis suppurativa (HS) (anti-acne); hypogammaglobulinemia; IgA nephropathy; IgA vasculitis; IgG4-related sclerosing disease; immune thrombocytopenic purpura (ITP); inclusion body myositis (IBM); interstitial cystitis (IC); juvenile arthritis; juvenile diabetes mellitus (type 1 diabetes); juvenile myositis (JM); Kawasaki disease; Lambert-Eaton syndrome; leukocytoclastic vasculitis; lichen planus; lichen sclerosus;Lignin conjunctivitis; Linear IgA disease (LAD); Lupus; Chronic Lyme disease; Meniere's disease; Microscopic polyangiitis (MPA); Mixed connective tissue disease (MCTD); Mooren's ulcer; Much-Habermann disease; Multifocal motor neuropathy (MMN) or MMNCB; Multiple sclerosis; Myasthenia gravis; Myositis; Narcolepsy; Neonatal lupus; Neuromyelitis optica; Neutropenia; Ocular cicatricial pemphigoid; Optic neuritis; Relapsing rheumatoid arthritis (PR); PANDAS; Paraneoplastic cerebellar degeneration (PCD); Paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; Parsplanitis (peripheral uveitis); Parsonage-Turner syndrome; Pemphigus, pemphigus vulgaris; Peripheral neuropathy; Perivenous encephalomyelitis; Pernicious anemia (PA); POEMS syndrome; Polyarteritis nodosa; Polyglandular syndrome types I, II, and III; Polymyalgia rheumatica; Polymyositis; Post-myocardial infarction syndrome ;Postpericardiotomy syndrome;Primary biliary cirrhosis;Primary sclerosing cholangitis;Progestational dermatitis;Psoriasis;Psoriatic arthritis;Pure red cell aplasia (PRCA);Pyoderma gangrenosum;Raynaud's phenomenon;Reactive arthritis;Reflex sympathetic dystrophy;Relapsing polychondritis;Restless legs syndrome (RLS);Retroperitoneal fibrosis;Rheumatic fever;Rheumatoid arthritis;Sarcoidosis;Schmidt's syndrome;Scleritis;Scleroderma;Sjogren's syndrome;Sperm and These include: uterine and testicular autoimmunity; stiff-body syndrome (SPS); subacute bacterial endocarditis (SBE); Susac syndrome; sympathetic ophthalmia (SO); Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS); transverse myelitis; type 1 diabetes; ulcerative colitis (UC); undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vitiligo or Vogt-Koyanagi-Harada syndrome;

[0374] In some embodiments, the provided immunomodulatory proteins (e.g., TACI-Fc) can be used to treat scleroderma, myasthenia gravis, GVHD (including acute GVHD or chronic GVHD), immune responses associated with transplantation; antiphospholipid syndrome; multiple sclerosis; Sjogren's syndrome; IgG4-related disease; type I diabetes; rheumatoid arthritis, including glucocorticoid therapy (GC) RA, or acute lupus nephritis.

[0375] In some embodiments, the provided immunomodulatory proteins (e.g., TACI-Fc) can be used to treat amyotrophic lateral sclerosis, neuromyelitis optica, transverse myelitis, CNS autoimmunity, Guillain-Barré syndrome, neurocysticercosis, sarcoidosis (T / seroneg), Churg-Strauss syndrome, Hashimoto's thyroiditis, Graves' disease, immune thrombocytopenia (ITP), Addison's disease, polymyositis, or dermatomyositis.

[0376] In some embodiments, the provided immunomodulatory proteins (e.g., TACI-Fc) can be used to treat IgA nephropathy, chronic inflammatory demyelinating polyneuropathy (CIDP), antisynthetase diseases such as Jo-1 syndrome, or ANCA vasculitis.

[0377] In some embodiments, the provided immunomodulatory proteins (e.g., TACI-Fc) can be used to treat B-cell cancer. In some embodiments, the B-cell cancer is a cancer in which BAFF and APRIL are involved or involved in providing an autocrine survival loop to B cells. In some embodiments, the cancer is B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, or myeloma. In some embodiments, the cancer is myeloma.

[0378] In some embodiments, a therapeutic amount of pharmaceutical composition is administered.Typically, the exact amount of the composition of the present invention to be administered can be determined by a doctor, taking into account the age, weight, degree of infection and individual differences in condition of the patient (subject).The optimal dosage and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0379] The subject compositions can be administered in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In one embodiment, the therapeutic composition is administered to patients by intradermal or subcutaneous injection. In another embodiment, the therapeutic composition is administered by iv injection.

[0380] In some embodiments, the pharmaceutical composition is administered as monotherapy (i.e., as a single agent) or as combination therapy (i.e., in combination with one or more additional immunosuppressive agents). In some embodiments, the additional agent is a glucocorticoid (e.g., prednisone, dexamethasone, and hydrocortisone), a cytostatic agent, e.g., a cytostatic agent that affects T cell and / or B cell proliferation (e.g., a purine analog, an alkylating agent, or an antimetabolite), an antibody (e.g., an anti-CD20 monoclonal antibody, an anti-CD25 monoclonal antibody, or an anti-CD3 monoclonal antibody), cyclosporine, tacrolimus, sirolimus, everolimus, interferon, an opioid, a TNF-binding protein, mycophenolic acid, a small molecule biological agent, e.g., fingolimod or myriocin, a cytokine, e.g., interferon beta-1a, an integrin agonist, or an integrin antagonist.

[0381] VII. ARTICLES OF MANUFACTURE AND KITS Also provided herein is an article of manufacture that comprises the pharmaceutical composition described herein in suitable packaging.Suitable packaging for the compositions (such as ophthalmic compositions) described herein is known in the art, and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (such as sealed Mylar or plastic bags), etc.These articles of manufacture can also be sterilized and / or sealed.

[0382] Kits containing the pharmaceutical compositions (or articles of manufacture) described herein are further provided, and may further include instructions for using the compositions, such as the uses described herein. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein.

[0383] VIII. Illustrative Embodiments Among the aspects provided are the following: 1. An immunomodulatory protein comprising at least one TACI polypeptide, which is a truncated wild-type TACI extracellular domain or a variant thereof, a truncated wild-type TACI extracellular domain containing cysteine-rich domain 2 (CRD2) but lacking the entire cysteine-rich domain 1 (CRD1); the variant TACI polypeptide comprises one or more amino acid substitutions in a truncated wild-type TACI extracellular domain; Immunomodulatory protein. 2. An immunomodulatory protein comprising at least one TACI polypeptide, which is a truncated wild-type TACI extracellular domain or a variant thereof, the truncated wild-type TACI extracellular domain comprises the contiguous sequence contained within amino acid residues 67-118, which comprises amino acid residues 71-104, based on the positions set forth in SEQ ID NO: 122; the variant TACI polypeptide comprises one or more amino acid substitutions in a truncated wild-type TACI extracellular domain; Immunomodulatory protein. 3. The immunomodulatory protein of embodiment 1 or embodiment 2, wherein the truncated wild-type TACI extracellular domain is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51 amino acids in length. 4. The immunomodulatory protein of any of aspects 1-3, wherein the truncated wild-type TACI extracellular domain consists of amino acid residues 68-110 as set forth in SEQ ID NO:122. 5. The immunomodulatory protein of any of aspects 1 to 4, wherein the TACI polypeptide consists of the amino acid sequence set forth in SEQ ID NO:13; or a variant thereof containing one or more amino acid substitutions in the sequence set forth in SEQ ID NO:13. 6. An immunomodulatory protein comprising at least one TACI polypeptide that is a truncated TACI polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:13, or a variant thereof containing one or more amino acid substitutions in the sequence set forth in SEQ ID NO:13. 7. The immunomodulatory protein of any of embodiments 1-5, wherein the truncated TACI polypeptide or variant thereof binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. 8. The immunomodulatory protein of any of aspects 1-7, wherein the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence set forth in SEQ ID NO:1. 9. The immunomodulatory protein of any of aspects 1-7, wherein the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence set forth in SEQ ID NO:13. 10. An immunomodulatory protein comprising a truncated TACI polypeptide consisting of the sequence set forth in SEQ ID NO:13. 11. The immunomodulatory protein of any of aspects 1-7, wherein the TACI polypeptide is a variant TACI polypeptide, and the variant TACI polypeptide has increased binding affinity for one or both of APRIL and BAFF compared to the truncated TACI polypeptide. 12. The immunomodulatory protein of any of embodiments 1-7 and 11, wherein the variant TACI polypeptide comprises one or more amino acid substitutions at positions selected from among 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the numbering set forth in SEQ ID NO:122. 13. One or more amino acid substitutions are TIFF2025134866000027.tif19163, or a conservative amino acid substitution thereof. 14. The immunomodulatory protein of embodiment 12 or embodiment 13, wherein the one or more amino acid substitutions comprise at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. 15. One or more amino acid substitutions are 14. The immunomodulatory protein of any of embodiments 12 to 13, wherein the immunomodulatory protein is TIFF2025134866000028.tif26132. 16. The immunomodulatory protein of any of aspects 12-15, wherein the one or more amino acid substitutions are K77E / F78Y / Y102D. 17. The immunomodulatory protein of any of aspects 12-15, wherein the one or more amino acid substitutions are Q75E / R84Q. 18. The immunomodulatory protein of any of aspects 12-16, wherein the variant TACI polypeptide is set forth in SEQ ID NO:26. 19. The immunomodulatory protein of any of embodiments 12-15 and 17, wherein the variant TACI polypeptide is set forth in SEQ ID NO:27. 20. The immunomodulatory protein of embodiment 1, wherein the TACI polypeptide is a variant TACI polypeptide comprising one or more amino acid substitutions at positions selected from among 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 in the extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof, corresponding to the numbering of the positions set forth in SEQ ID NO:122. 21. An immunomodulatory protein comprising at least one variant TACI polypeptide, the at least one variant TACI polypeptide comprises one or more amino acid substitutions in the extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof at positions selected from among 40, 59, 60, 61, 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the numbering of positions set forth in SEQ ID NO: 122; Immunomodulatory protein. 22. The immunomodulatory protein of embodiment 20 or embodiment 21, wherein the reference TACI polypeptide is a truncated polypeptide consisting of the extracellular domain of TACI that binds to APRIL, BAFF, or the BAFF / APRIL heterotrimer, or a specific binding portion thereof. 23. The immunomodulatory protein of any of embodiments 20-22, wherein the reference TACI polypeptide comprises: (i) the amino acid sequence set forth in SEQ ID NO:122; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:122; or (iii) a portion of (i) or (ii), including one or both of the CRD1 and CRD2 domains, that bind to APRIL, BAFF, or the BAFF / APRIL heterotrimer. 24. The immunomodulatory protein of any of embodiments 20-23, wherein the reference TACI polypeptide lacks an N-terminal methionine. 25. The immunomodulatory protein of any of embodiments 20-24, wherein the reference TACI polypeptide comprises a CRD1 domain and a CRD2 domain. 26. The immunomodulatory protein of any of embodiments 20-25, wherein the reference TACI polypeptide comprises the sequence set forth in SEQ ID NO:1. 27. The immunomodulatory protein of any of embodiments 20-25, wherein the reference TACI polypeptide consists of the sequence set forth in SEQ ID NO:1. 28. The immunomodulatory protein of any of embodiments 20-24, wherein the reference TACI polypeptide consists essentially of the CRD2 domain. 29. The immunomodulatory protein of any of embodiments 20-24 and 28, wherein the reference TACI polypeptide comprises the sequence set forth in SEQ ID NO:13. 30. The immunomodulatory protein of any of embodiments 20-24 and 28, wherein the reference TACI polypeptide consists of the sequence set forth in SEQ ID NO:13. 31. One or more amino acid substitutions are: TIFF2025134866000029.tif25160, or a conservative amino acid substitution thereof. 32. The immunomodulatory protein of any of aspects 20-31, wherein the one or more amino acid substitutions comprise at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. 33. The immunomodulatory protein of any of aspects 20-32, wherein the one or more amino acid substitutions include at least the amino acid substitution K77E. 34. The immunomodulatory protein of any of aspects 20-32, wherein the one or more amino acid substitutions include at least the amino acid substitution R84G. 35. The immunomodulatory protein of any of aspects 20-32, wherein the one or more amino acid substitutions include at least the amino acid substitution R84Q. 36. One or more amino acid substitutions are The immunomodulatory protein of any of embodiments 20 to 35, wherein the immunomodulatory protein is TIFF2025134866000030.tif33164. 37. The immunomodulatory protein of any of aspects 20-32, 33, and 36, wherein the one or more amino acid substitutions are K77E / F78Y / Y102D. 38. The immunomodulatory protein of any of aspects 20-32, 35, and 36, wherein the one or more amino acid substitutions are Q75E / R84Q. 39. The immunomodulatory protein of any of embodiments 20-38, wherein the variant TACI polypeptide has increased binding affinity for one or both of APRIL and BAFF compared to the reference TACI polypeptide. 40. The immunomodulatory protein of embodiment 11 or embodiment 39, wherein the variant TACI polypeptide has increased binding affinity for APRIL. 41. The immunomodulatory protein of embodiment 11 or embodiment 39, wherein the variant TACI polypeptide has increased binding affinity for ...

Claims

1. (i) a variant TACI polypeptide comprising the extracellular domain (ECD) of TACI or a portion of the ECD of TACI, comprising the amino acid substitutions K77E, F78Y, and Y102D, corresponding to the numbering of the positions set forth in SEQ ID NO:122; and (ii) Fc region 1. A TACI-Fc fusion protein comprising: the variant TACI polypeptide has an increased binding affinity for one or both of APRIL and BAFF compared to a TACI reference polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13; and the variant TACI polypeptide has at least 90% sequence identity to SEQ ID NO: 13 or at least 95% sequence identity to SEQ ID NO: 1; The TACI-Fc fusion protein. (a) the variant TACI polypeptide comprises cysteine-rich domain 1 (CRD1) and cysteine-rich domain 2 (CRD2) of the ECD of TACI; (b) the variant TACI polypeptide comprises CRD2 of the ECD of TACI but lacks the entire CRD1; or (c) the variant TACI polypeptide comprises amino acid residues 68 to 110 set forth in SEQ ID NO:122; The TACI-Fc fusion protein.

3. A TACI-Fc fusion protein as described in claim 1 or 2, wherein the variant TACI polypeptide comprises the sequence shown in SEQ ID NO:

26.

4. A TACI-Fc fusion protein as described in claim 1 or 2, wherein the variant TACI polypeptide comprises the sequence shown in SEQ ID NO:

111.

5. A TACI-Fc fusion protein described in any one of claims 1 to 3, wherein the variant TACI polypeptide is linked to the Fc region via a linker.

6. The TACI-Fc fusion protein of claim 5, wherein the linker comprises a peptide linker selected from GSGGS (SEQ ID NO: 76), GGGGS (G4S; SEQ ID NO: 77), GSGGGGS (SEQ ID NO: 74), GGGGSGGGGS (2xGGGGS; SEQ ID NO: 78), GGGSGGGGSGGGGGS (3xGGGGS; SEQ ID NO: 79), GGGSGGGGSGGGGSGGGGS (4xGGGGS, SEQ ID NO: 84), GGGSGGGGSGGGGSGGGGSGGGGS (5xGGGGS, SEQ ID NO: 91), GGGGSSA (SEQ ID NO: 80), or GSGGGGSGGGGS (SEQ ID NO: 194), or a combination thereof.

7. A TACI-Fc fusion protein described in claim 5 or 6, wherein the linker is GSGGGGS (SEQ ID NO: 74).

8. A TACI-Fc fusion protein described in any one of claims 1 to 7, wherein the Fc region is a human IgG1 Fc domain, a variant Fc domain of human IgG1 immunoglobulin with reduced effector function, an IgG2 Fc domain, an IgG4 Fc domain, or a variant Fc domain of IgG4 containing the S228P mutation according to EU numbering.

9. The TACI-Fc fusion protein of claim 8, wherein the Fc region is a variant Fc domain of human IgG1 immunoglobulin having reduced effector function.

10. The TACI-Fc fusion protein of claim 8, wherein the Fc region is a variant IgG1 Fc domain containing one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to EU numbering.

11. The TACI-Fc fusion protein of claim 10, wherein the Fc region comprises amino acid substitutions of L234A, L235E, and G237A according to EU numbering.

12. A TACI-Fc fusion protein described in any one of claims 1 to 8, wherein the Fc region comprises an amino acid sequence shown in any of SEQ ID NO:73, 75, 83, 136, or 221.

13. The TACI-Fc fusion protein described in claim 12, wherein the Fc region comprises the amino acid sequence shown in SEQ ID NO:

73.

14. A TACI-Fc fusion protein described in any one of claims 1 to 13, wherein the TACI-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO:

167.

15. A TACI-Fc fusion protein described in any one of claims 1 to 14, which is a homodimer of two identical polypeptides, each of which is a variant TACI polypeptide and a polypeptide comprising an Fc region.

16. The TACI-Fc fusion protein of claim 15, wherein the homodimer is linked by a covalent disulfide bond.

17. A nucleic acid molecule encoding the TACI-Fc fusion protein of any one of claims 1 to 14.

18. A vector comprising the nucleic acid molecule of claim 17.

19. 20. The vector of claim 18, which is an expression vector.

20. 20. A cell comprising the nucleic acid of claim 17 or the vector of claim 18 or claim 19.

21. 1. A method for producing a TACI-Fc fusion protein, comprising: introducing into a host cell the nucleic acid molecule of claim 17 or the vector of claim 18 or claim 19 under conditions that allow expression of the TACI-Fc fusion protein in the host cell; and Optionally further isolating or purifying the TACI-Fc fusion protein from said cells. The method comprising:

22. A pharmaceutical composition comprising the TACI-Fc fusion protein of any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

23. 23. An article of manufacture comprising the pharmaceutical composition of claim 22 in a vial.

24. 24. A kit comprising the pharmaceutical composition of claim 22 or the article of manufacture of claim 23 and instructions for use.

25. 23. The pharmaceutical composition of claim 22 for use in reducing an immune response in a subject.

26. 23. The pharmaceutical composition of claim 22 for use in treating a disease, disorder, or condition in a subject.

27. 27. The pharmaceutical composition of claim 26, wherein the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, kidney disease, transplant rejection, graft-versus-host disease, a viral infection, glomerulonephritis, renal ANCA vasculitis, immune thrombocytopenia, cold agglutinin disease, bullous pemphigoid, or myasthenia gravis.

28. The pharmaceutical composition of claim 27, wherein the disease, disorder, or condition is glomerulonephritis, and the glomerulonephritis is associated with membranous nephropathy, IgA nephropathy, IgM nephropathy, IgA vasculitis, Goodpasture's disease, post-infectious glomerulonephritis, mesangial proliferative disease, chronic lymphocytic leukemia, or minimal change nephrotic syndrome.

29. A pharmaceutical composition described in claim 26 or claim 27, wherein the disease, disorder, or condition is systemic lupus erythematosus (SLE), Sjogren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), autoimmune polyglandular syndrome type II (APSII), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, or pemphigus vulgaris.

30. 23. Use of the pharmaceutical composition of claim 22 in the manufacture of a medicament for reducing an immune response in a subject.

31. 23. Use of the pharmaceutical composition of claim 22 in the manufacture of a medicament for treating a disease, disorder, or condition in a subject.

32. 32. The use of claim 31, wherein the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, kidney disease, transplant rejection, graft-versus-host disease, a viral infection, glomerulonephritis, renal ANCA vasculitis, immune thrombocytopenia, cold agglutinin disease, bullous pemphigoid, or myasthenia gravis.

33. The use of claim 32, wherein the disease, disorder, or condition is glomerulonephritis, and the glomerulonephritis is associated with membranous nephropathy, IgA nephropathy, IgM nephropathy, IgA vasculitis, Goodpasture's disease, post-infectious glomerulonephritis, mesangial proliferative disease, chronic lymphocytic leukemia, or minimal change nephrotic syndrome.

34. The use of claim 31 or 32, wherein the disease, disorder, or condition is systemic lupus erythematosus (SLE), Sjogren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), autoimmune polyglandular syndrome type II (APSII), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, or pemphigus vulgaris.

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