APRIL and BAFF inhibitory immunomodulatory proteins, and methods for using the same.

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 of related applications This application incorporates, by reference, the entire contents of each of the following U.S. Provisional Applications: U.S. Provisional Application No. 63 / 022,373, filed on 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 on June 3, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF"; and September 18, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND We claim priority from U.S. Provisional Application No. 63 / 080,643, titled "METHODS OF USE THEREOF".

[0002] Inclusion by referencing sequence listings This application is filed together with an electronic sequence listing. The sequence listing is provided as a 278,660-byte file titled 761612003840SeqList.TXT, created on May 4, 2021. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] field This disclosure provides immunomodulatory proteins that exhibit neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers). The immunomodulatory proteins contain variant domains of transmembrane activators and CAML interactors (TACI). Among the immunomodulatory proteins provided are TACI-Fc fusion proteins. This disclosure also provides nucleic acid molecules encoding immunomodulatory proteins. Immunomodulatory proteins offer therapeutic utility for various immunological diseases, disorders, or conditions. Compositions and methods for producing and using such proteins are provided. [Background technology]

[0004] background There is 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 those targeting a single cell surface molecule or a soluble receptor (e.g., Fc-CTLA-4). There is a need for improved therapeutic agents that can modulate immune responses, particularly B-cell immune responses. Embodiments that meet such needs are provided. [Overview of the Initiative]

[0005] overview The immunomodulatory protein provided herein comprises at least one TACI polypeptide, which is either a cleaved wild-type TACI extracellular domain or a variant thereof, wherein the cleaved wild-type TACI extracellular domain contains cysteine-rich domain 2 (CRD2) but lacks the entirety of cysteine-rich domain 1 (CRD1), and the variant TACI polypeptide contains one or more amino acid substitutions within the cleaved wild-type TACI extracellular domain.

[0006] The immunomodulatory protein provided herein contains at least one TACI polypeptide, which is either a cleaved wild-type TACI extracellular domain or a variant thereof, wherein the cleaved wild-type TACI extracellular domain consists of a continuous sequence contained within amino acid residues 67-118, comprising amino acid residues 71-104 relative to the position shown in SEQ ID NO:122, and the variant TACI polypeptide contains one or more amino acid substitutions within the cleaved wild-type TACI extracellular domain. In some of the embodiments, the cleaved 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 acid lengths. In some of the embodiments, the cleaved wild-type TACI extracellular domain consists of amino acid residues 68-110, as shown in SEQ ID NO:122. In some of the various embodiments, the TACI polypeptide consists of the amino acid sequence shown in SEQ ID NO:13; or is a variant thereof containing one or more amino acid substitutions in the sequence shown in SEQ ID NO:13.

[0007] An immunomodulatory protein is provided herein that contains a cleaved TACI polypeptide comprising the amino acid sequence shown in SEQ ID NO:13, or at least one TACI polypeptide which is a variant thereof containing one or more amino acid substitutions in the sequence shown in SEQ ID NO:13. In some of the embodiments, the cleaved TACI polypeptide or its variant binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. In some of the embodiments, the TACI polypeptide is a cleaved wild-type TACI extracellular domain comprising the sequence shown in SEQ ID NO:1. In some of the embodiments, the TACI polypeptide is a cleaved wild-type TACI extracellular domain comprising the sequence shown in SEQ ID NO:13.

[0008] An immunomodulatory protein containing a cleaved TACI polypeptide consisting of the sequence shown in SEQ ID NO:13 is provided herein. In some of the embodiments, the TACI polypeptide is a variant TACI polypeptide, which has increased binding affinity to one or both of APRIL and BAFF compared to the cleaved TACI polypeptide. In some of the embodiments, the variant TACI polypeptide contains one or more amino acid substitutions at positions selected from 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 shown in SEQ ID NO:122.

[0009] In some of the various embodiments, one or more amino acid substitutions are TIFF0007866668000001.tif19157, or selected from its conserved amino acid substitutions. In some of the arbitrary embodiments, 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 arbitrary embodiments, one or more amino acid substitutions are The sequence is TIFF0007866668000002.tif26132. In some embodiments, one or more amino acid substitutions are K77E / F78Y / Y102D. In some embodiments, one or more amino acid substitutions are Q75E / R84Q. In some embodiments, the variant TACI polypeptide is shown as SEQ ID NO:26. In some embodiments, the variant TACI polypeptide is shown as SEQ ID NO:27.

[0010] In some of the various embodiments, the TACI polypeptide is a variant TACI polypeptide that contains one or more amino acid substitutions at positions selected from 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 position numbering shown in SEQ ID NO:122, in the extracellular domain (ECD) or specific binding fragment of the reference TACI polypeptide.

[0011] The immunomodulatory protein comprising at least one variant TACI polypeptide is provided herein, wherein at least one variant TACI polypeptide comprises one or more amino acid substitutions at positions selected from 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 position numbering shown in SEQ ID NO:122, in the extracellular domain (ECD) or specific binding fragment of the reference TACI polypeptide.

[0012] An immunomodulatory protein is provided herein, which is a variant TACI Fc fusion protein comprising a variant TACI polypeptide, an Fc region, and a linker between the TACI polypeptide and the Fc region, wherein the variant TACI polypeptide comprises one or more amino acid substitutions at positions selected from 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) or specific binding fragment of a reference TACI polypeptide, corresponding to the position numbering shown in SEQ ID NO:122.

[0013] In some of the various embodiments, the reference TACI polypeptide is a cleavable polypeptide comprising a TACI extracellular domain or a specific binding site of the TACI extracellular domain that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer.

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

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

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

[0017] In some of the various embodiments, the reference TACI polypeptide contains the sequence shown in SEQ ID NO:1. In some of the various embodiments, the reference TACI polypeptide consists of the sequence shown in SEQ ID NO:1.

[0018] In some of the various embodiments, the reference TACI polypeptide is essentially derived from the CRD2 domain.

[0019] In some of the various embodiments, the reference TACI polypeptide contains the sequence shown in SEQ ID NO:13. In some of the various embodiments, the reference TACI polypeptide consists of the sequence shown in SEQ ID NO:13.

[0020] In some of the various embodiments, one or more amino acid substitutions are TIFF0007866668000003.tif19163, or selected from its conserved amino acid substitutions.

[0021] In some of the various embodiments, 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 various embodiments, one or more amino acid substitutions include amino acid substitutions selected from the group consisting of Q75E, K77E, F78Y, R84G, R84Q, A101D, and Y102D, or any combination thereof.

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

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

[0025] In some of the various embodiments, one or more amino acid substitutions are The file is TIFF0007866668000004.tif33146.

[0026] In some of the various embodiments, one or more amino acid substitutions are The filename is TIFF0007866668000005.tif19158.

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

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

[0029] In some of the various embodiments, 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 various embodiments, the variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:122; or has its specific binding fragment containing the CRD1 domain and / or the CRD2 domain. In some embodiments, the variant TACI polypeptide has at least 95% sequence identity with SEQ ID NO:122; or has its specific binding fragment containing the CRD1 domain and / or the CRD2 domain. In some embodiments, the specific binding fragment is indicated by SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131.

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

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

[0034] In some of the arbitrary embodiments, the increased binding affinity to BAFF or APRIL is independently greater than approximately 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, or 60 times.

[0035] In some of the various embodiments, the variant TACI polypeptide contains the sequence shown in one of SEQ ID NO: 2-12, 21, 22, or 101-120, or the variant TACI polypeptide contains the sequence shown in one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192.

[0036] In some of the various embodiments, the variant TACI polypeptide consists of, or is essentially derived from, the sequence shown in any one of SEQ ID NO: 2-12, 21, 22, or 101-120, or the variant TACI polypeptide consists of, or is essentially derived from, the sequence shown in any one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192.

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

[0038] In some of the various embodiments, the linker includes a peptide linker, and the peptide linker is Selected from TIFF0007866668000006.tif33164 or a combination thereof.

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

[0040] In some embodiments, the immunomodulatory proteins provided herein do not contain TACI polypeptides linked to another target-directed moiety that binds to a molecule on the cell surface. In some embodiments, the immunomodulatory proteins provided herein do not contain TACI polypeptides linked to a target-directed moiety that is a binding partner of a T cell stimulating receptor or a ligand of a T cell stimulating receptor. In some embodiments, the immunomodulatory proteins provided herein do not contain TACI polypeptides linked to a target-directed 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 contain TACI polypeptides linked to the CTLA-4 polypeptide or the extracellular domain or binding moiety of CTLA-4, or variants thereof. For example, in the aspects provided herein, the immunomodulatory proteins provided herein do not contain TACI polypeptides linked to the wild-type CTLA-4 polypeptide or its extracellular domain or binding moiety. In the context provided, the immunomodulatory proteins provided herein do not include variant CTLA-4 polypeptides such as variant CTLA-4 or its binding moiety, or TACI polypeptides linked to its extracellular domain or binding moiety, such as variant CTLA-4 containing one or more amino acid modifications (e.g., substitutions) in the extracellular domain of CTLA-4 to enhance binding affinity to one or more cognitive binding partners.

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

[0042] In some of the various embodiments, the Fc fusion protein of TACI-Fc is dimerized. In some of the various embodiments, the immunoglobulin Fc region is homodimerized Fc region.

[0043] In some of the embodiments, immunoglobulin Fc is an IgG1 Fc domain; or optionally, a variant Fc exhibiting reduced binding affinity and / or reduced effector function to the Fc receptor compared to a wild-type IgG1 Fc domain. In some of the embodiments, immunoglobulin Fc is shown in SEQ ID NO:71. In some embodiments, immunoglobulin Fc is an IgG1 Fc domain, and Fc contains the amino acid sequence shown in SEQ ID NO:81. In some of the embodiments, 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 by EU numbering. In some of the 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 contains amino acid substitutions L234A, L235E, and G237A, according to EU numbering. In some embodiments, the Fc region is indicated by SEQ ID NO: 73, 75, 83, 136, or 221. In some embodiments, the immunoglobulin Fc region further contains amino acid substitutions A330S and P331S. In some embodiments, the immunoglobulin Fc region is indicated by SEQ ID NO: 175 or SEQ ID NO: 176.

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

[0045] In some of the arbitrary 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 within the wild-type Fc domain in order to form a heterodimer between polypeptides. In some of the arbitrary embodiments, the wild-type immunoglobulin Fc is the IgG1 Fc domain. In some of the arbitrary embodiments, one or more amino acid modifications are selected from knob-into-hole modifications and charge mutations that reduce or prevent self-association by charge repulsion.

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

[0047] In some of the various embodiments, the TACI-Fc fusion protein has the following structure: TACI polypeptide (TACI)-linker-Fc region This includes. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:168. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:170. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:167. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:169. In some embodiments, the immunomodulatory protein is a homodimer containing two identical copies of the TACI-Fc fusion protein.

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

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

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

[0051] This specification provides an immunomodulatory TACI-Fc fusion protein that is a homodimer containing 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 various embodiments, the TACI-Fc fusion protein has the following structure: (TACI)-linker-Fc region-linker-(TACI) This includes. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:201. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:202. In some embodiments, the immunomodulatory protein is a homodimer containing two identical copies of the TACI-Fc fusion protein.

[0053] In some of the various embodiments, the TACI-Fc fusion protein has the following structure: (TACI)-linker-(TACI)-linker-Fc region This includes the following. In some embodiments, the TACI-Fc fusion protein is shown as SEQ ID NO:198. In some embodiments, the immunomodulatory protein is a homodimer containing two identical copies of the TACI-Fc fusion protein.

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

[0055] In some of the various embodiments, immunomodulatory proteins (e.g., Fc fusion proteins) reduce or inhibit the maturation, differentiation, and proliferation of B cells.

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

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

[0059] Cells containing nucleic acids or vectors of any of the embodiments described herein are provided herein. In some of the embodiments, the cells are mammalian cells. In some of the embodiments, the cells are human cells.

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

[0061] An immunomodulatory protein (e.g., an Fc fusion protein) produced by any of the methods described herein is provided herein.

[0062] A pharmaceutical composition containing an immunomodulatory protein (e.g., an Fc fusion protein) in any of the embodiments described herein is provided 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] A manufactured article containing a pharmaceutical composition in any embodiment described herein, in a vial or container, is provided herein. In some of the embodiments, the vial or container is sealed.

[0064] A kit comprising a pharmaceutical composition of any embodiment provided herein and instructions for use is provided herein. In some of the arbitrary embodiments, the kit comprises a manufactured article of any embodiment described herein and instructions for use.

[0065] A method for reducing an immune response in a subject, comprising the step of administering an immunomodulatory protein of any form described herein to a subject in need thereof, is provided herein.

[0066] A method for reducing an immune response in a subject, comprising the step of administering an Fc fusion protein of any form described herein to a subject in need thereof, is provided herein.

[0067] A method for reducing the immune response in a subject, comprising the step of administering a pharmaceutical composition of any embodiment described herein to a subject requiring such reduction. In some of the embodiments, the B-cell immune response is reduced in the subject, thereby reducing or inhibiting the maturation, differentiation, and / or proliferation of B cells. In some of the embodiments, the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer are reduced in a control.

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

[0069] A method for treating a disease, disorder, or condition in a subject, comprising the step of administering an immunomodulatory protein of any form described herein to a subject in need thereof, is provided herein.

[0070] A method for treating a disease, disorder, or condition in a subject, comprising the step of administering an Fc fusion protein of any embodiment described herein to a subject in need thereof, is provided herein.

[0071] A method for treating a disease, disorder, or condition in a subject, comprising the step of administering a pharmaceutical composition of any embodiment described herein to a subject in need thereof, is provided herein. In some of the arbitrary embodiments, the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell carcinoma, an antibody-mediated condition, a kidney disease, a graft rejection, a graft-versus-host disease, or a viral infection. In some of the arbitrary embodiments, the disease, disorder, or condition is selected from the group consisting of systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, 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 the various aspects, the disease, disorder, or condition is B-cell carcinoma, and the cancer is myeloma.

[0072] Pharmaceutical compositions for use in reducing the immune response in subjects are also provided herein.

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

[0074] In some embodiments of the pharmaceutical compositions for use or uses provided herein, the immune response is a B-cell immune response, and by reducing the immune response, the maturation, differentiation, and / or proliferation of B cells are reduced or inhibited. In some embodiments, by reducing the immune response, the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimers in the subject are reduced. In some embodiments, by reducing the immune response, a disease, disorder, or condition in the subject is treated.

[0075] Pharmaceutical compositions for use in the treatment of diseases, disorders, or conditions in the subject are also provided herein.

[0076] The use of any of the provided immunomodulatory proteins or pharmaceutical compositions in the manufacture of medical agents for treating diseases, disorders, or conditions in subjects is also provided herein.

[0077] In some aspects of any embodiment of the pharmaceutical compositions for use or use 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 graft 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 mellitus, 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 various embodiments, the types of myeloma include multiple myeloma, plasmacytoma, multiple plasmacytoma, and / or extramedullary myeloma. In some of the various embodiments, the types of myeloma include light chain myeloma, nonsecretory myeloma, and / or IgD myeloma or IgE myeloma. [Invention 1001] An immunomodulatory protein comprising at least one variant TACI polypeptide, The at least one variant TACI polypeptide contains one or more amino acid substitutions at positions selected from 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 the reference TACI polypeptide, corresponding to the position numbering shown in SEQ ID NO:122. Immunomodulatory proteins. [Invention 1002] An immunomodulatory protein comprising a variant TACI-Fc fusion protein comprising a variant TACI polypeptide, an Fc region, and a linker between the TACI polypeptide and the Fc region, The variant TACI polypeptide contains one or more amino acid substitutions at positions selected from 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 the reference TACI polypeptide, corresponding to the position numbering shown in SEQ ID NO:122. Immunomodulatory proteins. [Invention 1003] An immunomodulatory protein according to Invention 1001 or Invention 1002, wherein the reference TACI polypeptide is a cleaved polypeptide consisting of a TACI extracellular domain or its specific binding site that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. [Invention 1004] An immunomodulatory protein according to any of Invention 1001 to 1003, wherein the reference TACI polypeptide comprises the amino acid sequence shown in SEQ ID NO:122, or a portion thereof that includes one or both of the CRD1 domain and the CRD2 domain that bind to APRIL, BAFF, or BAFF / APRIL heterotrimer. [Invention 1005] The reference TACI polypeptide is an immunomodulatory protein according to any of the present invention 1001 to 1004, lacking N-terminal methionine. [Invention 1006] An immunomodulatory protein according to any of invention 1001 to 1005, wherein the reference TACI polypeptide comprises a CRD1 domain and a CRD2 domain. [Invention 1007] An immunomodulatory protein according to any of the present invention 1001 to 1006, wherein the reference TACI polypeptide contains the sequence shown in SEQ ID NO:1. [Invention 1008] An immunomodulatory protein according to any of the present invention 1001 to 1006, wherein the reference TACI polypeptide consists of the sequence shown in SEQ ID NO:1. [Invention 1009] The reference TACI polypeptide is a cleaved wild-type TACI extracellular domain that contains cysteine-rich domain 2 (CRD2) but lacks the entirety of cysteine-rich domain 1 (CRD1). The variant TACI polypeptide contains one or more amino acid substitutions in the cleaved wild-type TACI extracellular domain. An immunomodulatory protein according to any of invention 1001 to 1005. [Invention 1010] The reference TACI polypeptide is a cleaved wild-type TACI extracellular domain consisting of a continuous sequence contained within amino acid residues 67-118, including amino acid residues 71-104, relative to the position indicated by SEQ ID NO:122. The variant TACI polypeptide contains one or more amino acid substitutions in the cleaved wild-type TACI extracellular domain. An immunomodulatory protein according to any of the present invention 1001-1005 and 1009. [Invention 1011] An immunomodulatory protein of Invention 1009 or Invention 1010, wherein the reference TACI polypeptide is a cleaved wild-type TACI extracellular domain having an amino acid length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51 amino acids. [Invention 1012] An immunomodulatory protein according to any of invention 1001-1005 and 1009-1011, wherein the reference TACI polypeptide essentially consists of a CRD2 domain. [Invention 1013] An immunomodulatory protein according to any of the present invention 1001-1005 and 1009-1012, wherein the reference TACI polypeptide is a cleaved wild-type TACI extracellular domain consisting of amino acid residues 68-110 as shown in SEQ ID NO:122. [Invention 1014] An immunomodulatory protein according to any of the present invention 1001-1005 and 1009-1013, wherein the reference TACI polypeptide contains the sequence shown in SEQ ID NO:13. [Invention 1015] An immunomodulatory protein according to any of the present invention 1001-1005 and 1009-1013, wherein the reference TACI polypeptide consists of the sequence shown in SEQ ID NO:13. [Invention 1016] One or more amino acid substitutions TIFF0007866668000007.tif19161 or any of the immunomodulatory proteins of the present invention 1001 to 1015, selected from the conservative amino acid substitutions thereof. [Invention 1017] An immunomodulatory protein according to any of invention 1001 to 1016, wherein one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. [Invention 1018] An immunomodulatory protein according to any of the invention 1001 to 1016, wherein one or more amino acid substitutions are selected from the group consisting of Q75E, K77E, F78Y, R84G, R84Q, A101D, and Y102D, or any combination thereof. [Invention 1019] An immunomodulatory protein according to any of inventions 1001 to 1016 and 1018, wherein one or more amino acid substitutions include at least amino acid substitution Q75E. [Invention 1020] An immunomodulatory protein according to any of the inventions 1001 to 1019, wherein one or more amino acid substitutions include at least amino acid substitution K77E. [Invention 1021] An immunomodulatory protein according to any of invention 1001-1016 and 1018-1020, wherein one or more amino acid substitutions include at least one amino acid substitution F78Y. [Invention 1022] An immunomodulatory protein according to any of invention 1001-1017 and 1019-1021, wherein one or more amino acid substitutions include at least one amino acid substitution R84G. [Invention 1023] An immunomodulatory protein according to any of the invention 1001 to 1021, wherein one or more amino acid substitutions include at least the amino acid substitution R84Q. [Invention 1024] An immunomodulatory protein according to any of invention 1001-1016 and 1018-1023, wherein one or more amino acid substitutions include at least amino acid substitution A101D. [Invention 1025] One or more amino acid substitutions TIFF0007866668000008.tif19145 An immunomodulatory protein according to any of the invention 1001 to 1023, including the above. [Invention 1026] One or more amino acid substitutions TIFF0007866668000009.tif33160 An immunomodulatory protein according to any of the present invention 1001 to 1025. [Invention 1027] One or more amino acid substitutions TIFF0007866668000010.tif19156 An immunomodulatory protein according to any of the present invention 1001 to 1025. [Invention 1028] An immunomodulatory protein according to any of the present invention 1001-1018, 1020, 1021, and 1025-1027, wherein one or more amino acid substitutions are K77E / F78Y / Y102D. [Invention 1029] An immunomodulatory protein according to any of the invention 1001-1019, 1023, and 1025-1027, wherein one or more amino acid substitutions are Q75E / R84Q. [Invention 1030] An immunomodulatory protein according to any of the present invention 1001-1018, 1020, and 1024-1027, wherein one or more amino acid substitutions are K77E / A101D / Y102D. [Invention 1031] An immunomodulatory protein according to any of the Invention 1001-1030, wherein the variant TACI polypeptide has increased binding affinity to one or both APRIL and BAFF compared to the reference TACI polypeptide. [Invention 1032] An immunomodulatory protein according to any of the invention 1001 to 1031, wherein the variant TACI polypeptide has increased binding affinity to APRIL. [Invention 1033] An immunomodulatory protein according to any of the invention 1001 to 1032, wherein the variant TACI polypeptide has increased binding affinity to BAFF. [Invention 1034] An immunomodulatory protein according to any of the present invention 1001 to 1033, wherein the variant TACI polypeptide has increased binding affinity to APRIL and BAFF. [Invention 1035] An immunomodulatory protein of any of the present invention 1030-1034, wherein the increased binding affinity to BAFF or APRIL is independently greater than 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, or 60 times. [Invention 1036] An immunomodulatory protein according to any of invention 1030 to 1035, wherein the variant TACI polypeptide has up to 10 amino acid modifications compared to the reference TACI polypeptide. [Invention 1037] An immunomodulatory protein according to any of invention 1030 to 1035, wherein the variant TACI polypeptide has up to 5 amino acid modifications compared to the reference TACI polypeptide. [Invention 1038] An immunomodulatory protein according to any of the Invention 1001-1037, wherein the variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:122; or has a specific binding fragment thereof containing the CRD1 domain and / or the CRD2 domain. [Invention 1039] An immunomodulatory protein according to any of the Invention 1001-1037, wherein the variant TACI polypeptide has at least 95% sequence identity to SEQ ID NO:122; or has a specific binding fragment thereof containing the CRD1 domain and / or the CRD2 domain. [Invention 1040] An immunomodulatory protein of Invention 1038 or Invention 1039, wherein the specific binding fragment is indicated by SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131. [Invention 1041] An immunomodulatory protein according to any of the present invention 1001 to 1040, wherein the variant TACI polypeptide has at least 90% sequence identity with respect to SEQ ID NO:13. [Invention 1042] An immunomodulatory protein according to any of the present invention 1001 to 1040, wherein the variant TACI polypeptide has at least 95% sequence identity with respect to SEQ ID NO:13. [Invention 1043] The variant TACI polypeptide contains the sequence shown in one of the following SEQ ID NO: 2-12, 21, 22, and 101-120, or The variant TACI polypeptide contains one of the sequences shown in SEQ ID NO: 14-20, 23-35, 92-100, and 177-192. An immunomodulatory protein according to any of invention 1001 to 1042. [Invention 1044] The variant TACI polypeptide consists of or is essentially derived from one of the sequences shown in SEQ ID NO: 2-12, 21, 22, and 101-120, or The variant TACI polypeptide consists of or is essentially derived from one of the sequences shown in SEQ ID NO: 14-20, 23-35, 92-100, and 177-192. An immunomodulatory protein according to any of invention 1001 to 1043. [Invention 1045] The variant TACI polypeptide is an immunomodulatory protein of any of the present invention 1001 to 1044, as shown in SEQ ID NO:26. [Invention 1046] The variant TACI polypeptide is an immunomodulatory protein of any of the present invention 1001-1044, as shown in SEQ ID NO:27. [Invention 1047] At least one TACI polypeptide, which is a cleaved wild-type TACI extracellular domain containing cysteine-rich domain 2 (CRD2) but lacking the entirety of cysteine-rich domain 1 (CRD1). Immunomodulatory proteins, including those mentioned above. [Invention 1048] An immunomodulatory protein comprising at least one TACI polypeptide which is a cleaved wild-type TACI extracellular domain, The cleaved wild-type TACI extracellular domain consists of a continuous sequence of amino acid residues 67-118, with reference to the position indicated by SEQ ID NO:122, comprising amino acid residues 71-104. Immunomodulatory proteins. [Invention 1049] An immunomodulatory method comprising a TACI Fc fusion protein containing a cleaved wild-type TACI extracellular domain, an Fc region, and a linker between the TACI polypeptide and the Fc region, The cleaved wild-type TACI extracellular domain contains cysteine-rich domain 2 (CRD2) but lacks the entirety of cysteine-rich domain 1 (CRD1). immunomodulation. [Invention 1050] An immunomodulatory method comprising a TACI Fc fusion protein containing a cleaved wild-type TACI extracellular domain, an Fc region, and a linker between the TACI polypeptide and the Fc region, The cleaved wild-type TACI extracellular domain consists of a continuous sequence of amino acid residues 67-118, with reference to the position indicated by SEQ ID NO:122, comprising amino acid residues 71-104. immunomodulation. [Invention 1051] An immunomodulatory protein according to any of the Invention 1047-1050, wherein the cleaved wild-type TACI extracellular domain has an amino acid length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51. [Invention 1052] An immunomodulatory protein according to any of the present invention 1047-1051, wherein the cleaved wild-type TACI extracellular domain consists of amino acid residues 68-110 as shown in SEQ ID NO:122. [Invention 1053] An immunomodulatory protein according to any of the present invention 1047-1051, wherein the cleaved wild-type TACI extracellular domain consists of the amino acid sequence shown in SEQ ID NO:13. [Invention 1054] An immunomodulatory protein containing at least one TACI polypeptide, which is a cleaved TACI polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13. [Invention 1055] An immunomodulatory protein according to any of the present invention 1047-1054, wherein a cleaved TACI polypeptide is bound to APRIL, BAFF, or a BAFF / APRIL heterotrimer. [Invention 1056] An immunomodulatory protein according to any of the present invention 1001, 1003-1048, and 1051-1055, comprising a heterologous moiety optionally linked via a linker to at least one TACI polypeptide. [Invention 1057] The immunomodulatory protein of the present invention 1056, wherein the heterogeneous portion is a half-life extension portion, a multimerization domain, a target-directed portion that binds to molecules on the cell surface, or a detectable label. [Invention 1058] The immunomodulatory protein of Invention 1057, wherein the half-life extension portion comprises a multimerizing domain, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the β-subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, or a combination thereof. [Invention 1059] It is a TACI Fc fusion protein, At least one TACI polypeptide is optionally linked to the Fc region of the immunoglobulin via a linker. An immunomodulatory protein according to any of the present invention 1001, 1003-1048, and 1051-1058. [Invention 1060] The linker contains a peptide linker, and the peptide linker is TIFF0007866668000011.tif41164 Alternatively, an immunomodulatory protein selected from any combination thereof, one of the immunomodulatory proteins 1002, 1049, 1050, and 1056-1059 of the present invention. [Invention 1061] Immunoglobulin Fc, It is an IgG1 Fc domain; or, Optionally, a variant Fc that exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the wild-type IgG1 Fc domain. An immunomodulatory protein according to any of the present invention 1002, 1049, 1050, 1059, and 1060. [Invention 1062] An immunomodulatory protein according to any of the present inventions 1002, 1049, 1050, and 1059-1061, wherein the immunoglobulin Fc is an IgG1 Fc domain, and the Fc contains the amino acid sequence shown in SEQ ID NO:81. [Invention 1063] An immunomodulatory protein according to any of the Invention 1002, 1049, 1050, and 1059-1061, wherein 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. [Invention 1064] The immunomodulatory protein of the present invention 1063, wherein the immunoglobulin Fc region comprises the amino acid substitutions L234A, L235E, and G237A according to EU numbering, and optionally the Fc region is indicated by SEQ ID NO: 73, 75, 83, 136, or 221. [Invention 1065] An immunomodulatory protein of the present invention 1063 or 1064, wherein the immunoglobulin Fc region further comprises amino acid substitutions A330S and P331S, and optionally, the Fc region is indicated by SEQ ID NO:175 or SEQ ID NO:176. [Invention 1066] TACI-Fc fusion protein, structure: TACI polypeptide (TACI)-linker-Fc region An immunomodulatory protein comprising any of the present invention 1002, 1049, 1050, and 1059-1065. [Invention 1067] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present inventions 1002, 1049, 1050, 1059-1062, and 1066, as shown in SEQ ID NO:168. [Invention 1068] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present inventions 1002, 1049, 1050, 1059-1062, and 1066, as shown in SEQ ID NO:170. [Invention 1069] An immunomodulatory protein according to any of the present invention 1002, 1049, 1050, 1059-1061, and 1063-1066, wherein the Fc is a variant Fc containing the amino acid sequence shown in SEQ ID NO:73. [Invention 1070] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present invention 1002, 1049, 1050, 1059-1061, 1063-1066, and 1069, as shown in SEQ ID NO:167. [Invention 1071] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present invention 1002, 1049, 1050, 1059-1061, 1063-1066, and 1069, as shown in SEQ ID NO:169. [Invention 1072] An immunomodulatory protein according to any of the present inventions 1002, 1049, 1050, and 1059-1071, which is a homodimer containing two identical copies of a TACI-Fc fusion protein. [Invention 1073] An immunomodulatory protein that is a homodimer containing two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:167, linked by a covalent disulfide bond. [Invention 1074] An immunomodulatory protein that is a homodimer containing two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:168, linked by a covalent disulfide bond. [Invention 1075] An immunomodulatory protein that is a homodimer containing two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:169, linked by a covalent disulfide bond. [Invention 1076] An immunomodulatory protein that is a homodimer containing two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:170, linked by a covalent disulfide bond. [Invention 1077] TACI-Fc fusion protein, structure: (TACI)-linker-Fc region-linker-(TACI) An immunomodulatory protein comprising any of the present invention 1002, 1049, 1050, and 1059-1065. [Invention 1078] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present inventions 1002, 1049, 1050, 1059-1065, and 1077, as shown in SEQ ID NO:201. [Invention 1079] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present inventions 1002, 1049, 1050, 1059-1065, and 1077, as shown in SEQ ID NO:202. [Invention 1080] TACI-Fc fusion protein, structure: (TACI)-linker-(TACI)-linker-Fc region An immunomodulatory protein comprising any of the present invention 1002, 1049, 1050, and 1059-1065. [Invention 1081] The TACI-Fc fusion protein is an immunomodulatory protein of any of the present invention 1002, 1049, 1050, 1059-1065, and 1080, as shown in SEQ ID NO:198. [Invention 1082] An immunomodulatory protein according to any of the present invention 1002, 1049, 1050, 1059-1065, and 1077-1081, which is a homodimer containing two identical copies of a TACI-Fc fusion protein. [Invention 1083] An immunomodulatory protein according to any of the present inventions 1002, 1049, 1050, and 1059-1082, wherein the Fc fusion protein neutralizes APRIL and BAFF. [Invention 1084] 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. An immunomodulatory protein according to any of the present invention 1002, 1049, 1050, and 1059-1083. [Invention 1085] The Fc fusion protein blocks the binding of APRIL, BAFF, or the APRIL / BAFF heterotrimer to BCMA or TACI, and / or The Fc fusion protein reduces the levels of circulating APRIL, BAFF, or APRIL / BAFF in the blood after administration to the target. An immunomodulatory protein according to any of invention 1001 to 1084. [Invention 1086] An immunomodulatory protein according to any of the Invention 1001-1085, wherein the immunomodulatory protein reduces or inhibits the maturation, differentiation, and / or proliferation of B cells. [Invention 1087] A nucleic acid molecule encoding any of the immunomodulatory proteins described in invention 1001 to 1086. [Invention 1088] A vector containing the nucleic acid molecule of the present invention 1087. [Invention 1089] The expression vector, vector 1088 of the present invention. [Invention 1090] A vector according to Invention 1088 or Invention 1089, which is a mammalian expression vector or a viral vector. [Invention 1091] A cell containing the nucleic acid of Invention 1087 or a vector of any of Invention 1088-1090. [Invention 1092] A mammalian cell, according to the present invention 1091. [Invention 1093] Human cells, according to Invention 1091 or Invention 1092. [Invention 1094] A method for producing immunomodulatory proteins, A step of introducing a nucleic acid molecule of Invention 1087 or a vector of Invention 1088 to 1090 into a host cell under conditions that cause the protein to be expressed in the cell. Methods that include... [Invention 1095] The method of the present invention 1094, further comprising the step of isolating or purifying immunomodulatory proteins from the cells. [Invention 1096] An immunomodulatory protein produced by the method of Invention 1094 or Invention 1095. [Invention 1097] A pharmaceutical composition comprising an immunomodulatory protein according to any of Invention 1001-1086 or Invention 1096. [Invention 1098] A pharmaceutical composition according to the present invention 1097, comprising pharmaceutically acceptable excipients. [Invention 1099] A manufactured article comprising a vial or container containing a pharmaceutical composition of Invention 1097 or Invention 1098. [Invention 1100] A kit comprising a pharmaceutical composition of Invention 1097 or Invention 1098 or a manufactured article of Invention 1099, and instructions for use. [Invention 1101] A method for reducing an immune response in a subject, comprising the step of administering to a subject in need of such reduction an immunomodulatory protein according to any of Invention 1001 to 1086 or a pharmaceutical composition according to Invention 1097 or Invention 1098. [Invention 1102] The method of the present invention 1101, wherein the B cell immune response is reduced in a subject, thereby reducing or inhibiting the maturation, differentiation, and / or proliferation of B cells. [Invention 1103] A method according to Invention 1101 or Invention 1102, wherein the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer are reduced in the subject. [Invention 1104] A method according to any one of the present invention 1101 to 1103, wherein a disease, disorder, or condition in a subject is treated by reducing the immune response. [Invention 1105] A method for reducing the circulation levels of APRIL, BAFF, or APRIL / BAFF heterotrimers in a target, A step of administering an immunomodulatory protein according to any of Invention 1001 to 1086 or a pharmaceutical composition according to Invention 1097 or Invention 1098 to a target. Methods that include... [Invention 1106] A method for treating a disease, disorder, or condition in a subject, comprising the step of administering to a subject in need of such treatment an immunomodulatory protein of any of Invention 1001 to 1086 or a pharmaceutical composition of Invention 1097 or Invention 1098. [Invention 1107] The method of Invention 1104 or Invention 1106, wherein the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, B-cell cancer, an antibody-mediated condition, kidney disease, graft rejection, graft-versus-host disease, or a viral infection. [Invention 1108] The method of Invention 1106 or Invention 1107, wherein the disease, disorder, or condition is selected from the group consisting of systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, 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. [Invention 1109] The method of Invention 1106 or Invention 1107, wherein the disease, disorder, or condition is B-cell carcinoma, and the cancer is myeloma. [Invention 1110] A pharmaceutical composition according to Invention 1097 or Invention 1098 for use in reducing the immune response in a subject. [Invention 1111] The use of any immunomodulatory protein according to Invention 1001 to 1086, or the use of a pharmaceutical composition according to Invention 1097 or Invention 1098, in the manufacture of a medical agent for reducing the immune response in a target. [Invention 1112] A pharmaceutical composition for use in the present invention 1110, or for use in the present invention 1111, wherein the immune response is a B-cell immune response, and by reducing the immune response, the maturation, differentiation, and / or proliferation of B cells is reduced or inhibited. [Invention 1113] A pharmaceutical composition for use, or use, according to any of Invention 1110 to 1112, which reduces the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer in a subject by reducing the immune response. [Invention 1114] A pharmaceutical composition for use, or use, according to any of the inventions 1110 to 1113, wherein a disease, disorder, or condition in a subject is treated by reducing the immune response. [Invention 1115] A pharmaceutical composition according to Invention 1097 or Invention 1098 for use in the treatment of a disease, disorder, or condition in a subject. [Invention 1116] The use of any immunomodulatory protein of Invention 1001 to 1086, or the use of a pharmaceutical composition of Invention 1097 or Invention 1098, in the manufacture of a medical agent for treating a disease, disorder, or condition in a subject. [Invention 1117] A pharmaceutical composition for use of Invention 1115, or use of Invention 1116, wherein the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, a kidney disease, a graft rejection, a graft-versus-host disease, or a viral infection. [Invention 1118] A pharmaceutical composition for use, or use, of any of the present inventions 1115 to 1117, for a disease, disorder, or condition selected from the group consisting of systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, 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. <原子力 [Invention 1119] <00原子力 A pharmaceutical composition for use, or use, according to any of the inventions 1115 to 1117, wherein the disease, disorder, or condition is B-cell carcinoma, and the cancer is myeloma.

Brief Description of the Drawings

[0078] [Figure 1] A schematic diagram of a functional inhibition assay involving recombinant APRIL and BAFF using TACI is shown. 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 regulates the 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 (at room temperature with shaking) and then added to wells containing 150,000 Jurkat / TACI / NFκB-luciferase cells for 5 hours. [Figure 3] This diagram illustrates the function of an exemplary TACI TD Fc fusion molecule in blocking APRIL (upper panel of this diagram) or BAFF (lower panel of this diagram). [Figure 4] This image shows human TACI TD Fc fusion molecules for blocking signaling mediated by mouse APRIL (left panel) and BAFF (right panel). [Figure 5] This shows human TACI TD Fc fusion molecules for blocking human APRIL (upper panel) and BAFF (lower panel)-mediated signaling 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 percentage change in body weight (Figure 6B), and percentage survival rate (Figure 6C) were evaluated from 20 weeks of age. Serum anti-double-stranded DNA IgG titer (Figure 6D) and blood urea nitrogen (BUN) (Figure 6E) were analyzed (for anti-dsDNA IgG, p<0.0001 by Student's t-test, **** vs. Fc; for BUN-4, p=0.0008 by Student's t-test, *** vs. Fc). Kidneys were processed and histologically analyzed in replicated periodate-Schiff (PAS) stained sections. Individual components and overall histological scores are shown in Figure 6F. The 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] Refer to the explanation in Figure 6A. [Figure 6C] Refer to the explanation in Figure 6A. [Figure 6D] Refer to the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 6F] See the explanation in Figure 6A. [Figure 6G] See the explanation in Figure 6A. [Figure 6H] See the explanation in Figure 6A. [Figure 6I] See the explanation in Figure 6A. [Figure 7] Quantified by luciferase production in Jurkat / NF-κB / TACI cells, the TACI mutation (K77E / F78Y / Y102D) demonstrates the ability to inhibit signaling mediated by APRIL (left panel) and BAFF (right panel). [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]Exemplary sequence alignments are shown to identify corresponding residues in a sequence compared to a reference sequence. The symbol "*" between two aligned amino acids indicates that the aligned amino acids are identical. The symbol "-" indicates a gap in the alignment. Exemplary, non-limiting positions for amino acid substitutions described herein are shown in bold. Based on the alignment of two similar sequences that have a common identical residue, those skilled in the art can identify the "corresponding" position in the sequence by comparing it to the reference sequence using the conserved identical amino acid residue as a guide. Figure 9 shows an exemplary alignment of the reference TACI extracellular domain sequence shown SEQ ID NO:122 (containing CRD1 and CRD2 and a complete extracellular domain with an initiating methionine residue) and the TACI extracellular domain sequence shown SEQ ID NO:13 (containing CRD2, which is just one CRD). When identical residues are aligned in a line, for example, amino acid residue E7 in SEQ ID NO:13 corresponds to residue E74 in SEQ ID NO:122, amino acid residue K10 in SEQ ID NO:13 corresponds to residue K77 in SEQ ID NO:122, amino acid residue Y12 in SEQ ID NO:13 corresponds to Y79 in SEQ ID NO:122, amino acid residue L15 in SEQ ID NO:13 corresponds to L82 in SEQ ID NO:122, amino acid residue R17 in SEQ ID NO:13 corresponds to R84 in SEQ ID NO:122, and amino acid residue D16 in SEQ ID NO:13 corresponds to D85 in SEQ ID NO:122. Performing similar alignments between two similar protein sequences to identify corresponding residues, including identifying corresponding residues based on the examples and descriptions herein, is within the scope of the skill of the art. [Figure 10]Figures 10A–10D show the analysis of parameters evaluated from a 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] Figures 11A–11B show the analysis of collected spleens evaluated from a mouse keyhole limpet hemocyanin (KLH) immunization model. The spleens were processed and analyzed by weight (Figure 11A) and total cell count (Figure 11B). [Figure 12] This figure illustrates the spleen analysis evaluating the cell subtype population composition from a mouse keyhole limpet hemocyanin (KLH) model, and shows the results of the B cell subset number compared to the group mean. [Figure 13] Figure 13 illustrates the spleen analysis used to evaluate the cellular subtype phenotypic composition from a mouse keyhole limpet hemocyanin (KLH) model, showing the results for germinal center B cells and plasma cells. [Figure 14] Figures 14A-D illustrate the number of T cells in the mouse keyhole limpet hemocyanin (KLH) model. Spleen CD3+, CD8+, CD4+, and follicular helper T cells are shown in Figures 14A, 14B, 14C, and 14D, respectively. [Figure 15] The Tcm cell population and Tem cell population in the mouse keyhole limpet hemocyanin (KLH) model are illustrated. [Figure 16] Figures 16A-16B illustrate the overall incidence and severity of sialadenitis (Figures 16A-16B) in diabetic mice treated with the test molecule. [Figure 17] Figures 17A-17B illustrate the overall incidence and severity of isletitis (Figures 17A-17B) in diabetic mice treated with the test molecule. [Modes for carrying out the invention]

[0079] Detailed explanation Immunomodulatory proteins are provided herein that bind to one or more ligands, for example, one or more ligands produced as soluble factors, in order to suppress or reduce the response or activity of B cells. Among the immunomodulatory proteins provided are proteins that bind to BAFF or APRIL ligands to neutralize their activity and block or antagonize the activity of B cell stimulating receptors, for example, TACI or BCMA. The immunomodulatory proteins provided may also be fusion proteins of the TACI extracellular domain or its binding site (hereinafter, TACI ECD) and a multimerization domain, for example, immunoglobulin Fc. For example, TACI-Fc fusion proteins are provided herein. In some embodiments, the immunomodulatory proteins provided herein can be used to treat diseases, disorders, or conditions associated with dysregulated immune responses, such as inflammatory diseases or autoimmune diseases, or diseases, disorders, or conditions associated with inflammatory or autoimmune symptoms.

[0080] The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and to protect tissues from excessive damage during an immune response, such as during an attack against a pathogen. However, in some cases, the immune system can become dysregulated, initiating an abnormal immune response against a normal body part or tissue, resulting in an autoimmune disease or condition or autoimmune symptoms. In other cases, an undesirable immune response may be initiated against foreign tissue, such as a graft, resulting in graft rejection.

[0081] In some cases, certain diseases, disorders, and conditions in which the immune response is dysregulated can be treated with immunotherapy that alters the activity of immune cells, such as B cell activity. In particular, inhibiting or attenuating immune responses, such as B cell response, may be desirable to reduce or prevent undesirable inflammation, autoimmune symptoms, and / or graft rejection. However, therapeutic approaches that attempt to modulate the interaction between ligands and their receptors that mediate 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 spatial conditions and size limitations imposed by the extent of immune synapses. In some cases, existing therapeutic agents, including antibody drugs, may not be able to interact simultaneously with multiple target proteins involved in modulating these interactions. For example, soluble receptors and antibodies generally do not bind competitively (e.g., they do not bind to multiple target species at once) and therefore lack the ability to bind to multiple targets simultaneously. Furthermore, pharmacokinetic differences between drugs that independently target one of these receptors can make it difficult to maintain the desired blood concentrations of drug combinations targeting two different receptors 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 work together 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-associated autoimmune diseases. Co-neutralizing them drastically reduces B cell function, including antibody production, while inhibiting either BAFF or APRIL alone mediates a relatively mild effect. Fc fusions of wild-type (WT) TACI (e.g., atacicept and teritacicept) 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 clearly demonstrate long-term and / or complete disease remission. B cell targeted therapies have demonstrated promising therapeutic potential, but are not entirely satisfactory. For example, soluble recombinant TACIs show great potential as therapeutic agents, but their usefulness appears to be hindered by their mild to moderate affinity for APRIL.

[0083] Among the embodiments provided are those resulting in improved neutralizing activity and suppression or reduction of B cell response. In some embodiments, the improvement in activity is mediated by increased or improved binding or interaction between the provided immunomodulatory protein (e.g., TACI-Fc fusion protein) and BAFF and / or APRIL. The provided immunomodulatory protein blocks or antagonizes the interaction between BAFF or APRIL, e.g., a homotrimer of BAFF or APRIL, a BAFF / APRIL heterotrimer, or a 60-mer of BAFF, and cognitive B cell stimulating receptors, thereby neutralizing the activity of BAFF and / or APRIL ligands. In some embodiments, the provided immunomodulatory protein reduces the response or activity of one or more B cells, including the ability of B cells to produce immunoglobulins. In some embodiments, the provided immunomodulatory protein (e.g., TACI-Fc fusion protein) reduces circulating serum immunoglobulins when administered to a subject. In some embodiments, the provided immunomodulatory protein reduces one or more of the maturation, differentiation, and proliferation of B cells. In the contexts provided, such activity is improved or superior to the activity achieved by WT TACI-Fc fusion proteins (e.g., teritacicept or atacicept). In some embodiments, the provided immunomodulatory proteins (TACI-Fc fusion proteins) are candidate therapeutic agents for treating several autoimmune and inflammatory diseases, particularly B-cell related diseases, such as SLE, SjS, and other connective tissue diseases.

[0084] The embodiments provided relate to the identification of variant TACI polypeptides engineered to exhibit improved affinity for APRIL and / or BAFF after random mutagenesis and directional evolution of the second cysteine-rich domain (CRD2) of TACI spanning residues 68–110. As shown herein, affinity maturation involved five selections in which APRIL and BAFF were alternately swapped while the selection reagent concentration was reduced to maintain selection pressure. The results demonstrated variant TACI polypeptides exhibiting substantially enhanced affinity for BAFF and APRIL compared to wild-type TACI. For example, 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 are provided herein. In particular, some embodiments provided result in an improved combination of BAFF inhibition and APRIL inhibition. Thus, the immunomodulatory proteins provided result in effective and long-lasting disease suppression in the treatment of autoimmune or inflammatory diseases, including severe B-cell-associated autoimmune diseases such as SLE.

[0085] For example, the provided embodiment is based on the finding that directional evolution through affinity modification of the TNFR domain (TD) of the TACI external domain has facilitated the development of molecules with improved affinity for APRIL and / or BAFF. Thus, affinity modification generates a variant TACI containing a variant TNFR domain (vTD). When such a molecule is fused with immunoglobulin Fc, an immunomodulatory protein that suppresses B cell activity and response is produced. For example, when the affinity-matured TACI variant output is reformatted as a soluble Fc fusion protein, inhibition of APRIL and BAFF in a TACI-dependent reporter assay, as shown herein, is lower IC than with wild-type TACI-Fc and belimumab control. 50The values ​​were shown. Furthermore, results from the evaluated animal models demonstrate rapid and significantly reduced key lymphocyte subsets, including plasma cells, germinal center B cells, and follicular T helper cells. In addition, the tested variant molecule showed 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 autoantibodies, serum urea nitrogen levels, proteinuria, sialadenitis, renal lesions, and renal immune complex deposition in an NZB / W lupus model. Furthermore, compared to wild-type TACI-Fc, the tested TACI Fc fusion significantly and sustainably reduced the titers of serum IgM, IgG, and IgA antibodies in mice. The findings herein demonstrate that these immunomodulatory proteins consistently exhibit potent immunosuppressive activity and efficacy in vitro and in vivo, and appear superior to existing and / or approved immunomodulatory agents such as belimumab, abatacept, atacicept, or teritacicept. Therefore, such biologics may be attractive development candidates for the treatment of B-cell related diseases, including severe autoimmune and / or inflammatory 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 whole for any purpose, as if each individual publication were incorporated by reference individually. If any definition provided herein conflicts with or otherwise contradicts any definition provided in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0087] The section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0088] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in which the claimed subject matter pertains. In some cases, terms that have a generally understood meaning are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from those generally understood in the art.

[0089] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0090] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. Any reference to any value or parameter “about” herein includes (and describes) aspects relating to the value or parameter itself. For example, a statement referring to “about X” includes a statement “X”.

[0091] The term "affinity-modified," as used in relation to protein domains, refers to a mammalian protein having an amino acid sequence modified in its extracellular domain or specific binding portion (compared to the corresponding wild-type parent domain or unmodified domain) to increase or decrease binding activity, such as binding affinity to at least one of its binding partners (or "counter-structures"), compared to the parent wild-type or unmodified (i.e., unaffinity-modified domain) protein. In some embodiments, an affinity-modified domain may include 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, compared to the wild-type or unmodified domain. Binding activity, e.g., increased or decreased 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 increased protein binding activity, e.g., affinity, to its binding partner is at least 10% greater than the wild-type control value, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type control value. The decreased protein binding activity, e.g., affinity, to at least one of its binding partners is 90% or less of the control value, but 10% or more of the wild-type control value, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type control value, but 10% or more. Affinity-modified proteins have altered primary amino acid sequences in their extracellular domain or specific binding sites, resulting from the substitution, addition, or deletion of amino acid residues. The term "affinity-modified" is not construed as imposing any conditions on any particular starting composition or method by which an affinity-modified protein was produced.Therefore, affinity-modified proteins are not limited to wild-type protein domains transformed into affinity-modified domains by any particular process of affinity modification. Affinity-modified domain polypeptides can be generated, for example, starting from wild-type mammalian domain sequence information, then modeled in silico for binding to their binding partners, and finally synthesized recombinantly or chemically, in order to obtain affinity-modified domain compositions of a substance. However, in an alternative example, affinity-modified domains can be produced by site-directed mutagenesis of wild-type domains. Thus, affinity-modified TD domains represent a certain product and do not necessarily represent a product produced by any given process. A variety of techniques can be used, including recombinant methods, chemical synthesis, or combinations thereof.

[0092] The term "affinity-modified TD domain" refers to the affinity-modified domain of a member of the tumor necrosis receptor superfamily (TNFRSF) protein or its TNF ligand, which has a modified amino acid sequence of either the TNFR domain or the TNF domain, respectively. For example, the affinity-modified TD domain of a TNFRSF protein has a modified amino acid sequence of the TNFR domain, which consists of at least one cysteine-rich domain (CRD) within the extracellular domain or specific binding region of the TNFRSF protein, so that binding activity, such as binding affinity to at least one of its binding partners (or "partner structures"), is increased or decreased compared to the parent wild-type or unmodified protein containing a non-affinity-modified TD domain or an unmodified TD domain.

[0093] "Affinity-modified TACI (also called variant TACI)" refers to a TACI protein molecule that antagonizes or blocks the activity of B cell stimulating receptors. For example, TACI binds to the B cell stimulating receptor B cell maturation antigen (BCMA), B cell activator receptor (BAFF-R), and APRIL and / or BAFF, which are ligands for transmembrane activators and calcium modulators and cyclophylline ligand-interactors (TACI). In certain embodiments, BIM includes the extracellular domain of TACI, or a portion of the extracellular domain of TACI, which binds to the congener ligand APRIL and / or BAFF, as well as the heterotrimer of APRIL and BAFF, or a TNF receptor family domain (e.g., TD, e.g., CRD). Affinity-modified variants of the extracellular domain or portion of TACI may include another amino acid modification (e.g., amino acid substitution) to TD that increases the binding affinity to the congener ligand (e.g., APRIL and / or BAFF, as well as the heterotrimer of APRIL and BAFF).

[0094] As used herein, “B cell stimulating receptor” refers to one or more of the related tumor necrosis factor (TNFR) superfamily receptors expressed on B cells, including B cell maturation antigens (BCMAs), B cell activator receptors (BAFF-Rs), and transmembrane activators and calcium-modulating and cyclophylline ligand-interactors (TACIs). The involvement or ligation of these related receptors by their congener ligands, BAFF and / or APRIL, or heterotrimers of APRIL and BAFF, modulates 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 domain, the cytoplasmic domain containing one or more TNF receptor-related 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 modulate B cell homeostasis.

[0095] As used herein, “to bind,” “bound,” or its grammatical variations thereof, refers to a situation in which one molecule engages in any attractive interaction with another molecule, resulting in a stable association in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds and covalent bonds (such as reversible and irreversible covalent bonds), and includes, but is not limited to, intermolecular interactions of proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as compounds including drugs.

[0096] As used herein, binding activity refers to the characteristics of a molecule, such as a polypeptide, relating to whether it binds to one or more binding partners and how it binds. Binding activity may include any measure of a molecule's binding to a binding partner. Binding activity may include the ability to bind to a binding partner, affinity to the binding partner (e.g., high affinity), avidity to the binding partner, strength of binding to the binding partner, and / or specificity or selectivity for binding to the binding partner.

[0097] As used herein, the term “binding affinity” refers to the specific binding affinity of a protein to its binding partner (i.e., its counterpart 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 a non-covalent interaction between two binding partners. The increase or decrease in binding affinity of an affinity-modified domain or an immunomodulatory protein containing an affinity-modified domain to a binding partner is determined by comparison with the binding affinity of the unmodified domain (e.g., the 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 immunoassays, ForteBio Octet, Biacore measurements, or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol 1(9): 793-801 (1994). In some embodiments, binding affinity can be measured by flow cytometry, 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 multiple affinities between proteins to their respective non-covalent interactions, such as 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 elimination, excretion, breakdown (e.g., enzymatic breakdown / digestion), or absorption and concentration within 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 a 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-lives are known in the art and include, but are not limited to, multimerizing domains (e.g., Fc immunoglobulin domains), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (elongated recombinant peptide; see International Publication No. 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylated).

[0100] As used herein, the term “cell surface partner” (or “cell surface binding partner”) refers to a partner (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 proteins such as receptors and soluble ligands, or extracellular domains or parts thereof or affinity-modified variants thereof, the terms “binding partner” or “mutant structure” refer 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-modified domain, or an immunomodulatory protein containing an affinity-modified domain, specifically binds to the binding partner of the corresponding domain of a native or wild-type protein, but with increased or decreased affinity. A “cell surface binding partner” is a binding partner expressed on a mammalian cell. Typically, a cell surface binding partner is a transmembrane protein. In some aspects, a cell surface binding partner is on cells such as mammalian cells that form immune synapses, e.g., immune cells, and is a receptor or ligand of a receptor expressed by immune cells.

[0102] In relation to binding to cell surface molecules, the term "cis" refers to the binding to two or more different cell surface molecules, each present on the surface of the same cell. In some aspects, cis means that two or more cell surface molecules are exclusively present on one of two mammalian cells forming an IS, or exclusively on the other (but not both).

[0103] As used herein, the term “conservative amino acid substitution” means an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of amino acids with side chains having 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. The conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

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

[0105] As used herein, a “domain” (typically a sequence of three or more amino acids, generally five or seven or more, e.g., 10 to 200 amino acid residues) refers to a portion of a molecule, such as a protein or coding nucleic acid, that is structurally and / or functionally distinct from the rest of the molecule and is identifiable. For example, a domain includes a portion of a polypeptide chain composed of one or more structural motifs, capable of forming independently folded structures within a protein, and / or recognized by functional activity such as binding activity. A protein may have one or more distinct domains. For example, a domain may be identified, defined, or distinguished by its primary sequence or structural homology to a related family member, e.g., homology to a motif. In another example, a domain may be distinguished by its function, e.g., its ability to interact with biomolecules such as congenital binding partners. Domains may independently exhibit biological function or activity, such as being able to perform activity such as binding, either independently or when fused to another molecule. A domain may be a linear or nonlinear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. Definitions are provided for illustrative purposes in this specification, but it will be understood that recognizing specific domains by name is well within the scope of those skilled in the art. Domains can be identified using appropriate software as needed. References to amino acids containing specific sequences, listed as SEQ ID NOs used to describe domain composition (e.g., TD domains), are for illustrative purposes only and are not intended to limit the scope of the embodiments provided. Descriptions of polypeptides and their domains are understood to be theoretically derived based on homology analysis and alignment with similar molecules. Additionally, in some cases, adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., TD) may also be included in the sequence, for example, to ensure proper domain folding when expressed.Therefore, the exact gene locus can vary and is not necessarily the same for each protein. For example, a particular TD domain, or a particular CRD domain, may be several amino acids longer or shorter (1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

[0106] In this specification, the terms “ectodomain,” “extracellular domain,” or “ECD” are used without distinction and refer to the region of a membrane protein, such as a transmembrane protein, that lies 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 the purposes of this specification, references to ECD are understood to refer to sequences and domains that constitute this region and do not require the protein containing the ECD to be a membrane protein or that the domain is located outside the cell. For example, soluble immunomodulatory proteins may contain an ECD sequence of a membrane protein fused to another part, e.g., a multimerization domain, e.g., an Fc region. Ectodomains often interact with specific ligands or specific cell surface receptors, for example, via binding domains that specifically bind to ligands or cell surface receptors. An example of a binding domain is the cysteine-rich domain (CRD). Ectodomains of members of the TNFR superfamily include TD domains (e.g., CRD domains). Therefore, references to ECD in this specification include the full-length sequence of the membrane protein ECD and its specific binding fragment, which includes a CRD that binds to a ligand or homologous partner.

[0107] The term “effective dose” or “therapeutic effective dose” 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 patient-derived cells) or in vivo (by administration to the patient), for example, by improving or eliminating the symptoms and / or causes of the disease, either alone (i.e., as monotherapy) or in combination with additional therapeutic agents, results in a statistically significant inhibition of disease progression. An effective dose for treating a disease, condition, or disorder, such as an immune system disease, immune system condition, or immune system disorder, may be an amount that reduces, mitigates, or alleviates at least one symptom or biological response or action associated with the disease, condition, or disorder, prevents the progression of the disease, condition, or disorder, or improves the patient’s physical function. In the case of cell therapy, the effective dose 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 means a polypeptide encoded by a nucleic acid sequence comprising the coding sequences of two or more proteins, possibly two, three, four, five or more proteins, such that the coding sequences are on the same reading frame so that when the fusion construct is transcribed and translated in a host cell, a protein comprising two or more proteins is produced. Each of the two or more proteins may be adjacent to another protein in the construct, or separated by a linker polypeptide comprising one, two, three or more, but typically fewer than 20, 15, 10, 9, 8, 7 or 6 amino acids. The protein product encoded by the fusion construct is called a fusion polypeptide. An example of a fusion protein according to the provided embodiment is an Fc fusion protein comprising an affinity modification domain linked to an immunoglobulin Fc domain (e.g., a variant or part of the extracellular domain of TACI containing a CRD).

[0109] The term “half-life extension portion” 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 with that portion. In some embodiments, the half-life is extended by more than 1.2 times, or about 1.2 times, about 1.5 times, about 2.0 times, about 3.0 times, about 4.0 times, about 5.0 times, or about 6.0 times. In some embodiments, 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 a protein without a half-life extension portion. Half-life refers to the amount of time it takes for a protein to lose half of its concentration, volume, or activity. Half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extension regions include the Fc domain, the polymerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see International Publication No. 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).

[0110] The Fc (crystalline fragment) region or Fc (crystalline fragment) domain (also called Fc polypeptide) of an immunoglobulin molecule primarily corresponds to the constant region of the immunoglobulin heavy chain and, in some cases, is responsible for a variety of functions, including antibody effector function. The Fc domain comprises some or all of the hinge domain of the immunoglobulin molecule, along with CH2 and CH3 domains. In some cases, all or part of the Fc hinge sequence may be deleted for inclusion in the provided fusion protein. The Fc domain can form a dimer of two polypeptide chains linked by one or more disulfide bonds. In some embodiments, Fc is a variant Fc exhibiting reduced activity (e.g., reduced by 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 by the EU numbering system unless otherwise stated based on a specific SEQ ID NO. EU numbering follows the publicly known and most recently 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 the EU index reported in Kabat, EA et al. Sequences of Proteins of Immunological Interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0111] An immunoglobulin Fc fusion ("Fc fusion"), such as an immunomodulatory Fc fusion protein, is a molecule comprising one or more polypeptides functionally linked to the Fc region of an immunoglobulin. An Fc fusion may, for example, comprise an Fc region functionally linked to a TACI extracellular domain or a portion thereof containing a CRD, including one of the affinity variants provided. The immunoglobulin Fc region may be linked indirectly or directly to one or more polypeptides. Various linkers, known in the art, may optionally be used to link Fc to a fusion partner to produce an Fc fusion. Fc fusions of the same species can be dimerized to form an Fc fusion homodimer. Fc fusions of different species (e.g., knob-into-hole operation) may be used to form an Fc fusion heterodimer. In some embodiments, Fc is a mammalian Fc, such as mouse Fc or 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 their derivatives growing in serum-free medium, such as the Veggie CHO cell line and related cell lines, or the DHFR-deficient CHO strain DX-B11.

[0113] As used herein, the term “immunological synapse” or “immune synapse” (abbreviated as “IS”) means the interface between mammalian cells expressing MHC I (major histocompatibility complex) or MHC II, such as antigen-presenting cells or tumor cells, and mammalian lymphocytes, such as effector T cells or natural killer (NK) cells.

[0114] As used herein, the term “immunoglobulin” (abbreviated as “Ig”) is synonymous with the term “antibody” (abbreviated as “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 immunoglobulins of less than full length, whether fully or partially synthesized (e.g., recombinant or chemosynthetic) or naturally produced, including any fragment thereof that includes at least a portion of the variable heavy (VH) chain region and / or variable light (VL) chain region of the immunoglobulin molecule, sufficient to form an antigen-binding site and to bind specifically to an antigen when constructed. Antibodies may also include all or part of the constant region. Such fragments include antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL, single-stranded variable fragments (scFv) containing linked VH and VL on a single chain, and other antibody V-region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments. Therefore, references to antibodies in this specification are understood to include full-length antibodies and antigen-binding fragments. The term antibody also includes antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-stranded molecules. Bispecific antibodies, homo-bispecificity, and hetero-bispecificity are included within the scope of the term. Antibodies include polyclonal antibodies or monoclonal antibodies. Antibodies also include synthetic antibodies or recombinantly produced antibodies. For information on the structures and properties of various classes of antibodies, see, for example, 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 an antibody in substantially intact form, as opposed to an antibody fragment. A full-length antibody is typically an antibody having 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.) and synthetically produced antibodies with the same domains. Specifically, whole antibodies include those having heavy and light chains containing an Fc region. The constant domain may be a native sequence constant domain (e.g., the human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0116] An "antibody fragment" includes a portion of an intact antibody, the antigen-binding region and / or variable region of an intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv(dsFv), Fd fragments, Fd' fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

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

[0117] "Fv" consists of one heavy-chain variable domain and one light-chain variable domain linked by non-covalent bonds. From the folding of these two domains, six complementarity-determining regions (CDRs) (three in each of the heavy and light chains) are generated 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 the Fv containing only the three CDRs specific for an antigen), although in some cases with a lower affinity than the entire binding site, has the ability to recognize and bind to the antigen.

[0118] "dsFv" refers to an Fv having engineered intermolecular disulfide bonds that stabilize the V H -V L pair.

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

[0120] "Fab fragment" is an antibody fragment resulting from digestion of a full-length immunoglobulin by papain or a fragment having the same structure produced synthetically, for example, by recombinant methods. The Fab fragment includes a light chain (including V L and C L ) and a separate chain including the variable domain (V H ) of the heavy chain and one constant domain (C H 1) of the heavy chain.

[0121] "F(ab')2 fragment" is an antibody fragment resulting from digestion of an immunoglobulin by pepsin at pH 4.0 - 4.5 or a fragment having the same structure produced synthetically, for example, by recombinant methods. The F(ab')2 fragment essentially includes two Fab fragments in which each heavy-chain portion includes several additional amino acids including cysteine residues that form a disulfide bond linking the two fragments.

[0122] "Fab' fragment" is a fragment that includes half of the F(ab')2 fragment (one heavy chain and one light chain).

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

[0124] The "Fv' fragment" is the V of the antibody molecule. H Domain and V L This is a fragment containing only the domain.

[0125] "scFv fragments" are variable light chains (V) covalently bonded by polypeptide linkers in any order. L ) and variable heavy chain (V H This refers to an antibody fragment containing a linker. The linker is of a length such that the two variable domains are cross-linked with virtually no interference. An exemplary linker has several Glu or Lys residues dispersed throughout to increase solubility (Gly-Ser). n There is a residue.

[0126] The "diabody" is a dimerized scFv. Diabodies typically have shorter peptide linkers than scFvs and preferentially dimerize.

[0127] As used herein, the term “immunological activity” refers to the activity of one or more immune cells, such as T cells or B cells, including, for example, activation, cell survival, cell proliferation, cytokine production (e.g., interferon-gamma), cytotoxic activity, or the ability to activate the NF-κB pathway or other signaling cascades resulting in the activation of transcription factors within immune cells. Assays for evaluating the immunomodulatory activity of immunomodulatory proteins may be compared to control proteins with known activity.

[0128] An "immunomodulatory protein" or "immunomodulatory polypeptide" is a protein that modulates immunological activity. "Modification" 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 the suppression of immunological activity of immune cells such as B cells or T cells. For example, a soluble Fc fusion protein as used herein may suppress the immunological activity of B cells. Immunomodulatory proteins 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 alteration of the amino acid sequence of a polypeptide or the nucleotide sequence within a nucleic acid molecule, and includes changes in the amino acids or nucleotides of the sequence, respectively. Amino acid modification or change may be a deletion, insertion, or substitution (substitution) of an amino acid or nucleotide, respectively. Methods for modifying polypeptides are common to those skilled in the art, such as by using recombinant DNA methods.

[0130] The term "multimerizing domain" refers to an amino acid sequence that facilitates the formation of a multimer of two or more polypeptides. A multimerizing domain includes sequences that facilitate stable interactions between a polypeptide molecule and one or more additional polypeptide molecules, each containing complementary multimerizing domains (e.g., a first multimerizing domain and a second multimerizing domain), which may be the same or different multimerizing domains. Interactions between complementary multimerizing domains, for example, between the first and second multimerizing domains, form a stable protein-protein interaction to produce a multimer of the polypeptide molecule and the additional polypeptide molecule. In some cases, the multimerizing domains are the same and interact with themselves to form a stable protein-protein interaction between two polypeptide chains. Generally, polypeptides can be directly or indirectly linked to multimerizing domains. Exemplary multimerizing domains include immunoglobulin sequences or parts 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 IgG-derived Fc domain or part thereof containing an IgG1 subtype, IgG2 subtype, IgG3 subtype, or IgG4 subtype, IgA, IgE, IgD, and IgM, or modified forms thereof.

[0131] The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to polymers 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 include known analogues of natural nucleotides, possess similar binding properties to natural nucleotides, and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue. The terms nucleic acid or polynucleotide encompass cDNA or mRNA encoded by genes.

[0132] As used herein, the terms “functional combination,” “functional order,” and “functionally linked” refer to the linking of nucleic acid sequences in a manner or orientation such that the segments are arranged to function in coordination for an intended purpose. In some embodiments, the terms refer to the linking of nucleic acids to produce a nucleic acid molecule capable of leading the transcription of a given gene and / or to produce a functionally desired protein molecule. For example, segments of a DNA sequence, e.g., coding sequences and regulatory sequences, are linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a transcription-activating protein) is bound to the regulatory sequence.

[0133] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammals, often in humans. A pharmaceutical composition typically comprises an effective amount of an active substance (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 herein without distinction and refer to molecular chains of two or more amino acids linked by peptide bonds. The terms do not refer to a specific length of the product. Therefore, “peptides” and “oligopeptides” are included within the definition of polypeptide. The terms include post-translational modifications of polypeptides, such as glycosylation, acetylation, and phosphorylation. The terms also include molecules that can be synthesized or recombinantly expressed using known protein manipulation techniques, which may include one or more amino acid analogs, or non-canonical or non-natural amino acids. Furthermore, proteins may be derivatized by known organic chemical techniques as described herein.

[0135] For example, the term “purified” applied to nucleic acids or proteins (e.g., immunomodulatory proteins) encoding immunomodulatory proteins generally indicates a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified polypeptides or polynucleotides form distinct bands in electrophoretic gels, chromatographic eluents, and / or media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that essentially produces one band in an electrophoretic gel is “purified.” Purified nucleic acids or proteins are at least about 50% pure, and typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99%, or more pure (e.g., by weight percentage or molar basis).

[0136] The term “recombinant” indicates that a material (e.g., nucleic acid or polypeptide) is artificially (i.e., unnaturally) modified by human intervention. The modification may be made to the material within its natural environment or state, or removed from its natural environment or state. For example, “recombinant nucleic acid” is produced, for example, by recombining nucleic acids during cloning, affinity modification, DNA shuffling, or other well-known molecular biological procedures. “Recombinant DNA molecule” consists of DNA segments linked together by such molecular biological techniques. As used herein, the terms “recombinant protein” or “recombinant polypeptide” refer to protein molecules (e.g., immunomodulatory proteins) expressed using recombinant DNA molecules. “Recombinant host cell” is a cell that contains and / or expresses recombinant nucleic acid, or has been genetically modified, such as by introducing nucleic acid molecules encoding recombinant proteins, such as immunomodulatory proteins provided herein. In eukaryotes, transcriptional regulatory signals include “promoter” elements and “enhancer” elements. Promoter and enhancer 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 (similar regulatory elements, i.e., promoters, are also found in prokaryotes). The choice of specific promoters and enhancers depends on which cell type is used to express the protein of interest.

[0137] As used herein, the term “recombinant expression vector” refers to a DNA molecule comprising a desired coding sequence (e.g., encoding an immunomodulatory protein) and appropriate nucleic acid sequences necessary for the expression of the functionally linked coding sequence within a particular cell. In prokaryotes, the nucleic acid sequences necessary for expression include a promoter, optionally an operator sequence, a ribosome binding site, and optionally other sequences. In eukaryotic cells, it is known that promoters, enhancers, and termination and polyadenylation signals are utilized. Secretory signal peptide sequences may also be optionally encoded by a recombinant expression vector and functionally linked to the coding sequence, for example, for their expression as a secretible protein if desired, or for easier isolation or purification of the immunomodulatory protein from the cell, so that the expressed protein can be secreted by a recombinant host cell. The term includes vectors as self-replicating nucleic acid structures, as well as vectors that are integrated 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 sequence identity between genes or proteins at the nucleotide level 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 sequences at a given position within each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequences 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 such methods 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, percentage sequence identity may be determined as the percentage of amino acid residues (or nucleotide residues) in a candidate sequence that are identical to amino acid residues (or nucleotide residues) in a reference sequence, after the sequences have been aligned and gaps introduced as necessary to achieve maximum percentage sequence identity. References to sequence identity include sequence identity across the entire length of each sequence being compared. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0139] As used herein with respect to proteins, the term "soluble" means that a protein is not a membrane protein or is not fixed to the cell membrane. A protein may be constructed as a soluble protein by containing only an extracellular domain or a portion thereof and not a transmembrane domain. In some cases, the solubility of a protein may be improved by linking or binding, directly or indirectly via linkers, to an Fc domain or other half-life-extending molecules, which may also improve the stability and / or half-life of the protein. In some aspects, a soluble protein is an Fc-fusion protein.

[0140] As used herein, the term “specifically binds” means the ability of a protein to bind to a target protein such that, under specific binding conditions, its affinity or avidity is at least 10 times, but optionally 50, 100, 250, or 500 times, or even at least 1000 times, the average affinity or avidity of the same protein to a sufficiently statistically sized aggregate of random peptides or polypeptides. A specifically binding protein does not need to bind exclusively to a single target molecule; it may bind specifically 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 structural conformation similar to that of the target protein. Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or to molecules with substantially similar epitopes to the target molecule (e.g., paralogs) is possible without compromising the specificity of binding determined compared to a unique, non-target, statistically effective aggregate (e.g., random polypeptide). Therefore, the immunomodulatory proteins of the present invention may, for cross-reactivity, specifically bind to target molecules of multiple different species. 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 × 10⁻¹⁶. -5 Less than M, often about 1 × 10⁻⁶-12 It has a low dissociation constant (Kd) of M. In certain aspects of this disclosure, the interaction between the two binding proteins is approximately 1 × 10⁻⁶. -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1x10 -11 It has a dissociation constant of M or less.

[0141] As used herein with respect to proteins, the terms “specific binding fragment” or “fragment” mean a polypeptide that is shorter than the full-length protein or its specific domain or region, and that specifically binds in vitro and / or in vivo to the binding partner of the full-length protein or its specific domain or region. A specific binding fragment relates to a fragment of the full-length extracellular domain of a polypeptide or the binding domain of a polypeptide, but still binds to the 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 its full-length TNFR domain (TD) (e.g., CRD), but still binds to the binding partner of the TNFR family member, or to the binding partner of the CRD of a TNFR family member. In some embodiments, a specific binding fragment is the full-length sequence of the extracellular domain, or 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 extracellular domain or the domain or region of the extracellular domain. In some embodiments, the specific binding fragment may have an amino acid length of at least 50 amino acids, for example, at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific binding fragment includes the CRD1 and / or CRD2 domains. In some embodiments, the specific binding fragment includes the CRD2 domain.

[0142] As used herein, “Subject” refers to a mammal such as a human or other animal, typically a human. A subject may be male or female and may be of any suitable age, including infants, young adults, adolescents, adults, and elderly subjects.

[0143] As used herein, for example, with respect to synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, “synthetic” refers to nucleic acid molecules or polypeptide molecules 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, all of which are type I (N-terminal extracellular) transmembrane glycoproteins, containing 1 to 6 cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on a shared structural feature that includes one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular region, often playing some role in the protein binding of their congeneral binding partners or ligands. TNFRSF proteins may have one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or ectodomain of a TNFRSF member contains 1 to 6 pseudo-repeats of a CRD. For example, the BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are usually trimer complexes or multimer complexes stabilized by their cysteine-disulfide bonds. When TNFRSF proteins bind to their ligands, they promote various intracellular biological activities, such as apoptosis (programmed cell death) or the induction of cell survival and proliferation.

[0145] The term "TD" refers to one or more structural domains of a TNFRSF protein or TNF family ligand. For example, the TD of a TNFRSF protein is a cysteine-rich domain (CRD) module of approximately 40 amino acids containing six conserved cysteines. Therefore, references to CRDs can also be used interchangeably with the term TD in relation 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 relation to the ECD of such a protein molecule. A reference to variant TD (vTD) refers to a variant or modified sequence of TD.

[0146] In relation 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 that, with respect to two different cell surface molecules, the first is exclusively present on one of two mammalian cells forming an IS, and the second is exclusively present on the other of the two mammalian cells forming the IS.

[0147] As used herein, the term “transmembrane protein” refers to a membrane protein that substantially or completely penetrates a lipid bilayer, such as a biological membrane, such as a mammalian cell membrane, or a lipid bilayer, such as those found in artificial constructs like liposomes. A transmembrane protein contains a transmembrane domain ("transmembrane domain") that incorporates the transmembrane protein into the lipid bilayer and makes this integration thermodynamically stable under physiological conditions. The transmembrane domain is generally predictable from its amino acid sequence via any number of commercially available bioinformatics software applications, based on its higher hydrophobicity compared to the protein region that interacts with aqueous environments (e.g., cytosol, extracellular fluid). The transmembrane domain is often a hydrophobic α-helix that penetrates the membrane. A transmembrane protein can penetrate both layers of a lipid bilayer once or multiple times.

[0148] As used herein, the terms “treating,” “treatment,” or “therapy” of a disease, condition, or disorder mean slowing, stopping, or reversing the progression of a disease or disorder by administering an immunomodulatory protein or the manipulated cells of the present invention alone or in combination with another compound described herein, as evidenced by a reduction, cessation, or elimination of any clinical or diagnostic symptom. “Treatment,” “treatment,” or “therapy” also means a reduction in the severity of symptoms in an acute or chronic disease, condition, or disorder, or a reduction in the relapse rate, such as in the course of an autoimmune disease or inflammatory state that relapses or goes into remission, or a reduction in inflammation, such as in the inflammatory phase of an autoimmune disease or inflammatory state. As used in the context of the present invention, “preventing,” “prophylaxis,” or “prevention” of a disease, condition, or disorder means administering the immunomodulatory proteins of the present invention, either alone or in combination with other compounds, to prevent the onset or development of some or all of the symptoms of a disease, condition, or disorder, or to reduce the likelihood of the development of a disease, condition, or disorder.

[0149] The term “variant” (and may also be used interchangeably with “modified” or “mutant”) as used in relation to variant proteins or variant polypeptides refers to proteins, such as mammalian (e.g., human or mouse) proteins, that have been produced by human intervention. A variant is a polypeptide having an altered or modified amino acid sequence compared to an unmodified or wild-type protein or its domain, such as by one or more amino acid substitutions, deletions, additions, or combinations thereof. Variant polypeptides may include differences of 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 acids, such as 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 higher sequence identity with the corresponding form of the wild-type or unmodified protein, e.g., its mature sequence (lacking a signal sequence) including the extracellular domain or its binding domain, or a portion thereof. Amino acids that do not exist naturally and naturally occurring amino acids are included within a range of acceptable substitutions or additions. Variant proteins are not limited to any particular method of production, including, for example, chemical synthesis, recombinant DNA technology, or a combination thereof. The variant proteins of the present invention specifically bind to at least one or more binding partners. In some embodiments, the modified amino acid sequence results in modified (i.e., increased or decreased) binding activity, e.g., binding affinity or binding affinity, to one or more binding partners. Thus, variant proteins may be "affinity-modified" proteins as described herein.

[0150] In this specification, the terms “wild-type” or “natural” or “native” are used interchangeably and are applied to biological materials such as nucleic acid molecules, proteins, and host cells that are found naturally and have not been modified by human intervention.

[0151] II. TACI immunomodulatory proteins and variant TACI polypeptides TACI immunomodulatory proteins are provided herein, comprising a portion of the extracellular domain (ECD) or a variant thereof of the TACI receptor that binds to at least one TACI congenital binding partner. Variant TACI polypeptides exhibiting modified (e.g., increased) binding activity or affinity for one or more of the TACI congenital binding partners are also provided herein. In some embodiments, the TACI congenital binding partners are one or more of BAFF or APRIL, or a BAFF / APRIL heterotrimer. The TACI immunomodulatory proteins and TACI immunomodulatory polypeptides provided comprise a soluble fusion protein in which the TACI portion of the extracellular domain or a variant thereof is ligated to another portion, e.g., immunoglobulin Fc or other multimerizing domain or half-life extension portion. Thus, in some embodiments, the immunomodulatory protein is a TACI-Fc fusion protein. In some embodiments, a TACI-Fc fusion protein is provided, comprising (1) a TACI polypeptide or a variant TACI polypeptide consisting of the extracellular domain or a portion thereof of a TACI receptor, which binds to at least one TACI homozygous binding partner, and (2) an Fc domain. The TACI polypeptide or variant TACI polypeptide may be linked directly or indirectly (e.g., via a peptide linker) to the Fc domain.

[0152] TACI is a member of the tumor necrosis factor receptor family characterized by having an extracellular domain (ECD) containing cysteine-rich pseudo-repeat domains (CRDs). TACI is a membrane-bound receptor having 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 cyclophylline ligand), an endogenous membrane protein located in intracellular vesicles, which is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with B cells and subsets of T cells. The TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family. One of the ligands is called BAFF (B cell Activating Factor of the TNF Family), as well as various other names such as ZTNF4, "Nutrokine-α", "BLyS", "TALL-1", and "THANK" (Yu et al., International Publication No. WO98 / 18921 (1998), Moore et al., Science 285:269 (1999); Mukhopadhyay et al., J. Biol. Chem. 274:15978 (1999); Schneider et al., J. Exp. Med. 189:1747 (1999); Shu et al., J. Leukoc. Biol. 65:680 (1999)). The other ligand is called APRIL, as well as various other names such 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 ligand, 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 described in SEQ ID NO:88. This 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 described in SEQ ID NO:88. The extracellular domain of TACI (amino acid residues 1-166 of SEQ ID NO:88; ECD described in SEQ ID NO:122) contains two cysteine-rich domains (CRDs, also called tumor necrosis family receptor domains or TDs) that exhibit affinity for binding to BAFF and APRIL, respectively. The first cysteine-rich domain (CRD1) contains amino acid residues 34-66 of the sequence described in SEQ ID NO:122. The second cysteine-rich domain (CRD2) corresponds to amino acids 71-104 of the sequence described in SEQ ID NO:122. TACI also includes a stalk region of approximately 60 amino acids following the second cysteine ​​repeat within the extracellular domain, corresponding to amino acid residues 105-165 of the sequence described in SEQ ID NO:122.

[0154] In some embodiments, the variant TACI polypeptides provided herein include one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions, or additions within the extracellular domain of a reference TACI polypeptide, e.g., a wild-type TACI polypeptide or an unmodified TACI polypeptide containing a CRD (hereinafter also referred to as TD). Thus, the provided variant TACI polypeptides are or include a variant TD ("vTD") in which one or more amino acid modifications (e.g., substitutions) are located in the CRD. In some embodiments, one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions, or additions are located in the CRD1 region. In some embodiments, one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions, or additions are located in the CRD2 region. In some embodiments, one or more amino acid modifications, such as one or more substitutions (or "mutations" or "replacements"), deletions, or additions, are located in amino acids within the CRD1 and CRD2 regions.

[0155] In some embodiments, the reference (e.g., unmodified) TACI sequence is either a wild-type TACI sequence or a portion thereof containing one or both CRDs. In some embodiments, the reference (e.g., unmodified) TACI is either a portion thereof containing the extracellular domain (ECD) of TACI, or one or both CRD domains, or includes them. In some embodiments, the extracellular domain of the reference (e.g., unmodified) TACI polypeptide contains CRD1 and CRD2; however, the variant TACI polypeptide does not need to contain both CRD1 and CRD2. In some embodiments, the variant TACI polypeptide contains or is essentially derived from CRD1 or its specific binding fragment. In some embodiments, the variant TACI polypeptide contains or is essentially derived from CRD2 or its specific binding fragment. In some embodiments, the variant TACI is a soluble polypeptide and lacks a transmembrane domain. In some embodiments, the variant TACI polypeptide further contains a transmembrane domain and, possibly, 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 may 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 described in SEQ ID NO:122.

[0157] In some embodiments, the reference (e.g., unmodified) TACI sequence has (i) the amino acid sequence described in SEQ ID NO:122, or the sequence lacking the N-terminal methionine; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to SEQ ID NO:122, and which binds to APRIL, BAFF, or the APRIL / BAFF heterotrimer; or (iii) is a fragment or portion of (i) or (ii) containing CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or the APRIL / BAFF heterotrimer. In some embodiments, the reference (e.g., unmodified) TACI sequence lacks the N-terminal methionine described in SEQ ID NO:122. TACI extracellular domain (ECD): SEQ ID NO: 122 TIFF0007866668000012.tif27155

[0158] In some embodiments, the reference (e.g., unmodified) TACI sequence is the extracellular domain sequence of TACI, which is part of the ECD including an N-terminal deletion compared to the amino acid sequence described in SEQ ID NO:122. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-28 corresponding to the residues described in SEQ ID NO:122. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-29 corresponding to the residues described in SEQ ID NO:122. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-30 corresponding to the residues described in SEQ ID NO:122. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-31 corresponding to the residues described in SEQ ID NO:122. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-32 corresponding to the residues described in SEQ ID NO:122. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-33, which correspond to the residues described in SEQ ID NO:122.

[0159] In some of the embodiments provided, the reference (e.g., unmodified) TACI sequence is an ECD portion containing the 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 beginning at residue 105, corresponding to the residue of the ECD sequence described in SEQ ID NO:122, up to amino acid residue 166, or lacking one or more consecutive C-terminal amino acid residues including amino acid residue 166. In some embodiments, the ECD sequence is deleted at positions 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.

[0160] In some embodiments, the reference (e.g., unmodified) TACI sequence includes an ECD portion having a continuous amino acid sequence, comprising CRD1 and / or CRD2 (e.g., CRD1 and CRD2, or CRD2 only) and only a segment or portion of the stalk sequence. A preferred stalk segment comprises one or more amino acids from amino acid residues 105-154 of SEQ ID NO:122. For example, the stalk segment, relative to SEQ ID NO:122, comprises amino acid residues 105, 105-106, 105-107, 105-108, 105-109, 105-110, 105-111, 105-112, 105-113, 105-114, 105-115, 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, 105-131, 105-132, 105-133, 105-134, 105-135, 105-136, 105-137, 105-138, 105-139, 105-140, 105-141, 105-1 It may consist of 42, amino acid residues 105-143, 105-144, 105-145, 105-146, 105-147, 105-148, 105-149, 105-150, 105-151, 105-152, 105-153, and 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 mutated potential furin cleavage sites. In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or moiety with a mutated arginine residue at position 119 (e.g., R119G). In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or moiety with a mutated glutamine residue at position 121 (e.g., Q121P). In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or moiety with a mutated arginine residue at position 122 (e.g., R122Q).

[0162] In some embodiments, the reference TACI sequence is a TACI ECD sequence described in International PCT Publication Numbers 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 described in SEQ ID NO:131. TACI ECD(CRD1 / CRD2):SEQ ID NO:131 TIFF0007866668000013.tif19141

[0164] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence described in SEQ ID NO:130. TACI ECD(CRD1 / CRD2):SEQ ID NO:130 TIFF0007866668000014.tif12153

[0165] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence described in SEQ ID NO:1 (encoded by the nucleotide sequence described in SEQ ID NO:36). TACI ECD(CRD1 / CRD2):SEQ ID NO:1 TIFF0007866668000015.tif12159

[0166] In some embodiments, the reference TACI sequence is essentially composed solely of the CRD2 sequence, with the entire CRD1 sequence and substantially the entire stalk region being deleted or absent, and this is the extracellular domain region of TACI. Previous studies have shown that residues within the stalk region may contain protease cleavage sites, but at least CRD1 and CRD2 were considered necessary for sufficient expression and / or binding activity of TACI to its homologous ligands. For example, in international PCT publication number WO2002 / 094852, it was 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 preceding N-terminal region of CRD1 is necessary for sufficient binding activity of TACI to its homologous ligand; see, for example, international publication number WO2008 / 154814, where 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 that the TACI extracellular region consisting essentially of only CRD2 with a small portion of the stalk region exhibits substantially improved homologous binding activity compared to the longer TACI molecule containing both CRD1 and CRD2 (e.g., Example 3).

[0167] For example, provided herein are immunomodulatory proteins (e.g., TACI-Fc fusion proteins) comprising a TACI polypeptide which is part of the TACI extracellular domain (ECD) region, comprising CRD2, with deletions of the N-terminal region and CRD1, and deletions of one or more residues in the stalk portion of the TACI extracellular domain, compared to the amino acid sequence described in SEQ ID NO:122. In some embodiments, the portion of the TACI extracellular domain comprising CRD2 comprises amino acid residues 71-104 corresponding to the residues described in SEQ ID NO:122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein comprises deletions of N-terminal amino acid residues 1-66 corresponding to the residues described in SEQ ID NO:122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein comprises deletions of N-terminal amino acid residues 1-67 corresponding to the residues described in SEQ ID NO:122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein comprises deletions of N-terminal amino acid residues 1-68 corresponding to the residues described in SEQ ID NO:122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein includes deletions of N-terminal amino acid residues 1-69 corresponding to the residue described in SEQ ID NO:122. In the embodiments provided, the TACI polypeptide of the immunomodulatory protein includes deletions of N-terminal amino acid residues 1-70 corresponding to the residue described in SEQ ID NO:122. In some of any such embodiments, the TACI polypeptide of the immunomodulatory protein lacks one or more consecutive C-terminal amino acid residues, starting from residue 105 and up to amino acid residue 166, or including amino acid residue 166, corresponding to the residue of the ECD sequence described in SEQ ID NO:122. In some embodiments, the ECD sequence is deleted at positions 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.

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

[0169] In particular, some of the TACI polypeptides provided herein have or consist of the sequence described in SEQ ID NO:13 (a TACI ECD sequence encoded by the nucleotide sequence described in SEQ ID NO:48). In some embodiments, the reference TACI sequence has or consists of the sequence described in SEQ ID NO:13, and the provided variant TACI polypeptide is modified by one or more amino acid modifications (e.g., substitutions) described herein compared to such a reference TACI sequence. TACI ECD sequence (CRD2): SEQ ID NO: 13 TIFF0007866668000016.tif5128

[0170] Among the TACI polypeptides provided are variant TACI polypeptides. Also provided are immunomodulatory proteins, e.g., TACI-Fc fusion proteins, comprising the provided TACI polypeptide and the provided TACI polypeptide. In any of the embodiments provided, the variant TACI sequence has the sequence of a reference (e.g., unmodified) TACI sequence, e.g., one of the 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, which comprises one or more amino acid substitutions in the TD domain of the reference (e.g., unmodified or wild-type) TACI polypeptide, so that the variant TACI polypeptide exhibits modified (e.g., increased) binding activity or binding affinity to one or both APRIL or BAFF compared to the reference (e.g., unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has a binding affinity to APRIL and / or BAFF that differs from the binding affinity to 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 to each of the congeneral binding partners is independent. That is, in some embodiments, the variant TACI polypeptide has an increased binding affinity to one or both APRIL and BAFF, and a decreased or unchanged binding affinity to the other of APRIL or BAFF, compared to the reference (e.g., unmodified or wild-type) TACI polypeptide.

[0171] In some embodiments, the variant TACI polypeptide has increased binding affinity to BAFF compared to the reference (unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has increased binding affinity to APRIL compared to the reference (unmodified or wild-type) TACI polypeptide. In some embodiments, the variant TACI polypeptide has increased binding affinity to both APRIL and BAFF compared to the reference (unmodified or wild-type) TACI polypeptide. The congener ligands BAFF and / or APRIL may be mammalian proteins, e.g., human or mouse proteins. In some embodiments, BAFF and / or APRIL are human-type. In some embodiments, the variant TACI polypeptide having increased or enhanced binding affinity to APRIL and / or BAFF has at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 35%, or 50% increased binding affinity compared to the 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 approximately 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, or 50 times. 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 contain one or more amino acid modifications (e.g., substitutions).

[0172] In some embodiments, the equilibrium dissociation constant (K) of any of the embodiments described above is used for BAFF. d ) is 1 × 10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1 × 10 -12It may be less than M. In some embodiments, K of any of the above embodiments relative to BAFF. d is 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 Less than M, or approximately 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 It is less than M. In some embodiments, K of any of the above embodiments relative to BAFF d is 1 × 10 -9 From M, 1 x 10 -12 M or approximately 1 x 10 -12 M is M. In some embodiments, K is one of the aforementioned embodiments relative to BAFF. d is 1 × 10 -9 M or approximately 1 x 10 -9 M, 2×10 -9 M or approximately 2 x 10 -9 M, 4×10 -9 M or approximately 4 x 10 -9 M, 6×10 -9 M or approximately 6 x 10 -9 M, 8×10 -9 M or approximately 8 x 10 -9 M, 1×10 -10 M or approximately 1 x 10 -10 M, 2×10 -10 M or approximately 2 x 10 -10 M, 4×10 -10 M or approximately 4 x 10 -10 M, 6×10 -10 M or approximately 6 x 10 -10 M, 8×10 -10 M or approximately 8 x 10 -10 M, 1×10 -11 M or approximately 1 x 10 -11 M, 2×10 -11 M or approximately 2 x 10 -11 M, 4×10 -11 M or approximately 4 x 10 -11 M, 6×10 -11 M or approximately 6 x 10 -11 M, 8×10 -11 M or approximately 8 x 10-11 M or 1×10 -12 M or approximately 1×10 -12 M, or any value between any of the foregoing. In some embodiments, the provided embodiment comprises the variant TACI polypeptide described above, and the K d for BAFF is more than 1.5-fold, or approximately more than 1.5-fold, e.g., more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more, or approximately more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more (increase in binding affinity).

[0173] In some embodiments, the equilibrium dissociation constant (K d ) for any of the foregoing embodiments with respect to APRIL is 1×10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1×10 -12 M may be less than. In some embodiments, the K d for any of the foregoing embodiments with respect to APRIL is 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1×10 -12 M is less than, or approximately 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1×10 -12 M is less than. In some embodiments, the K d for any of the foregoing embodiments with respect to APRIL is 1×10 -9 M to 1×10 -12 M or approximately 1×10 -12 M. In some embodiments, the K d for any of the foregoing embodiments with respect to APRIL is 1×10 -9 M or approximately 1×10 -9 M, 2×10 -9 M or approximately 2×10 -9 M, 4×10-9 M or approximately 4 x 10 -9 M, 6×10 -9 M or approximately 6 x 10 -9 M, 8×10 -9 M or approximately 8 x 10 -9 M, 1×10 -10 M or approximately 1 x 10 -10 M, 2×10 -10 M or approximately 2 x 10 -10 M, 4×10 -10 M or approximately 4 x 10 -10 M, 6×10 -10 M or approximately 6 x 10 -10 M, 8×10 -10 M or approximately 8 x 10 -10 M, 1×10 -11 M or approximately 1 x 10 -11 M, 2×10 -11 M or approximately 2 x 10 -11 M, 4×10 -11 M or approximately 4 x 10 -11 M, 6×10 -11 M or approximately 6 x 10 -11 M, 8×10 -11 M or approximately 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10 -12 M is any value between any of the above. In some embodiments, the provided embodiment comprises the above variant TACI polypeptide and K relative to APRIL. d The binding affinity decreases by more than 1.5 times, or by approximately more than 1.5 times, for example, by more than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, or by more than approximately 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more (increase in binding affinity).

[0174] A reference (e.g., unmodified or wild-type) TACI sequence does not necessarily have to be used as a starting composition for generating the variant TACI polypeptides described herein. Therefore, the use of the term “modified,” e.g., “substitution,” does not imply that this embodiment is limited to a specific method for producing variant TACI polypeptides or immunomodulatory proteins containing them. Variant TACI polypeptides can be produced, for example, by de novo peptide synthesis, and therefore do not necessarily require modifications such as “substitution,” in the sense of altering codons to encode substitutions. This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific method of production. The means for designing or producing variant TACI polypeptides are not limited to a specific method. However, in some embodiments, a reference (e.g., unmodified or wild-type) TACI coding nucleic acid is mutagenicated from a reference (e.g., unmodified or wild-type) TACI genetic material and screened for 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, as is the UniProtKB database mentioned above.

[0175] Unless otherwise indicated, as shown throughout this disclosure, amino acid modifications in variant TACI polypeptides are specified by amino acid position numbers corresponding to the position numbering of the reference ECD sequence listed in SEQ ID NO:122. Identifying the corresponding positions of modifications, such as amino acid substitutions, within a TACI polypeptide, including its TD (e.g., CRD1 and / or CRD2), by, for example, alignment of a reference sequence (e.g., SEQ ID NO:1 or 13) with SEQ ID NO:122, is within the scope of the art. An example of an alignment for identifying corresponding residues is shown in Figure 9. In the list of modifications throughout this disclosure, amino acid positions are shown in the center, with the corresponding reference (e.g., unmodified or wild-type) amino acids listed before the number, and the identified variant amino acid substitutions 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 their centrally located amino acid positions, the corresponding reference amino acids are listed before and after the numbers, and identified variant amino acid insertions are listed after the unmodified (e.g., wild-type) amino acids.

[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 of those described. One or more amino acid modifications, e.g., substitutions, may be located within the ectodomain (extracellular domain) of the reference (e.g., unmodified or wild-type) TACI sequence. In some embodiments, one or more amino acid modifications, e.g., substitutions, are located within the CRD1 domain or its specific binding fragment. In some embodiments, one or more amino acid modifications, e.g., substitutions, are located within the CRD2 domain or its specific binding fragment. In some embodiments of the variant TACI polypeptide, some of the one or more amino acid modifications, e.g., substitutions, are located within the CRD1 domain or its specific binding fragment, and some of the one or more amino acid modifications, e.g., substitutions, are located within the CRD2 domain or its specific binding fragment.

[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, may be located within the CRD1 domain or the CRD2 domain. In some embodiments, the variant TACI polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in the CRD1 domain or its specific binding fragment of the reference TACI sequence. 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 or its specific binding fragment of the reference TACI sequence.

[0178] 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 with the reference (e.g., unmodified or wild-type) TACI polypeptide shown in SEQ ID NO:122 or its specific binding fragment, which contains the CRD1 and / or CRD2 domains. In some embodiments, the specific binding fragment contains the CRD1 domain, for example, the specific binding fragment contains the sequence shown in amino acids 34-66 of SEQ ID NO:122. In some cases, the CRD1 domain is the only complete CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is the CRD2 domain or contains the CRD2 domain, for example, the specific binding fragment contains the sequence shown in amino acids 71-104 of SEQ ID NO:122. In some cases, the CRD2 domain is the only complete CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is either the CRD1 domain and the CRD2 domain, or contains the CRD1 domain and the CRD2 domain, for example, the specific binding fragment contains amino acids 34-104 of SEQ ID NO:122. In some embodiments, the specific binding fragment contains a contiguous portion of the stalk domain, for example, the specific binding fragment contains a contiguous portion of amino acids 105-165 of SEQ ID NO:122. In some of the arbitrary embodiments, the specific binding fragment of SEQ ID NO:122 is shorter than the full-length ECD shown in SEQ ID NO:122. In some embodiments, the specific binding fragment is shown in SEQ ID NO:1. In some embodiments, the specific binding fragment is shown in SEQ ID NO:13. In some embodiments, the specific binding fragment is shown in SEQ ID NO:130. In some embodiments, the specific binding fragment is shown in SEQ ID NO:131.

[0179] In some embodiments, a variant TACI polypeptide comprising 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 with a reference (e.g., unmodified or wild-type) TACI polypeptide or its specific binding fragment having an amino acid sequence such as SEQ ID NO: 1, 13, or 122.

[0180] In some embodiments, a variant TACI polypeptide comprising 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 with the amino acid sequence of SEQ ID NO:122.

[0181] In some embodiments, a variant TACI polypeptide comprising 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 with the amino acid sequence of SEQ ID NO:1.

[0182] In some embodiments, a variant TACI polypeptide comprising 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 with the amino acid sequence of SEQ ID NO:13.

[0183] In some embodiments, a variant TACI polypeptide comprising 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 with the amino acid sequence of SEQ ID NO:130.

[0184] In some embodiments, a variant TACI polypeptide comprising 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 with 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, on the reference TACI polypeptide or its specific binding fragments 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, relative to the numbering of SEQ ID NO:122. In some embodiments, the variant TACI polypeptide BIM has one or more amino acid modifications, such as 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 substitutions selected from their conserved amino acid substitutions. In some embodiments, the reference TACI polypeptide includes a CRD1 domain or a CRD2 domain, for example, the reference TACI polypeptide is described in SEQ ID NO:1 or SEQ ID NO:122.

[0186] In some embodiments, the amino acid substitutions are located only in the CRD2 domain. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications, such as substitutions, in the reference TACI polypeptide or its specific binding fragments 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, relative to the numbering of SEQ ID NO: 709. In some embodiments, the variant TACI polypeptide BIM has one or more amino acid modifications, such as substitutions selected from E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions. In some embodiments, the reference TACI polypeptide contains only the CRD2 domain of the CRD domain but lacks the CRD1 domain, for example, the reference TACI polypeptide described in SEQ ID NO:13. Therefore, in some embodiments, the variant TACI polypeptide includes a portion of the ECD sequence of a TACI polypeptide that contains a CRD2 domain but lacks a CRD1 domain.

[0187] Conservative amino acid modifications, such as substitutions, are any amino acid in the same class as the substituted amino acid, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, and tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartate, glutamate, asparagine, and glutamine).

[0188] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 75, relative to the numbering 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 an 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 a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 75 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 75 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 75 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 75 is glutamine (Gln, Q).

[0189] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 77 relative to the numbering 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 an amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or amide. In some embodiments, the substituted amino acid at position 77 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 77 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 77 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 77 is glutamine (Gln, Q).

[0190] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 78 relative to the numbering 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 a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 78 is phenylalanine (Phe, F). In some embodiments, the substituted amino acid at position 78 is tyrosine (Tyr, Y). In some embodiments, the substituted amino acid at position 78 is tryptophan (Trp, W).

[0191] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 84 relative to the numbering 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 an amino acid substitution. In some embodiments, the substituted amino acid at position 84 is an acidic amino acid or 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 contains at least one amino acid substitution at position 101 relative to the numbering 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 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 contains at least one amino acid substitution at position 102, relative to the numbering 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 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, the variant TACI polypeptide contains at least one amino acid substitution E74V. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution K77E. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution L82H. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution L82P. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution R84G. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution R84L. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution D85V. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution A101D. In some embodiments, the variant TACI polypeptide contains at least one amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide contains two or more of the aforementioned amino acid substitutions. In some embodiments, the variant TACI polypeptide contains one or more amino acid substitutions that are any of the aforementioned conservative amino acid substitutions. In the embodiments provided, the variant TACI polypeptide contains at least one amino acid substitution in any of the described reference TACI polypeptide sequences. In some embodiments, at least one amino acid substitution is in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, at least one amino acid substitution is in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, at least one amino acid substitution is in the reference TACI sequence shown in SEQ ID NO:130.In some embodiments, at least one amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

[0195] In some embodiments, the variant TACI polypeptide contains the amino acid substitution E74V. In some embodiments, the variant TACI polypeptide contains the amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide contains the amino acid substitution K77E. In some embodiments, the variant TACI polypeptide contains the amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide contains the amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide contains the amino acid substitution L82H. In some embodiments, the variant TACI polypeptide contains the amino acid substitution L82P. In some embodiments, the variant TACI polypeptide contains the amino acid substitution R84G. In some embodiments, the variant TACI polypeptide contains the amino acid substitution R84L. In some embodiments, the variant TACI polypeptide contains the amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide contains the amino acid substitution D85V. In some embodiments, the variant TACI polypeptide contains the amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide contains the amino acid substitution A102D. In some embodiments, the variant TACI polypeptide contains the amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide contains two or more of the aforementioned amino acid substitutions. In some embodiments, the variant TACI polypeptide contains one or more amino acid substitutions which are any of the aforementioned conservative amino acid substitutions. In the embodiments provided, the variant TACI polypeptide contains an amino acid substitution located in any of the described reference TACI polypeptide sequences. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

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

[0197] In some of the various embodiments, the variant TACI polypeptide contains 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 contains 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, one or more amino acid substitutions include Q75E / R84Q. In some of the embodiments, one or more amino acid substitutions include Q75E / K77E. In some of the embodiments, one or more amino acid substitutions include Q75E / F78Y. In some of the embodiments, one or more amino acid substitutions include Q75E / A101D. In some of the embodiments, one or more amino acid substitutions include Q75E / Y102D. In some of the embodiments, one or more amino acid substitutions include F77E / F78Y. In some of the embodiments, one or more amino acid substitutions include K77E / R84Q. In some of the embodiments, one or more amino acid substitutions include K77E / A101D. In some of the embodiments, one or more amino acid substitutions include K77E / Y102D. In some of the embodiments, one or more amino acid substitutions include F78Y / R84Q. In some of the embodiments, one or more amino acid substitutions include F78Y / A101D. In some of the embodiments, one or more amino acid substitutions include F78Y / Y102D. In some of the embodiments, one or more amino acid substitutions include R84Q / A101D. In some of the embodiments, one or more amino acid substitutions include R84Q / Y102D. In some of the embodiments, one or more amino acid substitutions include A101D / Y102D. In the embodiments provided, the variant TACI polypeptide includes amino acid substitutions in any of the described reference TACI polypeptide sequences, for example, in the sequences shown 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 is an amino acid substitution. This includes TIFF0007866668000017.tif19158. In the provided embodiment, the variant TACI polypeptide includes amino acid substitutions in any of the described reference TACI polypeptide sequences, for example, in the sequences shown 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 the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions present in any of the described reference TACI polypeptide sequences. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:131.

[0201] In some embodiments, the variant TACI polypeptide comprises the amino acid substitutions K77E and Y102D (K77E / Y102D). In the embodiments provided, the variant TACI polypeptide comprises the amino acid substitutions present in any of the described reference TACI polypeptide sequences. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

[0202] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions F78Y and Y102D (F78Y / Y012D). In the embodiments provided, the variant TACI polypeptide contains the amino acid substitutions present in any of the described reference TACI polypeptide sequences. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:131.

[0203] In some embodiments, the variant TACI polypeptide contains the amino acid substitutions K77E, F78Y, and Y102D (K77E / F78Y / Y102D). In the embodiments provided, the variant TACI polypeptide contains the amino acid substitutions present in any of the described reference TACI polypeptide sequences. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitutions are located in the reference TACI sequence shown in SEQ ID NO:131.

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

[0205] In some embodiments, variant TACI polypeptides may contain any of the mutations listed in Table 1. Table 1 also shows exemplary sequences by referencing the SEQ ID NOs of the reference (e.g., unmodified) TACI polypeptide and the exemplary variant TACI polypeptide. As shown, the exact locus or residues corresponding to a particular domain may vary, for example, depending on the method used to identify or classify the domain. Also, in some cases, adjacent N-terminal and / or C-terminal amino acids of a particular domain (e.g., CRD) may be included in the variant TACI polypeptide sequence, for example, to ensure correct domain folding during expression. Therefore, the examples of SEQ ID NOs in Table 1 should not be interpreted as limiting. For example, a particular domain of a variant TACI polypeptide, e.g., the ECD domain or part thereof containing only CRD1 / CRD2 or CRD2, may be several amino acids longer or shorter than the amino acid sequence shown in its respective SEQ ID NO, for example, by 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0206] In some embodiments, the variant TACI polypeptide contains one of the mutations (amino acid substitutions) listed in Table 1. In one example, a mutation (amino acid substitution) is made to a reference TACI containing the amino acid sequence shown in SEQ ID NO:122. In another example, a mutation (amino acid substitution) is made to a reference TACI containing the CRD1 and CRD2 domains of TACI, as shown in SEQ ID NO:1. In yet another example, a mutation (amino acid substitution) is made to a reference TACI that has been further cleaved by deleting the N-terminal and C-terminal amino acid residues to retain CRD2, as shown in SEQ ID NO:13.

[0207] The use of terms such as “substitution” or “mutation” as “modification” does not mean that this embodiment is limited to a specific method for producing immunomodulatory proteins. Variant TACI polypeptides may be produced, for example, by novel peptide synthesis, and therefore do not necessarily require modification, such as “substitution” in the sense of changing codons to encode substitutions. This principle also extends to the terms “addition” and “deletion” of amino acid residues, and likewise does not mean a specific method of production. The means by which vTDs are designed or produced are not limited to any specific method. However, in some embodiments, nucleic acids encoding wild-type or unmodified TDs are mutagenesized from wild-type or unmodified TD genetic material and screened for desired specific binding activity, e.g., binding affinity, and / or changes in NF-κB modulation or other functional activity. In some embodiments, vTDs are novelly synthesized using protein or nucleic acid sequences available in any number of publicly available databases and then screened. Such information is provided by the National Center for Biotechnology Information, whose website is publicly available via the internet, such as the UniProtKB database.

[0208] In some embodiments, the variant TACI polypeptide comprises a variant TACI polypeptide represented by one of the extracellular domain (ECD) sequences containing CRD1 and CRD2, e.g., SEQ ID NO: 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 one of the SEQ ID NO: 2-12, 21, 22, 101-120, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide comprises one specific binding fragment with sequence ID numbers 2-12, 21, 22, and 101-120, the specific binding fragment being bound to BAFF, APRIL, or a BAFF / APRIL heterotrimer and containing a continuous sequence that includes amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0209] In some embodiments, the variant TACI polypeptide consists of or is essentially derived from a variant TACI extracellular domain (ECD) sequence shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120. In some embodiments, the variant TACI polypeptide consists of or is essentially derived from a polypeptide sequence that exhibits at least about 90% identity to any one of SEQ ID NO: 2-12, 21, 22, 101-120, 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, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or is essentially composed of one specific binding fragment from SEQ ID NO: 2-12, 21, 22, 101-120, the specific binding fragment being bound to BAFF, APRIL, or APRIL / BAFF heterotrimer and containing a continuous sequence that includes amino acid modifications, e.g., substitutions, not present in the reference (e.g., unmodified or wild-type) TACI.

[0210] In some embodiments, the variant TACI polypeptide comprises a variant TACI polypeptide represented by one of the following SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192, which contains CRD2 but lacks CRD1 of the reference TACI polypeptide. 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, for example, at least about 96% identity, 97% identity, 98% identity, or 99% identity to one of the following SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192, and retains amino acid modifications, such as substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide comprises one specific binding fragment from among SEQ ID NO: 14-20, 23-35, 92-100, and 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and contains a continuous sequence containing amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0211] In some embodiments, the variant TACI polypeptide consists of or is essentially derived from the sequence shown in one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192. In some embodiments, the variant TACI polypeptide consists of or is essentially derived from the 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 one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of, or is essentially composed of, one specific binding fragment with SEQ ID NO: 14-20, 23-35, 92-100, or 177-192, the specific binding fragment being bound to BAFF, APRIL, or a BAFF / APRIL heterotrimer and containing a continuous sequence that includes amino acid modifications, e.g., substitutions, not present in the reference (e.g., unmodified or wild-type) TACI.

[0212] In some embodiments, the variant TACI polypeptide contains the sequence shown in SEQ ID NO:20. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence shown in SEQ ID NO:20. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO:20.

[0213] In some embodiments, the variant TACI polypeptide contains the sequence shown in SEQ ID NO:26. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence shown in SEQ ID NO:26. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO:26.

[0214] In some embodiments, the variant TACI polypeptide contains the sequence shown in SEQ ID NO:27. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence shown in SEQ ID NO:27. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO:27.

[0215] In some embodiments, the variant TACI polypeptide contains the sequence shown in SEQ ID NO:107. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence shown in SEQ ID NO:107. In some embodiments, the variant TACI polypeptide consists of the sequence shown in SEQ ID NO:107.

[0216] In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence shown in any of SEQ ID NO: 37-47, 56, or 57. 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 NO: 37-47, 56, or 57, and is encoded by a nucleotide sequence that retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. Nucleic acids are also provided herein that contain sequences shown in any of SEQ ID NO:37-47, 56, or 57, or sequences showing at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO:37-47, 56, or 57.

[0217] In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence shown in either SEQ ID NO: 49-55 or 58-70. In some embodiments, the variant TACI polypeptide exhibits at least about 90% identity to one of SEQ ID NO: 49-55 or 58-70, 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, and is encoded by a nucleotide sequence that retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. Nucleic acids are also provided herein that contain sequences that exhibit at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 49-55 or 58-70.

[0218] (Table 1) Exemplary variant TACI TIFF0007866668000018.tif116165TIFF0007866668000019.tif230165TIFF0007866668000020.tif207165

[0219] In some embodiments, TACI ECD fusion sequences are also provided herein in which any of the above-mentioned TACI ECD sequences are linked or fused with a multimerization domain, for example, any multimerization domain described herein.

[0220] The interaction of two or more polypeptides of immunomodulatory proteins can be facilitated by directly or indirectly linking them to other polypeptides that can interact with any portion or themselves to form a stable structure. For example, separate encoding polypeptide chains can be linked by polymerization, which is mediated by polymerizing domains. Typically, polymerizing domains create 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 together by multimerization, for example, as a dimeric, trimer, tetramer, or pentamer molecule. In some cases, the individual polypeptides are identical. For example, a trimer may be formed from three copies of the same individual polypeptide. In other examples, a tetramer is made from four copies of the same individual polypeptide. In further examples, a pentamer is made from five copies of the same individual polypeptide. The multimerization domain may also be a domain that facilitates dimerization, trimerization, tetramerization, or pentamerization of the polypeptide chain.

[0222] In some embodiments, immunomodulatory proteins form multimers, such as dimers. In some embodiments, the dimer is a homodimer, where the two polypeptides of the immunomodulatory protein are the same. In some embodiments, the dimer is a heterodimer, where the two polypeptides of the immunomodulatory protein are different.

[0223] In some embodiments, the multimerization domain includes any one capable of forming stable protein-protein interactions. The multimerization domain can interact via immunoglobulin sequences (e.g., Fc domains; see, for example, 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); leucine zippers (e.g., derived from nuclear transforming proteins fos and jun, or from the proto-oncogene c-myc or General Control of Nitrogen (GCN4)) (see, for example, Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz ​​et al., (1989) Science, 243:1695-1699); hydrophobic regions; hydrophilic regions; or via free thiols that form intermolecular disulfide bonds between homomultimer or heteromultimer chimeric molecules. Furthermore, the multimerizing domain may include an amino acid sequence containing a protrusion complementary to the hole-containing amino acid sequence, as described, for example, in U.S. Patent No. 5,731,168; International Patent Publication Nos. WO98 / 50431 and WO2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such multimerizing regions can be manipulated so that steric interactions not only promote stable interactions but also further promote the formation of more heterodimers than homodimers from a mixture of chimeric monomers. Generally, the protrusion is constructed by exchanging a small amino acid side chain originating 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 at the interface of the second polypeptide by exchanging the larger amino acid side chain with a smaller side chain (e.g., alanine or threonine). Exemplary multimerizing domains are described below.

[0224] A TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) can be ligated anywhere, but typically, it can be ligated via its N-terminus or C-terminus to the N-terminus or C-terminus of a multimerizing domain to form a chimeric polypeptide. The ligation may be direct or indirect via a linker. The chimeric polypeptide may also be a fusion protein, which may be formed by chemical ligation, for example, via covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerizing domain, a nucleic acid encoding all or part of a TACI polypeptide sequence, such as any described TACI ECD containing a variant TACI polypeptide sequence, may be functionally ligated directly or indirectly, or optionally via a linker domain, to the nucleic acid encoding the multimerizing domain sequence. In some cases, the construct encodes a chimeric protein in which the C-terminus of the TACI polypeptide sequence is ligated to the N-terminus of a multimerizing domain. In some cases, the construct encodes a chimeric protein in which the N-terminus of the TACI polypeptide sequence is ligated to the N-terminus or C-terminus of a multimerizing domain.

[0225] Polypeptide multimers contain two chimeric proteins created by directly or indirectly linking two identical or different TACI polypeptide sequences (e.g., two identical or different variant TACI polypeptide sequences) to the multimerizing domain. In some cases where the multimerizing domain is a polypeptide, a gene fusion encoding the TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) and the multimerizing domain is inserted into a suitable expression vector. The resulting chimeric or fusion protein can be expressed and assembled in host cells transformed with a recombinant expression vector to construct a multimer. In this case, the multimerizing domains interact to form a polyvalent polypeptide. The multimerizing 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 multimer formed therefrom, can be purified by any suitable method, for example, affinity chromatography using a protein A column or a protein G column. When two nucleic acid molecules encoding different polypeptides are introduced into cells by transformation, homodimer and heterodimer formation occurs. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

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

[0228] In some embodiments, the polymerizing domain is an immunoglobulin (e.g., IgG1) Fc region, and the fusion protein is a TACI-Fc comprising (1) a TACI sequence containing or consisting of any of the provided TACI ECD sequences, and (2) an immunoglobulin Fc region. Thus, among the embodiments provided, there is a TACI-Fc fusion protein comprising (1) a TACI sequence containing or consisting of any of the above TACI ECD polypeptide sequences, e.g., a variant TACI polypeptide, and (2) an immunoglobulin Fc region.

[0229] In some embodiments, a TACI-Fc fusion protein is provided herein that comprises (1) a TACI ECD sequence including the sequence shown in SEQ ID NO:13, and (2) an immunoglobulin Fc region. In some embodiments, a TACI-Fc fusion protein is provided herein that comprises (1) a TACI ECD sequence consisting of or essentially the sequence shown 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 comprising, or consisting of, one of the above-described variant TACI polypeptides and an immunoglobulin Fc region.

[0231] In some embodiments, variant TACI-Fc fusion sequences are provided herein that include (1) a TACI ECD sequence containing CRD1 and CRD2, for example, a TACI sequence containing one of the sequences shown in SEQ ID NO: 2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region. In some embodiments, variant TACI-Fc fusion sequences are provided herein that include (1) a TACI ECD sequence containing CRD1 and CRD2, for example, a TACI sequence consisting of or essentially derived from one of the sequences shown in SEQ ID NO: 2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region.

[0232] In some embodiments, variant TACI-Fc fusion sequences are provided herein that contain (1) a TACI ECD sequence containing a CRD2 domain but lacking a CRD1 domain, for example, a sequence shown as one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192, and (2) an immunoglobulin Fc region. In some embodiments, variant TACI-Fc fusion sequences are provided herein that contain (1) a TACI sequence consisting of, or essentially derived from, a sequence shown as one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192, but lacking a CRD2 domain, and (2) an immunoglobulin Fc region.

[0233] In the provided embodiment of TACI-Fc, the immunoglobulin Fc region may be a wild-type immunoglobulin Fc, e.g., IgG1 Fc. In some cases, the Fc region may be a variant Fc lacking effector function (also called "effectorless Fc"). Exemplary Fc regions and their variants found in the provided TACI-Fc fusion protein are described below.

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

[0235] In some embodiments, the Fc region is or includes the sequence shown in any one of SEQ ID NO: 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 originates from IgG1, for example, IgG1 shown in any one of SEQ ID NO: 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 derived from IgG2, e.g., any IgG2 shown in SEQ ID NO: 138 or 219. In some embodiments, the Fc region is or derived from IgG4, e.g., any IgG4 shown in SEQ ID NO: 139, 140, or 220. In some embodiments, the Fc region in the Fc fusion protein provided herein may also include Fc regions that exhibit at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of any of the above-mentioned Fc regions.

[0236] In some embodiments, Fc is derived from IgG1, for example, human IgG1. In some embodiments, Fc is IgG1 Fc shown in SEQ ID NO:71, having an allotype containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbering. In some embodiments, Fc contains the amino acid sequence shown in SEQ ID NO:71, 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 greater sequence identity with respect to SEQ ID NO:71. In other embodiments, Fc is IgG1 Fc containing amino acids of the human G1m1 allotype, for example, residues containing Asp(D) and Leu(L) at positions 356 and 358, for example, the residues shown in SEQ ID NO:81. Accordingly, in some cases, the Fc provided herein may contain amino acid substitutions E356D and M358L to reconstitute the residue of allotype G1m1. In some embodiments, the Fc contains the amino acid sequence shown in SEQ ID NO:81, or contains an amino acid sequence exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity with respect to SEQ ID NO:81.

[0237] In some embodiments, the Fc region has the amino acid sequence shown in SEQ ID NO:81. TIFF0007866668000021.tif33160

[0238] In some embodiments, variant Fc includes the sequence shown in SEQ ID NO:173. In some embodiments, variant Fc includes the sequence shown in SEQ ID NO:174. In some embodiments, the Fc region used in the constructs provided herein may further lack a C-terminal lysine residue.

[0239] In some embodiments, Fc is derived from IgG2, such as human IgG2. In some embodiments, Fc contains the amino acid sequence shown in SEQ ID NO:138, or contains an amino acid sequence exhibiting at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity with respect to SEQ ID NO:138. In some embodiments, the Fc region is an IgG2 Fc region having the sequence shown in SEQ ID NO:138. In some embodiments, the Fc region is an IgG2 Fc region having the sequence shown in SEQ ID NO:219.

[0240] In some embodiments, Fc is derived from IgG4, for example, human IgG4. In some embodiments, Fc includes the amino acid sequence shown in SEQ ID NO:139, or includes an amino acid sequence exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity with respect to SEQ ID NO:139. In some embodiments, IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced with the CH3 domain of human IgG1, exhibiting 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, as indicated by the EU index proposed by Kabat et al., is replaced with lysine, exhibiting inhibited aggregate formation (see, for example, U.S. Patent No. 8,911,726). In some embodiments, Fc is IgG4 containing the S228P mutation, which has been shown to inhibit recombination between the therapeutic antibody and endogenous IgG4 by Fab arm exchange (see, for example, Labrijin et al. (2009) Nat. Biotechnol., 27(8): 767-71). In some embodiments, Fc includes the amino acid sequence shown in SEQ ID NO:140, or an amino acid sequence exhibiting at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity with respect to SEQ ID NO:140. In some embodiments, the Fc region is the IgG4 Fc region shown in SEQ ID NO:140. In some embodiments, the Fc region is the IgG4 Fc region shown in SEQ ID NO:220.

[0241] In some embodiments, the Fc region is a variant Fc region in which the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or to inactivate Fc for its 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. Generally, in addition to antigen-binding ability, which is the primary function of immunoglobulins, the Fc region is responsible for effector functions, such as complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by extending the in vivo half-life through conjugation to the in vivo FcRn receptor. In some embodiments, such functions may be reduced or modified in the Fc region for use with the provided Fc fusion protein.

[0243] In some embodiments, one or more amino acid modifications may be introduced into the Fc region to create 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 incorporated herein by reference as expressly as possible.

[0244] In some embodiments, the immunomodulatory proteins offered exhibit reduced effector function, for which the in vivo half-life of the immunomodulatory protein is important, but which contain an Fc region that is a desirable candidate for applications where certain effector functions (e.g., CDC and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the immunomodulatory protein lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, which are primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and U.S. Patent 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, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, Calif.) and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that immunomodulatory proteins cannot bind to C1q and therefore lack CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC assays are sometimes performed to evaluate complement activation (see, for example, 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, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0245] Immunomodulatory 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 the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine, and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, according to EU numbering (U.S. Patent No. 7,332,581).

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

[0247] Certain Fc variants exhibiting improved or reduced binding to FcR have been described (see, for example, 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 include a variant Fc region comprising one or more amino acid substitutions that extend the half-life and / or improve binding to the fetal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO2015107026. These antibodies include an Fc region with one or more substitutions that improve binding between the Fc region and FcRn. Such Fc variants include those with substitutions of one or more 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, for example, substitution of 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 WO94 / 29351.

[0250] In some embodiments, modifications are made in the Fc region to reduce C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0251] In some embodiments, the variant Fc region, which includes one or more amino acid modifications (e.g., amino acid substitutions), is derived from wild-type IgG1, e.g., wild-type human IgG1. In some embodiments, the wild-type IgG1 Fc may also be the Fc shown in SEQ ID NO:71, which has an allotype containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbering. In some embodiments, the variant Fc region is derived from the amino acid sequence shown in SEQ ID NO:71. In other embodiments, the wild-type IgG1 Fc contains amino acids from the human G1m1 allotype, e.g., residues containing Asp(D) and Leu(L) at positions 356 and 358, e.g., as shown in SEQ ID NO:81. Thus, in some cases, the variant Fc is derived from the amino acid sequence shown 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 in EU numbering).

[0253] In some embodiments, the variant Fc region includes a C5S amino acid modification of the wild-type or unmodified Fc region, numbered SEQ ID NO:71 (corresponding to C220S in EU numbering).

[0254] In some embodiments, the Fc region is a variant Fc containing at least one amino acid substitution that is N82G, numbered as SEQ ID NO:71 (corresponding to N297G in EU numbering). In some embodiments, Fc further contains at least one amino acid substitution that is R77C or V87C, numbered as SEQ ID NO:71 (corresponding to R292C or V302C in EU numbering). In some embodiments, the variant Fc region further contains a C5S amino acid modification, numbered as SEQ ID NO:71 (corresponding to C220S in EU numbering). For example, in some embodiments, the variant Fc region includes the following amino acid modification: N297G and one or more of the following amino acid modifications C220S, R292C, or V302C according to EU numbering (corresponding to N82G and one or more of the following amino acid modifications C5S, R77C, or V87C based on SEQ ID NO: 71), for example, the Fc region includes the sequence shown in SEQ ID NO: 82.

[0255] In some embodiments, variant Fc contains the amino acid substitution L234A / L235E / G237A according to EU numbering. In some embodiments, variant Fc contains the amino acid substitution A330S / P331S according to EU numbering. In some embodiments, variant Fc contains the amino acid substitution L234A / L235E / G237A / A330S / P331S (Gross et al. (2001) Immunity 15:289). In some embodiments, variant Fc contains the sequence shown in SEQ ID NO:175. In some embodiments, variant Fc contains the sequence shown in SEQ ID NO:176. In some embodiments, the Fc region used in the constructs provided herein may further lack a C-terminal lysine residue.

[0256] In some embodiments, the Fc region is a variant Fc containing the mutations L234A, L235E, and G237A, as indicated by EU numbering. In some embodiments, the wild-type Fc is further modified by the removal of one or more cysteine ​​residues, for example, by the exchange of a cysteine ​​residue for a serine residue at position 220 (C220S) as indicated by EU numbering. Exemplary inactive Fc regions with reduced effector function are shown in SEQ ID NO:83 and SEQ ID NO:75, which are based on the allotype shown in SEQ ID NO:71 or SEQ ID NO:81, respectively. In some embodiments, the Fc region may further lack a C-terminal lysine residue. In some embodiments, the variant Fc region contains one or more amino acid modifications C220S, L234A, L235E, or G237A, for example, the Fc region contains the sequence shown in SEQ ID NO:73, 75, 83, or 136. In some embodiments, variant Fc includes the sequence shown in SEQ ID NO:73. In some embodiments, variant Fc includes the sequence shown in SEQ ID NO:75. In some embodiments, variant Fc includes the sequence shown in SEQ ID NO:83. In some embodiments, variant Fc includes the sequence shown in SEQ ID NO:136.

[0257] In some embodiments, the Fc region is variant Fc having the sequence shown in SEQ ID NO:73. TIFF0007866668000022.tif33159

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

[0259] In some embodiments, the Fc region is variant Fc having the sequence shown in SEQ ID NO:221. TIFF0007866668000023.tif33160

[0260] In some embodiments, the Fc region is a variant Fc region containing one or more amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, for example, the Fc region contains the sequence shown in SEQ ID NO:134. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del in EU numbering).

[0261] In some embodiments, the Fc region is a variant Fc region containing one or more amino acid modifications C220S, R292C, N297G, and V302C. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del in EU numbering). An exemplary variant Fc region is shown in SEQ ID NO:135.

[0262] In some embodiments, the variant Fc region contains one or more amino acid modifications C220S / E233P / L234V / L235A / G236del / S267K. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO:71 (corresponding to K447del in EU numbering). An exemplary variant Fc region is shown in SEQ ID NO:137.

[0263] Table 2 shows examples of such Fc regions suitable for inclusion in immunomodulatory polypeptides.

[0264] (Table 2) Exemplary IgG1 Fc region, wild type or variant (effectorless) TIFF0007866668000024.tif127160

[0265] In some embodiments, the Fc region is a variant Fc region containing any combination of the Fc mutations listed in Table 2. In some embodiments, the Fc region is a variant Fc region having the sequence shown in any one of the SEQ ID NOs listed in Table 2.

[0266] For example, the variant Fc region may be an effectorless Fc exhibiting reduced effector activity compared to wild-type IgG1 shown in SEQ ID NO:71 or SEQ ID NO:81. In some embodiments, the variant Fc includes 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 greater sequence identity with any of SEQ ID NO:75, 82, 83, 134, 73, 135, 136, or 137. In some embodiments, the variant Fc has the sequence shown in SEQ ID NO:73. In this embodiment, when produced and expressed from cells, the immunomodulatory protein provided (e.g., TACI-Fc fusion) 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 also 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 include modifications (e.g., substitutions) such that the interface of the Fc molecule is modified to facilitate and / or promote heterodimerization. Methods for promoting heterodimerization of the Fc chain include mutagenesis of the Fc region, for example, by including a series of "knob-into-hole" mutations, or by including mutations that cause electrostatic steering of Fc and promote attractive interactions between different polypeptide chains. In some embodiments, the Fc region of the heterodimer molecule may further include one or more other Fc mutations, e.g., any of the above. In some embodiments, the heterodimer molecule includes an Fc region having a mutation that reduces effector function. In some embodiments, such an Fc region includes mutations C220S, L234A, L235E and / or G237A, according to EU numbering. In some embodiments, any of the above mutations in the Fc skeleton can be generated within an allotype containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbering. In other embodiments, any of the above mutations in the Fc skeleton can be generated within an allotype containing residues Asp(D) and Leu(L) at positions 356 and 358 according to EU numbering.

[0269] In some embodiments, the modification involves introducing a knob into the first Fc polypeptide and a hole into the second Fc polypeptide so that the knob can be positioned within a cavity to facilitate complexation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create a knob or cavity within the polypeptide are typically interfacial amino acids that interact with or come into contact with 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 involves substituting a native or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide and is therefore locatable in a compensatory cavity (hole) at an adjacent interface of the second polypeptide. In most cases, the substituted amino acid has a larger side chain volume than the original amino acid residue. Those skilled in the art know how to characterize and / or evaluate amino acid residues to identify an amino acid residue that is an ideal substitution amino acid for forming a knob. In some embodiments, the substituted residues for forming the knob are naturally occurring amino acid residues, which include, 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 modification of a second polypeptide to contain a cavity (hole) involves substituting a native or original amino acid with an amino acid having at least one side chain that is recessed from the interface of the second polypeptide and thus capable of accommodating a corresponding projection 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 characterize and / or evaluate amino acid residues to identify amino acid residues that are ideal substitution residues for cavity formation. Generally, substitution residues for cavity formation are naturally occurring amino acids, which include, 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 embedded 1090 Å2 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 are described, e.g., 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 cases, modifications to the CH3 domain to create protrusions or cavities typically target residues located on two central antiparallel β-strands. The goal is to minimize the risk that the created protrusions may be accommodated by protruding into the surrounding solvent rather than being housed by a compensatory cavity within a partner CH3 domain.

[0273] In some embodiments, the heterodimer molecule contains the T366W mutation in the CH3 domain of the "knob" chain and the T366S, L368A, Y407V mutation in the CH3 domain of the "hole" chain. In some cases, additional interchain disulfide bridges between CH3 domains can also be used, for example, by introducing the Y349C mutation into the CH3 domain of the "knob" or "hole" chain and the E356C or S354C mutation into the CH3 domain of the other chain (Merchant, AM, et al., Nature Biotech. 16(1998) 677-681). In some embodiments, the heterodimer molecule contains the S354C, T366W mutation in one of the two CH3 domains and the Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. For example, the knob Fc may include the sequence described in SEQ ID NO:89, which includes S354C and T366W, and the hole Fc may include the sequence described in SEQ ID NO:90, which includes the mutations Y349C, T366S, L368A and Y407V. In some embodiments, the heterodimer molecule includes the E356C, T366W mutation in one of the two CH3 domains and the Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimer molecule includes the Y349C, T366W mutation in one of the two CH3 domains and the E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimer molecule contains the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. Other examples of knob-in-hole techniques are known in the art, for example, as described in EP1870459A1.

[0274] In some embodiments, an Fc variant containing a CH3 protrusion (knob) or cavity (hole) modification may be ligated to the N-terminus or C-terminus of one or more TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) at any location, typically via its N-terminus or C-terminus, to form a fusion polypeptide, for example. The ligation may be direct or indirect via a linker. Typically, the knob and hole molecules are generated by co-expression of a first immunomodulatory polypeptide ligated to an Fc variant containing a CH3 protrusion modification and a second immunomodulatory polypeptide ligated to an Fc variant containing a CH3 cavity modification.

[0275] Exemplary sequences of the knob and whole Fc polypeptides are described in SEQ ID NO: 123 and 129, respectively. In some embodiments, the knob or whole Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type Fc or unmodified Fc described in SEQ ID NO: 71 (corresponding to K447del in EU numbering). Exemplary sequences of the knob and whole Fc polypeptides are described in SEQ ID NO: 89 and 90, respectively.

[0276] In some embodiments, individual polypeptides of a multidomain polypeptide, or individual polypeptides of a single-domain polypeptide, are linked to a multimerizing domain that forms an immunomodulatory protein which is a trimer, tetramer, or pentamer. In some embodiments, the individual polypeptides of such a molecule are identical. In some embodiments, such a multimerizing domain is a cartilage oligomeric substrate protein (COMP) assembly domain, a vasodilator-stimulated phosphorylated protein (VASP) tetramerizing domain, or a ZymoZipper (ZZ) 12.6 domain.

[0277] In some embodiments, the multimerization domain is part of the cartilage oligomeric substrate protein (COMP) assembly domain (Voulgaraki et al., Immunology (2005) 115(3):337-346. In some examples, COMP is or contains the amino acid sequence described in SEQ ID NO:146 (e.g., amino acids 29-72 of full-length COMP, Uniprot accession number P49747), or a sequence having approximately 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with SEQ ID NO:146.

[0278] In some embodiments, the multimerization domain is a vasodilator-stimulated phosphorylated protein (VASP) tetramerization domain (Bachmann et al., J Biol Chem (1999) 274(33):23549-23557). In some embodiments, VASP is the amino acid sequence described in SEQ ID NO:147 (e.g., amino acids 343-375 of full-length VASP; Uniprot accession number P50552), or a sequence having approximately 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:147, or includes such a sequence.

[0279] In some embodiments, a TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) is linked to a polymerization domain (e.g., an Fc region) via a linker, e.g., a peptide linker. In some embodiments, the peptide linker may be at least one amino acid residue in length. In some embodiments, the peptide linker has at least one amino acid residue, but its length 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.

[0280] In some embodiments, the linker is (in single-letter amino acid code): GGGGS ("4GS"; SEQ ID NO: 77) or a polymer of the 4GS linker, 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 contain a series of alanine residues alone or in addition to another peptide linker (e.g., a 4GS linker or a polymer thereof). In some embodiments, the linker is (in single-letter amino acid code) GSGGGGS (SEQ ID NO: 74) or GGGSSA (SEQ ID NO: 80). In some examples, the linker is 2xGGGGS followed by 3 alanines (GGGGSGGGGSAAA; SEQ ID NO: 133). In some examples, the linker is shown as SEQ ID NO: 194 or 195.

[0281] In some embodiments, a TACI polypeptide, such as a variant TACI polypeptide, is directly linked to the Fc sequence. In some embodiments, a TACI polypeptide, such as a variant TACI polypeptide, is indirectly linked to the Fc sequence, for example, via a linker. In some embodiments, one or more "peptide linkers" link the TACI polypeptide (e.g., a variant TACI polypeptide) to the Fc region. In some embodiments, the peptide linker may have a length of one amino acid residue or more. In some embodiments, the peptide linker has at least one amino acid residue, but its length is 20 amino acid residues, 19 amino acid residues, 18 amino acid residues, 17 amino acid residues, 16 amino acid residues, 15 amino acid residues, 14 amino acid residues, 13 amino acid residues, 12 amino acid residues, 11 amino acid residues, 10 amino acid residues, 9 amino acid residues, 8 amino acid residues, 7 amino acid residues, 6 amino acid residues, 5 amino acid residues, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue or less. Exemplary linkers include any linkers described herein.

[0282] In some embodiments, the TACI-Fc fusion protein has the following 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 disulfide covalent bond, resulting in a dimer molecule containing two TACI polypeptides (e.g., two variant TACI polypeptides).

[0283] In some embodiments, a TACI-Fc fusion protein is provided, comprising a TACI polypeptide, e.g., any of the above-described TACI polypeptides, a linker, and an Fc region in that order. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a cleaved wild-type TACI polypeptide, e.g., any of the described cleaved wild-type TACI polypeptides. In some embodiments, the TACI polypeptide of the TACI-Fc fusion is shown in SEQ ID NO:13. The linker may be any of the described linkers. In some embodiments, the linker is GSGGGGS (SEQ ID NO:74). In some embodiments, the linker is GS(G4S)2 (SEQ ID NO:194). The Fc region may be any of the described Fc regions. In some embodiments, the Fc region is wild-type IgG1 Fc shown in SEQ ID NO:81. In some embodiments, the Fc region is variant Fc shown in SEQ ID NO:73.

[0284] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:171. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:197. In some embodiments, the TACI-Fc fusion is encoded by the sequence shown in SEQ ID NO:208. TIFF0007866668000025.tif41160

[0285] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:172. TIFF0007866668000026.tif41160

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

[0287] In some embodiments, the TACI polypeptide is a variant TACI polypeptide. In some embodiments, a variant TACI-Fc fusion protein is provided, comprising a variant TACI polypeptide, e.g., any of the above variant TACI polypeptides, a linker, and an Fc region in that order. In some embodiments, the TACI polypeptide of the TACI-Fc fusion is a variant TACI polypeptide, e.g., any of the described variant TACI polypeptides. In some embodiments, the variant TACI of the variant TACI-Fc fusion is represented by one of SEQ ID NO: 2-12, 21, 22, or 101-120. In some embodiments, the variant TACI of the variant TACI-Fc fusion is represented by one of SEQ ID NO: 14-20, 23-35, 92-100, or 177-192. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). In some embodiments, the linker is GS(G4S)2 (SEQ ID NO: 194). In some embodiments, the Fc region is wild-type IgG1 Fc as shown in SEQ ID NO:81. In some embodiments, the Fc region is variant Fc as shown in SEQ ID NO:73.

[0288] In some embodiments, the TACI-Fc fusion protein has the amino acid sequence shown in one of the following 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. TIFF0007866668000027.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. TIFF0007866668000028.tif41154

[0292] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:169. TIFF0007866668000029.tif41159

[0293] In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:170. TIFF0007866668000030.tif41154

[0294] In some embodiments, the TACI-Fc fusion protein contains multiple copies of TACI polypeptide sequences (e.g., variant TACI polypeptide sequences), for example, two, three, or four TACI polypeptide sequences. In some embodiments, the TACI-Fc fusion protein contains two TACI polypeptide sequences (e.g., two variant TACI polypeptide sequences). In some cases, the TACI polypeptide sequences may be directly linked or indirectly linked via a linker, for example, a peptide linker containing any of the peptide linkers described. In such examples, one of the TACI polypeptide sequences is attached or linked to an Fc region, for example, either the N-terminus or C-terminus of an Fc region. In other cases, the TACI polypeptide sequences may be separated from each other by Fc regions, each individually attached to the N-terminus or C-terminus of an Fc region. Linking to the Fc regions may be direct or indirect via a linker, for example, a peptide linker containing any of the peptide linkers described.

[0295] In some embodiments, the TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) may be arranged sequentially in tandem within the fusion protein (hereinafter referred to as the "tandem" Fc fusion construct). In some embodiments, the TACI-Fc fusion protein has the following structure: (TACI)-linker-(TACI)-linker-Fc region In some embodiments, the immunomodulatory protein is a tetravalent molecule 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 forms a disulfide covalent bond, resulting in a dimer 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 TACI polypeptide described above; a linker; another TACI polypeptide, e.g., any TACI polypeptide described above; and an Fc region. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a cleaved wild-type TACI polypeptide, e.g., any cleaved wild-type TACI polypeptide described above. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is indicated by SEQ ID NO: 13. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a variant TACI polypeptide, e.g., any variant TACI polypeptide described above. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a variant TACI indicated by SEQ ID NO: 2-12, 21, 22, or 101-120. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a variant TACI shown in one of the following SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linker may be any of the described linkers. In some embodiments, the linker is GSGGGGS (SEQ ID NO: 74). The Fc region may be any of the described Fc regions. In some embodiments, the Fc region is wild-type IgG1 Fc shown in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc shown in SEQ ID NO: 73. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 198 and is encoded by the sequence shown in SEQ ID NO: 209.

[0297] In some embodiments, the TACI polypeptide sequence (e.g., variant TACI polypeptide sequence) may be separated by an Fc region in the fusion protein, with the Fc region positioned between the two TACI polypeptide sequences (hereinafter referred to as the "barbell" Fc fusion construct). In some embodiments, the TACI-Fc fusion protein has the following structure: (TACI)-linker-Fc region-linker-(TACI) It has the following characteristics. In some embodiments, the linkers may be the same or different. In some embodiments, the immunomodulatory protein is a tetravalent molecule which is a homodimer of two identical copies of the TACI-Fc fusion protein. For example, interaction between the Fc regions of two identical polypeptide fusions forms a disulfide covalent bond, resulting in a dimer molecule containing four TACI polypeptides (e.g., four variant TACI polypeptides).

[0298] In some embodiments, a TACI-Fc fusion protein is provided, containing a TACI polypeptide, e.g., any TACI polypeptide described above; a linker; an Fc region; another linker; and another TACI polypeptide, e.g., any TACI polypeptide described above, in that order. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a cleaved wild-type TACI polypeptide, e.g., any cleaved wild-type TACI polypeptide described above. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is indicated by SEQ ID NO: 13. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a variant TACI polypeptide, e.g., any variant TACI polypeptide described above. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a variant TACI indicated by SEQ ID NO: 2-2, 21, 22, or 101-120. In some embodiments, each TACI polypeptide of the TACI-Fc fusion is a variant TACI shown in one of the following SEQ ID NOs: 14-20, 23-35, 92-100, or 177-192. The linker may be any of the described linkers, and may be the same or different. In some embodiments, the first linker is GSGGGGS (SEQ ID NO: 74) and the second linker is (GGGGS)4 (SEQ ID NO: 84). The Fc region may be any of the described Fc regions. In some embodiments, the Fc region is wild-type IgG1 Fc shown in SEQ ID NO: 81. In some embodiments, the Fc region is a variant Fc shown in SEQ ID NO: 73. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO: 201 and is encoded by the sequence shown in SEQ ID NO: 212. In some embodiments, the TACI-Fc fusion protein has the sequence shown in SEQ ID NO:202 and is encoded by the sequence shown in SEQ ID NO:213.

[0299] In some embodiments, a TACI-Fc fusion protein is provided, which is a dimer formed by two identical TACI polypeptides described (e.g., variant TACI polypeptides) linked to an Fc domain. In some embodiments, either of the provided TACI-Fc fusion polypeptides of the same species (also called copies), e.g., the variant TACI-Fc fusion, is dimerized to produce a homodimer. In some embodiments, the dimer is a homodimer in which the two TACI-Fc polypeptides, e.g., the variant TACI-Fc polypeptide, are identical. When constructing a homodimer Fc molecule, the Fc region is an Fc region that can form a homodimer using the corresponding Fc region by co-expressing the individual Fc regions in a cell. In some embodiments, dimerization is mediated by a disulfide covalent 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 an immunomodulatory protein, the nucleic acid molecule encoding the immunomodulatory protein is inserted into a suitable expression vector. The resulting immunomodulatory protein can be expressed in host cells transformed by the expression, where, within the host cells, interchain disulfide bonds formed between Fc portions cause aggregation of Fc domains to occur, resulting in a dimer, for example, a bivalent immunomodulatory protein.

[0301] Nucleic acid molecules encoding TACI-Fc fusion proteins, such as variant TACI-Fc fusion proteins, are also provided. In some embodiments, when producing an Fc fusion protein, the nucleic acid molecule encoding the TACI-Fc fusion protein, such as variant TACI-Fc fusion protein, is inserted into a suitable expression vector. The resulting TACI-Fc fusion protein, such as variant TACI-Fc fusion protein, can be expressed in host cells transformed by the expression, where the Fc domains assemble due to interchain disulfide bonds formed between the Fc portions, resulting in a dimer, such as a bivalent TACI-Fc fusion protein. The resulting Fc fusion protein can be easily purified by affinity chromatography using a protein A column or a protein G column. When producing heterodimers, additional steps may be required for purification. For example, when two nucleic acids encoding different immunomodulatory proteins are introduced into cells by transformation, the immunomodulatory proteins possessing Fc domains are also expressed as disulfide-linked homodimers, so heterodimers must be biochemically formed. Therefore, homodimers can be reduced under conditions that favor the disruption of interchain disulfides but do not generate 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 chromatography. Alternatively, the formation of this type of heterodimer can be biased by genetically engineering and expressing immunomodulatory proteins containing one or more TACI variant-containing Fc fusion molecules using the knob-into-hole method described.

[0302] In one embodiment, when produced and expressed from cells, the immunomodulatory protein provided, such as TACI-Fc (e.g., variant TACI-Fc), is a homodimer containing two identical polypeptide chains. Figures 8A and 8B illustrate the structures 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, designed to neutralize the B-cell stimulating 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 disulfide covalent bond, each having the 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, designed to neutralize the B-cell stimulating 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 disulfide covalent bond, each having the 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 stimulating 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 disulfide covalent bond, each having 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 stimulating 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 disulfide covalent bond, each having 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 protein, e.g., its homodimer, exhibits an IC50 of less than 400 pM in BAFF neutralization. 50 This indicates that, in some embodiments, the IC50 of BAFF neutralization is 1 pM to 400 pM, for example, 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, and 20 pM to 100 pM. These ranges are 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. In some embodiments, BAFF neutralization IC 50 This 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 approximately 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 protein, e.g., its homodimer, exhibits an IC50 of less than 400 pM in APRIL neutralization. 50 This indicates that in some embodiments, IC50 for APRIL neutralization is 0.5 pM to 100 pM, for example, 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 This 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 approximately 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. Methods for producing nucleic acids, vectors, and polypeptides or cells Isolated nucleic acids or recombinant nucleic acids, collectively referred to as “nucleic acids,” that encode any of the immunomodulatory proteins provided herein are provided herein. In some embodiments, including all of the following, 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 the following, the nucleic acids provided herein are useful for the expression of the immunomodulatory proteins provided herein, for example, the TACI fusion proteins provided herein. The nucleic acids provided herein may be in the form of RNA or DNA, and include mRNA, cRNA, recombinant RNA or synthetic RNA, and recombinant DNA 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, containing any of the nucleotide sequences of the present invention are also provided.

[0310] In some cases, heterologous (non-natural) signal peptides can be added to nucleic acids encoding immunomodulatory proteins. This may be desired, for example, in the case of expressing TACI fusion proteins that do not contain an amino-terminal signal sequence. In some embodiments, the signal peptide is derived from immunoglobulins (such as IgG heavy chains or IgG-kappa light chains), cytokines (e.g., interleukin-2 (IL-2) or CD33), serum albumin proteins (e.g., HSA or albumin), human azurosidin preprotein signal sequences, luciferases, trypsinogens (e.g., chymotrypsinogen or trypsinogen), or other signal peptides that can efficiently express proteins from cells and, in some aspects, secrete proteins from cells. Exemplary signal peptides include any of those listed in Table 3.

[0311] (Table 3) Exemplary signal peptides TIFF0007866668000031.tif119166

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

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

[0314] In any of the embodiments provided above, nucleic acids encoding immunomodulatory polypeptides provided herein can be introduced into cells using recombinant DNA and cloning techniques. For this purpose, recombinant DNA molecules encoding 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 produced by polymerase chain reaction (PCR). DNA inserts encoding immunomodulatory proteins can be cloned into suitable transduction / transfection vectors, as is known to those skilled in the art. Expression vectors containing nucleic acid molecules are also provided.

[0315] In some embodiments, expression vectors can enable the expression of immunomodulatory proteins in appropriate cells under conditions suitable for protein expression. In some aspects, the nucleic acid molecule or expression vector contains a DNA molecule encoding an immunomodulatory protein functionally linked to an appropriate regulatory sequence. Methods for performing this functional linkage, either before or after the DNA molecule is inserted into the vector, are well known. Regulatory sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the regulation of transcription or translation.

[0316] In some embodiments, the expression of immunomodulatory proteins is controlled by promoters or enhancers to regulate or modulate their expression. Promoters are functionally ligated to a portion of a nucleic acid molecule encoding a variant polypeptide or immunomodulatory protein.

[0317] The resulting recombinant expression vector, having a 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 secreted peptide or signal peptide functionally linked to the nucleic acid encoding the immunomodulatory polypeptide, so that the resulting soluble immunomodulatory polypeptide is recovered from the culture medium, host cells, or host cell periplasm. In other embodiments, a suitable expression regulatory signal is selected to enable membrane expression of the immunomodulatory polypeptide. Furthermore, the engineered cells or recombinant host cells provided herein can also be prepared using commercially available kits and contract manufacturers.

[0318] In some embodiments, the resulting expression vector having a DNA molecule thereon is used to transform, for example, transduce, suitable cells. Transduction may be carried out using methods well known in the art. Exemplary methods include methods for the transduction of 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 transduced into cells by lentiviral transduction or retroviral transduction.

[0319] In the preparation of polypeptides or manipulated cells, any of the many publicly available and well-known mammalian host cells, including mammalian T cells or APCs, can be used. Cell selection depends on numerous 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, biosafety, and cost. The balance of these factors must be agreed upon with the understanding that not all cells can 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 fetal 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 ovarian 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 liver cancer cells (H-4-II-E; ATCC CRL 1548), and SV40-transformed monkey kidney cells (COS-1; ATCC CRL Examples include 1650) and mouse embryonic cells (NIH-3T3; ATCC CRL 1658).

[0321] In some embodiments, the host cell may be a variety of eukaryotic cells (e.g., in yeast cells) or a mammalian cell such as Chinese hamster ovary (CHO) cells or HEK293 cells. In some embodiments, the host cell is a suspension cell, and the polypeptide is manipulated or produced in a culture suspension, for example, in cultured 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 cell, such as suspension CHO cells, may be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

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

[0323] In some embodiments, recombinant vectors are viral vectors. Exemplary recombinant viral vectors include lentiviral vector genomes, poxvirus vector genomes, vaccinia virus vector genomes, adenovirus vector genomes, adenovirus-associated viral vector genomes, herpesvirus vector genomes, and alphavirus vector genomes. Viral vectors may be living, attenuated, replication-conditionally or replication-deficient, non-pathogenic (deficient), replication-competent viral vectors, and / or modified to express heterologous gene products, e.g., variant immunomodulatory polypeptides provided herein. Vectors for generating viruses may also be modified to alter the attenuation of the virus, including any method of increasing or decreasing the transcriptional or translational loading.

[0324] Examples of viral vectors that may be used include modified vaccinia virus vectors (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); see U.S. Patent Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615 and 7,368,116); adenovirus vectors or adenovirus-related viral vectors (e.g., Molin et al.) See also al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93 (2004); U.S. Patent No. 6,143,290; U.S. Patent No. 6,596,535; U.S. Patent No. 6,855,317; U.S. Patent No. 6,936,257; U.S. Patent No. 7,125,717; U.S. Patent No. 7,378,087; U.S. Patent No. 7,550,296); retroviral vectors including those based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), ecotropic retrovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., Buchscher et al.,J.Virol.66:2731-39(1992);Johann et al.,J.Virol.66:1635-40(1992);Sommerfelt et al.,Virology 176:58-59(1990);Wilson et al.,J.Virol.63:2374-78(1989);Miller et al. al., J. Virol. 65:2220-24 (1991); Miller et al., Mol. Cell Biol.See 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 (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. This includes (see al., J.Virol.69:2729,1995; Nightingale et al., Mol.Therapy,13:1121,2006; Brown et al., J.Virol.73:9011(1999); International Publication No. 2009 / 076524; International Publication No. 2012 / 141984; International Publication No. 2016 / 011083; McWilliams et al., J.Virol.77:11150,2003; Powell et al., J.Virol.70:5288,1996), or any variant thereof, as well as / or vectors that may be used to generate any of the above viruses. In some embodiments, recombinant vectors may include regulatory sequences, such as promoter sequences or enhancer sequences, that can regulate the expression of the viral genome within a packaging cell line, for example, in the case of RNA viruses (see, for example, U.S. Patents 5,385,839 and 5,168,062).

[0325] In some cases, nucleic acids or expression vectors contain nucleic acid sequences encoding immunomodulatory proteins functionally linked to appropriate regulatory sequences. Methods for performing this functional linkage, either before or after insertion of the immunomodulatory protein-encoding nucleic acid sequence into a vector, are well known. Regulatory sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the regulation of transcription or translation. Promoters can be functionally linked to portions of the immunomodulatory protein-encoding nucleic acid sequence.

[0326] The transcriptional regulatory sequence contains a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the mouse metallothionein I gene promoter (Hamer et al, J. Molec. Appl Genet. 1:273 (1982)), the herpesvirus TK promoter (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 cancer virus promoter (generally, see Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages...). See 163-181 (John Wiley & Sons, Inc. 1996). One useful combination of promoter and enhancer is provided by a myeloproliferative sarcoma virus promoter and a human cytomegalovirus enhancer.

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

[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, and electroporation. Transfected cells can be selected and grown to provide recombinant host cells containing expression vectors stably integrated into the host cell genome. Techniques for introducing vectors into eukaryotic cells, and techniques 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. The selection system can also be used to increase the expression level of the gene of interest; this process is called "amplification." Amplification is performed by culturing transfectants in the presence of a low level of the selector, and then increasing the amount of the selector to select cells that produce high levels of the product of the introduced gene. A suitable amplifiable selectable marker is dihydrofolate reductase that confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used. Alternatively, markers that introduce the altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, class I MHC, or placental alkaline phosphatase, may be used to separate transfected cells from untransfected cells by means of FACS sorting or magnetic bead separation techniques.

[0330] In some embodiments, the polypeptides provided herein may also be prepared by synthetic methods. Solid-phase synthesis is a preferred technique for preparing individual peptides because it is the most cost-effective method for producing small peptides. For example, well-known solid-phase synthesis techniques include the use of protecting groups, linkers and solid 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 Compositions containing any of the immunomodulatory proteins described herein are provided herein. The pharmaceutical composition may further contain pharmaceutically acceptable excipients. For example, the pharmaceutical composition may contain one or more excipients for modifying, maintaining, or preserving, for example, the pH, molar osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption or osmosis of the composition. Such a composition may include buffers, e.g., neutral buffered saline, phosphate-buffered saline; carbohydrates, e.g., glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., 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 before administration.

[0333] In some embodiments, the pharmaceutical composition is an immunomodulatory protein dissolved in a liquid, such as an aqueous solution (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 includes pharmaceutically acceptable excipients, such as fillers, binders, coatings, preservatives, lubricants, flavorings, sweeteners, colorants, solvents, buffers, chelating agents, or stabilizers. Examples of pharmaceutically acceptable fillers include cellulose, dicalcium phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Examples of pharmaceutically acceptable coatings include hydroxypropyl methylcellulose (HPMC), shellac, 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 carbonates, citrates, glucons, acetates, phosphates, or tartarates.

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

[0336] In some embodiments, pharmaceutical compositions are sterile. Sterilization can be achieved by filtration through a sterile filtration membrane or by radiation. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Parenteral compositions can be stored in lyophilized form or in solution. Furthermore, parenteral compositions are generally placed in containers with a sterile access port, such as intravenous solution bags or vials with stoppers that can be punctured with a subcutaneous needle.

[0337] A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle. For example, a carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or any combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation. It must also be suitable for contact with any tissue, organ, or body part it may encounter, meaning it must not carry a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that would unduely 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, in accordance with standard pharmaceutical practice. For example, the therapeutically effective dose may be initially estimated in a cell culture assay or in an animal model such as a mouse, rat, rabbit, dog, pig, or monkey. The animal model may be used to determine an appropriate concentration range and route of administration. Such information can then be used to determine a useful dose and route for administration to humans. The exact dosage is determined by taking into account factors relevant to the subject requiring treatment. The dosage and administration are adjusted to provide a sufficient level of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the general health status of the subject, the age, weight, and sex of the subject, the time and frequency of administration, the combination of drugs, sensitivity to response, and response to treatment.

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

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

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

[0342] In some embodiments, the dosage of the pharmaceutical composition is a single dose or a repeated dose. In some embodiments, the dose is administered to a subject once, twice, three times, or four or more times a day. In some embodiments, a dose of approximately one or more (e.g., approximately two or more, approximately three or more, approximately four or more, approximately five or more, approximately six or more, or approximately seven or more) is administered per week. In some embodiments, multiple doses are administered over several days, weeks, months, or years. In some embodiments, a series of treatments consists of approximately one or more doses (e.g., approximately two or more, approximately three or more, approximately four or more, approximately five or more, approximately seven or more, approximately ten or more, approximately fifteen or more, approximately 25 or more, approximately 40 or more, approximately 50 or more, or approximately 100 or more).

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

[0344] V. Methods for evaluating the activity and immunomodulation of immunomodulatory proteins In some embodiments, the immunomodulatory proteins provided, such as the TACI fusion proteins provided herein, exhibit immunomodulatory activity. The immunomodulatory proteins provided, such as the TACI fusion proteins, can regulate one or more of B cell activities, such as B cell proliferation, differentiation, or survival.

[0345] The function of immunomodulatory proteins can be examined using various methods to assess the ability of proteins to bind to congenerally linked 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., an immunomodulatory protein) specifically binds to a particular binding partner. For example, determining the binding affinity of a binding molecule, e.g., an immunomodulatory protein, to a binding partner, e.g., APRIL or BAFF, using any of the many binding assays known in the art is within the scope of the art. Various binding assays are known, including, but are not limited to, those described herein, such as ELISA K DThis includes methods involving KinExA, flow cytometry, and / or surface plasmon resonance (SPR) instruments. Such methods include, but are not limited to, methods involving BIAcore®, Octet®, or flow cytometry. For example, in some embodiments, BIAcore® instruments can be used to determine the binding kinetics and binding constants of a complex between two proteins using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. NYAcad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patents 5,283,173, 5,468,614, or equivalents). SPR measures the change in molecular concentration at the sensor surface as the molecule binds to or dissociates from the surface. Changes in the SPR signal are directly proportional to changes in mass concentration near the surface, thereby enabling the measurement of binding kinetics between the two molecules. The dissociation constant of the complex can be determined by monitoring the change in refractive index with respect to time as the buffer passes over the chip. Other suitable assays for measuring the binding of one protein to another include immunoassays such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring changes in the spectroscopic or optical properties of the protein by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays, without limitation, include Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting the binding of expressed polynucleotides or proteins.

[0346] The immunomodulatory proteins provided can also be evaluated in any of a variety of assessments to evaluate the modulation of B cell activity. One such assay is a cell proliferation assay. Cells are cultured in or out of the presence of the test compound (e.g., immunomodulatory protein), and cell proliferation is detected, for example, by measuring the uptake of tritium-labeled thymidine or by a colorimetric assay based on the metabolic degradation of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosman, J.Immunol.Meth.65:55-63, 1983). In an alternative assay format, cells further engineered to express a reporter gene are used. The reporter gene is linked to a promoter element that is responsive to the receptor junction pathway, and the assay detects activation of the transcription of the reporter gene. Numerous reporter genes readily assayable in cell extracts, such as *E. coli* lacZ, chloramphenicol acetyltransferase (CAT), and serum response elements (SREs), are known in the art (see, e.g., 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). Luciferase gene expression 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 immunomodulatory proteins provided may be characterized by their ability to inhibit the stimulation of human B cells by soluble APRIL or soluble BAFF, as described by Gross et al., international publication number WO00 / 40716. In summary, 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 stimulating conditions, for example, in the presence of soluble APRIL, and further in the presence of immunomodulatory proteins at gradually increasing concentrations. B cells can be labeled with a growth dye or 1 μCi to measure their proliferation. 3 They can be labeled with H-thymidine. The number of B cells can be determined over time.

[0348] Transcription factors, for example, NF- K Reporter cell lines expressing reporter genes under the functional control of B, NFAT-1, and AP-1 can be constructed to express TACI or BCMA. For example, reporter cells may include Jurkat cell lines and other B lymphoma cell lines. Incubation of these cells with soluble BAFF ligand or soluble APRIL ligand transmits signals via the reporter gene within these constructs. The effects of provided immunomodulatory proteins on regulating this signaling can then be evaluated.

[0349] Well-established animal models are available to test the in vivo efficacy of immunomodulatory proteins in certain disease states, including those involving autoimmune or inflammatory conditions. For example, animal models of autoimmune diseases include the MRL-lpr / lpr or NZB×NZW F1 congenic mouse line, which serves as a model for SLE (systemic lupus erythematosus). Such animal models are publicly known in the art; see, for example, Autoimmune Disease Models A Guidebook, Cohen and Miller eds. Academic Press. Offspring of a cross between New Zealand Black (NZB) mice and New Zealand White (NZW) mice develop a spontaneous form of SLE that closely resembles human SLE. The offspring mice, known as NZBWs, begin developing IgM autoantibodies against T cells at 1 month of age, and by 5-7 months of age, Ig anti-DNA autoantibodies become the dominant immunoglobulin. Polyclonal B cell hyperfunction leads to the overproduction of autoantibodies. The deposition of these autoantibodies, particularly autoantibodies against single-stranded DNA, is associated with the development of glomerulonephritis, which clinically manifests as proteinuria, azotemia, and death due to renal failure. In mice with spontaneously occurring SLE, renal failure is the leading cause of death, and in NZBW strains, this process is chronic and obstructive. The disease is more rapid and severe in females than in males, with an average survival time of only 245 days compared to 406 days in males. While most female mice become symptomatic (proteinuria) by 7-9 months of age, some female mice may be much younger or older when symptoms develop.Fatal immune nephritis observed in NZBW mice is remarkably similar to glomerulonephritis seen in human SLE, making this spontaneously occurring mouse model highly attractive for testing potential SLE treatments (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 immunomodulatory proteins provided to these mice can be evaluated to assess the effects of improving symptoms and changes in 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). Female IA bm12 B6(C)-H2-Ab1 bm12 Splenocyte suspension derived from / KhEgJ ("bm12") mice is adopted into female C57BL / 6NJ recipient mice. H2-Ab1 bm12 is H2-Ab1 bThis differs by three nucleotides, resulting in a three-amino acid change within the β-chain of the MHC class II IA molecule. Alloactivation of donor bm12 CD4+ T cells by recipient antigen-presenting cells leads to chronic GVHD with symptoms very similar to SLE, including autoantibody production, changes in immune cell subsets, and mild renal disease. Glomerulonephritis with immune complex deposition develops later in this model and consists mainly of autoantigens bound to IgG1, IgG2b, IgG2c, and IgG3 antibodies. Endpoints in this model may include the concentration of anti-dsDNA antibodies, selected IgG isotypes, blood urea nitrogen (BUN), and serum creatinine, immune cell subset composition in the spleen and cervical LN, and renal histological features.

[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 antibody can induce SjS disease and accelerated onset of diabetes in female, diabetic, non-obese diabetic (NOD) mice. Starting at 6 weeks of age, 100 μg of anti-PD-L1 antibody is intraperitoneally (IP) injected into mice on days 0, 2, 4, and 6 of the study, and mice are treated on various days with the provided immunomodulatory protein. Naive mice are included as controls for endpoint analysis. All mice are typically sacrificed on day 10 of the study, and the submandibular gland (SMG) and pancreas from each mouse are collected for histopathological evaluation to assess the signs and severity of sialadenitis and isletitis. 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 and produce demyelination as a result of T cell activation to neuronal proteins such as myelin basic protein (MBP) or proteolipid protein (PLP). Antigen inoculation leads to induction of CD4+, class II MHC-restricted T cells (Th1). By modifying the protocol for EAE, acute, chronic relapsing, or passive transfer variants of the model can be created (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 immunomodulatory proteins can be evaluated to improve symptoms and changes in the course of the disease.

[0353] In some embodiments, a collagen-induced arthritis (CIA) model can be used, in which mice develop chronic inflammatory arthritis that closely resembles human rheumatoid arthritis (RA). Because CIA shares similar immunological and pathological features with RA, it is an ideal model for screening potential human anti-inflammatory compounds. Another advantage of using the CIA model is that the pathogenesis is known. T-cell and B-cell epitopes on type II collagen have been identified, and various immunological parameters (delayed-type hypersensitivity and anti-collagen antibodies) and inflammatory parameters (cytokines, chemokines, and matrix-degrading enzymes) associated with immune-mediated arthritis have been determined and can be used to evaluate the efficacy 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). Administration of immunomodulatory proteins to improve symptoms and changes in the course of the disease can be evaluated.

[0354] In some embodiments, a model of bronchial infection, such as asthma, can be created by injecting mice with ovalbumin and restimulating them nasally with an antigen, thereby inducing an asthmatic response in the bronchi similar to that of asthma. The administration of provided immunomodulatory proteins can then be evaluated to improve symptoms and changes in the course of the disease.

[0355] In some aspects, myasthenia gravis (MG) is another autoimmune disease for which a mouse model is available. MG is a neuromuscular disorder involving the production of autoantibodies against nicotinic acetylcholine receptors (AChRs). MG can be acquired or inherited and is characterized by abnormal weakness and exercise-induced fatigue. 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 incomplete neuromuscular electrical impulses and decreased muscle strength. In the EAMG model, mice are immunized using nicotinic acetylcholine receptors. Clinical signs of MG become apparent several weeks after the second immunization. EAMG is assessed by several methods, including measurement of serum AChR antibody levels by radioimmunoassay (Christadoss and Dauphinee, J.Immunol.136:2437-40, 1986; and Lindstrom et al., Methods Enzymol.74:432-60, 1981), measurement of muscle AChR, or electromyography (Wu et al. Protocols in Immunology. Vol.3, Eds.Coligen, Kruisbeak, Margulies, Shevach, and Strober. John Wiley and Sons, New York, p.15.8.1, 1997).

[0356] Another use for in vivo models involves the delivery of antigen loadings to animals, followed by the administration of immunomodulatory proteins, and the measurement of 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, immune responses can be measured in animals subjected to regular antigen loadings (e.g., keyhole limpet hemocyanin (KLH), sheep erythrocytes (SRBCs), ovalbumin, or collagen) followed by administration of the provided immunomodulatory proteins.

[0357] The distribution and half-life of such polypeptides can be determined in vivo using pharmacokinetic studies in combination with radiolabeled immunomodulatory proteins.

[0358] VI. Therapeutic applications The pharmaceutical compositions described herein (including pharmaceutical compositions comprising immunomodulatory proteins described herein) may be used for a variety of therapeutic applications, such as the treatment of diseases. For example, in some embodiments, the pharmaceutical compositions may be used to treat inflammatory or autoimmune disorders, cancer, organ transplantation, viral infections, and / or bacterial infections in mammals. The pharmaceutical compositions may modulate (e.g., reduce) the immune response 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 manipulated cells are administered in an amount effective to treat the disease or disorder. Uses include the use of molecules containing immunomodulatory proteins and their use in preparing pharmaceuticals to carry 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 having a disease or condition. In some embodiments, the method thereby treats the disease or disorder or condition or disorder of the subject.

[0360] Exemplary subjects include mammalian subjects, such as livestock, domesticated animals, and human patients. In certain embodiments, the subject is a human subject.

[0361] The pharmaceutical compositions described herein may be used for a variety of therapeutic applications, such as the treatment of diseases. For example, in some embodiments, the pharmaceutical compositions may be used to treat inflammatory or autoimmune disorders, organ transplantation, viral infections, and / or bacterial i...

Claims

1. (i) Variant TACI polypeptides containing the extracellular domain (ECD) of TACI or a portion of the ECD of TACI, including amino acid substitutions K77E, F78Y, and Y102D corresponding to the position numbering shown in SEQ ID NO:122, and (ii) Fc region A TACI-Fc fusion protein containing, The variant TACI polypeptide has increased binding affinity to one or both APRIL and BAFF compared to the TACI reference polypeptide containing the amino acid sequence shown in SEQ ID NO:13, and The variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:13 or at least 95% sequence identity with SEQ ID NO:

1. The aforementioned TACI-Fc fusion protein.

2. (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 contains CRD2 of the ECD of TACI but lacks the entirety of CRD1, or (c) The variant TACI polypeptide comprises amino acid residues 68 to 110 shown in SEQ ID NO:122, The TACI-Fc fusion protein according to claim 1.

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

26.

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

111.

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

7. The TACI-Fc fusion protein according to claim 5 or 6, wherein the linker is GSGGGS (SEQ ID NO: 74).

8. The TACI-Fc fusion protein according to 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 having 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 according to 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 according to claim 8, wherein the Fc region is a variant IgG1 Fc domain comprising 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 according to claim 10, wherein the Fc region comprises amino acid substitutions of L234A, L235E, and G237A according to EU numbering.

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

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

73.

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

16. The TACI-Fc fusion protein according to claim 15, wherein the homodimers are linked by a covalent disulfide bond.

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

18. A vector comprising the nucleic acid molecule described in claim 17.

19. The vector according to claim 18, which is an expression vector.

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

21. A method for producing a TACI-Fc fusion protein, A step of introducing a nucleic acid molecule according to claim 17 or a vector according to claim 18 or claim 19 into a host cell under conditions that cause the TACI-Fc fusion protein to be expressed in the host cell, and Optionally, further steps include isolating or purifying the TACI-Fc fusion protein from the cells. The method, including the method described above.

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

23. A manufactured article comprising a vial containing the pharmaceutical composition described in claim 22.

24. A kit comprising the pharmaceutical composition according to claim 22 or the manufactured article according to claim 23, and instructions for use.

25. The pharmaceutical composition according to claim 22 for use in reducing the immune response in a subject.

26. The pharmaceutical composition according to claim 22 for use in the treatment of a disease, disorder, or condition in a subject.

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

28. The pharmaceutical composition according to 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 disease, post-infectious glomerulonephritis, mesangial proliferative disorder, chronic lymphocytic leukemia, or minimal change nephrotic syndrome.

29. The pharmaceutical composition according to claim 26 or 27, wherein the disease, disorder, or condition is systemic lupus erythematosus (SLE), Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, 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. Use of the pharmaceutical composition according to claim 22 in the manufacture of a pharmaceutical for reducing the immune response in a subject.

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

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

33. The use according to 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 disease, post-infectious glomerulonephritis, mesangial proliferative disorder, chronic lymphocytic leukemia, or minimal change nephrotic syndrome.

34. The use according to claim 31 or 32, wherein the disease, disorder, or condition is systemic lupus erythematosus (SLE), Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, 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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