HK40090097A - April and baff inhibitory immunomodulatory proteins and methods of use thereof - Google Patents

Description

APRIL and BAFF, inhibitory immunomodulatory proteins and their usage

Cross-references to related applications

This application claims U.S. Provisional Application 63/022,373, filed May 8, 2020, entitled "APRIL and BAFF Inhibitory Immunomodulatory Proteins with and without a T Cell Inhibitory Protein and Methods of Use Thereof," and U.S. Provisional Application 63/022,373, filed June 3, 2020, entitled "APRIL and BAFF Inhibitory Immunomodulatory Proteins with and without a T Cell Inhibitory Protein and Methods of Use Thereof." Priority is claimed to U.S. Provisional Application 63/034,361 entitled “APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITHAND WITHOUT A T CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF” filed on September 18, 2020, the contents of each of the applications of which are incorporated herein by reference in their entirety for all purposes.

By referencing and incorporating into the sequence list

This application is submitted together with the electronic sequence list. The sequence list is provided as a file named 761612003840SeqList.TXT, created on May 4, 2021, and is 278,660 bytes in size. Information from the electronic sequence list is incorporated herein by reference in its entirety.

Technical Field

This disclosure provides immunomodulatory proteins exhibiting neutralizing activity against BAFF and APRIL (or BAFF/APRIL heterotrimers). The immunomodulatory proteins include a variant domain of a transmembrane activator and a CAML interactor (TACI). The provided immunomodulatory proteins include TACI-Fc fusion proteins. This disclosure also provides nucleic acid molecules encoding the immunomodulatory proteins. The immunomodulatory proteins provide therapeutic efficacy against a variety of immune diseases, disorders, or conditions. Compositions and methods for preparing and using such proteins are provided.

Background Technology

Modulating immune responses through intervention in processes involving the interaction between soluble ligands and their receptors is of increasing medical interest. Currently, biopharmaceuticals for enhancing or suppressing immune responses are generally limited to antibodies (e.g., anti-PD-1 antibodies) or soluble receptors targeting single cell surface molecules (e.g., Fc-CTLA-4). There is a need for improved therapeutic agents that can modulate immune responses, particularly B-cell immune responses. Implementation schemes that meet this need are provided.

Summary of the Invention

This article provides an immunomodulatory protein containing at least one TACI polypeptide, which is a truncated wild-type TACI extracellular domain or a variant thereof, wherein the truncated wild-type TACI extracellular domain contains a cysteine-rich domain 2 (CRD2) but lacks the complete cysteine-rich domain 1 (CRD1), and wherein the variant TACI polypeptide comprises one or more amino acid substitutions in the truncated wild-type TACI extracellular domain.

This document provides an immunomodulatory protein containing at least one TACI polypeptide, which is a truncated wild-type TACI extracellular domain or a variant thereof, wherein, with respect to the position shown in SEQ ID NO:122, the truncated wild-type TACI extracellular domain consists of a continuous sequence of amino acid residues 71-104 within amino acid residues 67-118, wherein the variant TACI polypeptide comprises one or more amino acid substitutions in the truncated wild-type TACI extracellular domain. In some embodiments, the length of the truncated wild-type TACI extracellular domain is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51 amino acids. In some embodiments, the truncated wild-type TACI extracellular domain consists of amino acid residues 68-110 shown in SEQ ID NO:122. In some embodiments, the TACI polypeptide consists of the amino acid sequence shown in SEQ ID NO:13, or a variant thereof containing one or more amino acid substitutions from the sequence shown in SEQ ID NO:13.

This document provides an immunomodulatory protein comprising at least one TACI polypeptide, wherein the TACI polypeptide is a truncated TACI polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or a variant thereof containing one or more amino acid substitutions from the sequence shown in SEQ ID NO:13. In some embodiments, the truncated TACI polypeptide or a variant thereof binds to APRIL, BAFF, or a BAFF/APRIL heterotrimer. In some embodiments, the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence shown in SEQ ID NO:1. In some embodiments, the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence shown in SEQ ID NO:13.

This document provides an immunomodulatory protein comprising a truncated TACI polypeptide consisting of the sequence shown in SEQ ID NO:13. In some embodiments, the TACI polypeptide is a variant TACI polypeptide, wherein the variant TACI polypeptide has increased binding affinity for one or both of APRIL and BAFF compared to the truncated TACI polypeptide. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions at positions corresponding to those numbered in SEQ ID NO:122, selected from positions 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103.

In some embodiments, the one or more amino acid substitutions are 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 one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. In some embodiments, the one or more amino acid substitutions are D85E/K98T, I87L/K98T, L82P/I87L, G76S/P97S, K77E/R84L/F103Y, Y79F/Q99E, L83S/F103S, K77E/R84Q, K77E/A101D, K77E/F78Y/Y102D, Q75E/R84Q, Q75R/R84G/I92V, K77E/A101D/Y102D, R84Q/S88N/A101D, R84Q/F103V, K77E/Q95R/A101D, or I87M/A101D. In some embodiments, the one or more amino acid substitutions are K77E/F78Y/Y102D. In some embodiments, the one or more amino acid substitutions are Q75E/R84Q. In some embodiments, the variant TACI polypeptide is shown in SEQ ID NO:26. In some embodiments, the variant TACI polypeptide is shown in SEQ ID NO:27.

In some embodiments, the TACI polypeptide is a variant TACI polypeptide comprising one or more amino acid substitutions in the extracellular domain (ECD) of the reference TACI polypeptide or its specifically binding fragment thereof, the amino acid substitutions being located 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 numbers shown in SEQ ID NO:122.

This document provides an immunomodulatory protein containing at least one variant TACI polypeptide, wherein the at least one variant TACI polypeptide comprises one or more amino acid substitutions in the extracellular domain (ECD) of a reference TACI polypeptide or its specifically binding fragment thereof, the amino acid substitutions being located 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 numbers shown in SEQ ID NO:122.

This article provides an immunomodulatory protein, which is a variant TACI-Fc fusion protein containing a variant TACI polypeptide, an Fc region, and a linker between the TACI polypeptide and the Fc region. The variant TACI polypeptide contains one or more amino acid substitutions in the extracellular domain (ECD) of a reference TACI polypeptide or its specifically binding fragment thereof, wherein the amino acid substitutions are located 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 numbers shown in SEQ ID NO:122.

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

In some 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 containing one or both of the CRD1 and CRD2 domains, said portion being bound to APRIL, BAFF, or a BAFF/APRIL heterotrimer.

In some embodiments, the reference TACI polypeptide lacks an N-terminal methionine.

In some embodiments, the reference TACI polypeptide comprises the CRD1 domain and the CRD2 domain.

In some embodiments, the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1. In some embodiments, the reference TACI polypeptide consists of the sequence shown in SEQ ID NO:1.

In some embodiments, the reference TACI polypeptide is essentially composed of the CRD2 domain.

In some embodiments, the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13. In some embodiments, the reference TACI polypeptide consists of the sequence shown in SEQ ID NO:13.

In some embodiments, the one or more amino acid substitutions are selected from W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions.

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

In some embodiments, the one or more amino acid substitutions include amino acid substitutions selected from the following: Q75E, K77E, F78Y, R84G, R84Q, A101D and Y102D or any combination thereof.

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

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

In some embodiments, the one or more amino acid substitutions are D85E/K98T, I87L/K98T, R60G/Q75E/L82P, R60G/C86Y, W40R/L82P/F103Y, W40R/Q59R/T61P/K98T, L82P/I87L, G76S/P97S, K77E/R84L/F103Y, Y79F/Q 99E, L83S/F103S, K77E/R84Q, K77E/A101D, K77E/F78Y/Y102D, Q75E/R84Q, Q75R/R84G/I92V, K77E/A101D/Y102D, R84Q/S88N/A101D, R84Q/F103V, K77E/Q95R/A101D or I87M/A101D.

In some embodiments, the one or more amino acid substitutions are R84G, A101D, K77E/R84Q, K77E/A101D, K77E/F78Y, K77E/F78Y/Y102D, Q75E/R84Q, K77E/A101D/Y102D, R84Q, K77E, A101D, Q75E, K77E/F78Y/R84Q, F78Y, F78Y/R84Q, F78Y/A101D, F78Y/Y102D, or K77E/Y102D.

In some embodiments, the one or more amino acid substitutions are K77E/F78Y/Y102D.

In some embodiments, the one or more amino acid substitutions are Q75E/R84Q.

In some embodiments, the one or more amino acid substitutions are K77E/A101D/Y102D.

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

In some embodiments, the variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:122 or a specific binding fragment thereof comprising the CRD1 domain and/or the CRD2 domain. In some embodiments, the variant TACI polypeptide has at least 95% sequence identity with SEQ ID NO:122 or a specific binding fragment thereof comprising the CRD1 domain and/or the CRD2 domain. In some embodiments, the specific binding fragment is shown in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131.

In some embodiments, the variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:13. In some embodiments, the variant TACI polypeptide has at least 95% sequence identity with SEQ ID NO:13.

In some embodiments, the variant TACI peptide has increased binding affinity for one or both of APRIL and BAFF compared to the reference TACI peptide. In some embodiments, the variant TACI peptide has increased binding affinity for APRIL. In some embodiments, the variant TACI peptide has increased binding affinity for BAFF. In some embodiments, the variant TACI peptide has increased binding affinity for both APRIL and BAFF.

In some implementations, the increased binding affinity to BAFF or APRIL is independently increased by more than about 1.2 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, or about 60 times.

In some embodiments, the variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120; or the variant TACI polypeptide comprises the sequence shown in any 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 substantially composed of the sequences shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120; or the variant TACI polypeptide consists of or is substantially composed of the sequences shown in any 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 substantially composed of the sequence shown in SEQ ID NO:26. In some embodiments, the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:27. In some embodiments, the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:107. In some embodiments, the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:20.

In some embodiments, the adapter comprises a peptide adapter and the peptide adapter is selected from GSGGS (SEQ ID NO: 76), GGGGS (G4S; SEQ ID NO: 77), GSGGGGS (SEQ ID NO: 74), GGGGSGGGGS (2xGGGGS; SEQ ID NO: 78), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO: 79), GGGGSGGGGSGGGGSGGGGS (4xGGGGS; SEQ ID NO: 84), GGGGSGGGGSGGGGSGGGGS (5xGGGGS; SEQ ID NO: 91), GGGGSSA (SEQ ID NO: 80), or GSGGGGSGGGGS (SEQ ID NO: 194) or combinations thereof.

In some embodiments, the immunomodulatory protein contains a heterologous portion linked to the at least one TACI polypeptide. In some embodiments, the heterologous portion is a half-life extension portion, a polymerizing domain, a targeting portion that binds to molecules on the cell surface, or a detectable marker. In some embodiments, the half-life extension portion comprises a polymerizing domain, albumin, albumin-binding polypeptide, Pro/Ala/Ser (PAS), the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, polyethylene glycol (PEG), a long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, or combinations thereof. In some embodiments, the at least one TACI polypeptide is linked to the Fc region of an immunoglobulin. In some embodiments, any immunomodulatory protein of any embodiment provided herein as a TACI-Fc fusion protein comprises at least one TACI polypeptide linked to the Fc region of an immunoglobulin.

In some embodiments, the immunomodulatory proteins provided herein do not include a TACI polypeptide linked to another targeting moiety that binds to a molecule on the cell surface. In some embodiments, the immunomodulatory proteins provided herein do not include a TACI polypeptide linked to a targeting 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 include a TACI polypeptide linked to a targeting moiety that is a binding partner of CD28 or a partner of CD28 (e.g., CD80 or CD86). In some embodiments, the immunomodulatory proteins provided herein do not include a TACI polypeptide linked to a CTLA-4 polypeptide or its extracellular domain or binding moiety, or a variant thereof. For example, in the provided aspects, the immunomodulatory proteins provided herein do not include a TACI polypeptide linked to a wild-type CTLA-4 polypeptide or its extracellular domain or binding moiety. In the respect provided, the immunomodulatory proteins provided herein do not include TACI peptides linked to variant CTLA-4 peptides or their extracellular domains or binding portions (such as variant CTLA-4 or its binding portions containing one or more amino acid modifications (e.g., substitutions) in the extracellular domains of CTLA-4, for example to increase binding affinity to one or more homologous binding partners).

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

In some embodiments, the TACI-Fc Fc fusion protein is a dimer. In some embodiments, the immunoglobulin Fc region is a homodimeric Fc region.

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

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

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

In some embodiments, the immunomodulatory protein contains one or more amino acid substitutions to reduce binding affinity to the Fc receptor and/or reduce effector function, optionally as compared to the wild-type IgG1 Fc domain. In some embodiments, the one or more amino acid substitutions are selected according to EU designations L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C. In some embodiments, the immunoglobulin Fc region contains amino acid substitutions according to EU designations L234A, L235E, and G237A, or amino acid substitutions according to EU designations R292C, N297G, and V302C.

In some embodiments, the TACI-Fc fusion protein comprises the following structure: TACI polypeptide (TACI)-linker-Fc region. In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:168. In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:170. In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:167. In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:169. In some embodiments, the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein.

This article provides an immunomodulatory TACI-Fc fusion protein, which is a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:167, linked by covalent disulfide bonds.

This article provides an immunomodulatory TACI-Fc fusion protein, which is a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:168, linked by covalent disulfide bonds.

This article provides an immunomodulatory TACI-Fc fusion protein, which is a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:169, linked by covalent disulfide bonds.

This article provides an immunomodulatory TACI-Fc fusion protein, which is a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:170, linked by covalent disulfide bonds.

In some embodiments, the TACI-Fc fusion protein comprises the following structure: (TACI)-connector-Fc region-connector-(TACI). In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:201. In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:202. In some embodiments, the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein.

In some embodiments, the TACI-Fc fusion protein comprises the following structure: (TACI)-connector-(TACI)-connector-Fc region. In some embodiments, the TACI-Fc fusion protein is shown in SEQ ID NO:198. In some embodiments, the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein.

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

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

In some embodiments, the Fc fusion protein neutralizes APRIL and BAFF. In some embodiments, the IC50 for neutralizing APRIL 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 foregoing; and/or the IC50 for neutralizing BAFF 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 foregoing.

This document provides one or more nucleic acid molecules that encode an immunomodulatory protein (e.g., an Fc fusion protein) of any of the embodiments described herein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid molecule is cDNA.

This document provides a vector containing a nucleic acid molecule of any of the embodiments described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector or a viral vector.

This document provides a cell containing nucleic acids or vectors of any of the embodiments described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

This document provides a method for generating an immunomodulatory protein, the method comprising introducing a nucleic acid molecule of any embodiment described herein or a vector of any embodiment described herein into a host cell under conditions in which the protein is expressed. In some embodiments, the method comprises isolating or purifying the immunomodulatory protein (e.g., an Fc fusion protein) from the cell. This document provides a method for generating an Fc fusion protein, the method comprising introducing a nucleic acid molecule of any embodiment described herein or a vector of any embodiment described herein into a host cell under conditions in which the protein is expressed.

This document provides an immunomodulatory protein (e.g., an Fc fusion protein) generated by any of the embodiments described herein.

This document provides a pharmaceutical composition comprising an immunomodulatory protein (e.g., an Fc fusion protein) according to any of the embodiments described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

This document provides an article of manufacture comprising a pharmaceutical composition of any embodiment described herein in a vial or container. In some embodiments, the vial or container is sealed.

This document provides a kit containing a pharmaceutical composition and instructions for use of any of the embodiments described herein. In some embodiments, the kit includes an article of manufacture and instructions for use of any of the embodiments described herein.

This article provides a method for reducing the immune response of a subject, the method comprising administering an immunomodulatory protein of any of the embodiments described herein to a subject in need.

This article provides a method for reducing the immune response of a subject, the method comprising administering an Fc fusion protein of any of the embodiments described herein to a subject in need.

This document provides a method for reducing the immune response of a subject, the method comprising administering a pharmaceutical composition of any of the embodiments described herein to a subject in need. In some embodiments, a B-cell immune response is reduced in the subject, thereby reducing or inhibiting B-cell maturation, differentiation, and/or proliferation. In some embodiments, circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer are reduced in the subject.

This document provides a method for reducing circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer in a subject, the method comprising administering a pharmaceutical composition of any of the embodiments described herein to the subject. In some embodiments, a T-cell immune response is reduced in the subject, thereby reducing or suppressing T-cell co-stimulation. In some embodiments, the reduction of the immune response is used to treat a disease or condition in the subject.

This article provides a method for treating a disease, disorder, or condition in a subject, the method comprising administering an immunomodulatory protein of any of the embodiments described herein to the subject in need.

This article provides a method for treating a disease, disorder, or condition in a subject, the method comprising administering an Fc fusion protein of any of the embodiments described herein to the subject in need.

This document provides a method for treating a subject with a disease, disorder, or condition, the method comprising administering a pharmaceutical composition of any of the embodiments described herein to a subject in need. In some embodiments, the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, B-cell carcinoma, antibody-mediated disease, nephropathy, graft rejection, graft-versus-host disease, or viral infection. In some embodiments, the disease, disorder, or condition is selected from: systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris. In some embodiments, the disease, disorder, or condition is B-cell carcinoma and the carcinoma is myeloma.

This article also provides a pharmaceutical composition for reducing the immune response in a subject.

This document also provides the use of any provided immunomodulatory protein (e.g., Fc fusion protein) or any provided pharmaceutical composition in the manufacture of a medicament for reducing the immune response of a subject.

In some embodiments of the pharmaceutical composition used for the purposes described herein, or for the purposes described herein, the immune response is a B-cell immune response, wherein reducing the immune response reduces or inhibits B-cell maturation, differentiation, and/or proliferation. In some embodiments, reducing the immune response reduces the circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer in the subject. In some embodiments, reducing the immune response treats a disease, disorder, or condition in the subject.

This article also provides a pharmaceutical composition for treating a subject’s disease, disorder, or condition.

This document also provides information on the use of any of the provided immunomodulatory proteins or pharmaceutical compositions in the manufacture of a medicament for the treatment of a subject’s disease, disorder, or condition.

In any embodiment of the pharmaceutical composition used for the purposes described herein, or in any embodiment of the purposes described herein, the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, B-cell carcinoma, antibody-mediated disease, nephropathy, graft rejection, graft-versus-host disease, or viral infection. In some embodiments, the disease, disorder, or condition is selected from: systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris. In some embodiments, the disease, disorder, or condition is B-cell carcinoma and the cancer is myeloma. In any embodiment, the type of myeloma includes multiple myeloma, plasmacytoma, multiple solitary plasmacytoma, and/or extramedullary myeloma. In some implementations, the types of myeloma include light chain myeloma, non-secretory myeloma, and/or IgD or IgE myeloma.

Attached Figure Description

Figure 1 shows a schematic representation of a functional inhibition assay involving recombinant APRIL and BAFF performed via TACI. In this assay, Jurkat cells were transduced with a luciferase-based NF-κB reporter and used to stably express mouse or human TACI based on cell surface expression. Upon activation by recombinant APRIL or BAFF, the endogenous NF-κB transcription factor binds to the DNA response element controlling the transcription of the firefly luciferase gene. Luteinase expression can be monitored, for example, by detection with Bio-Glo ^™ reagents and measurement using a Cytation 3 reader.

Figure 2 shows an exemplary human TACITD Fc fusion molecule used to block signal transduction mediated by human APRIL (top) and BAFF (bottom). The TACITD Fc fusion was incubated with APRIL or BAFF for 20 min (room temperature and shaking) and then added to wells containing 150,000 Jurkat/TACI/NFκB-luciferase cells for 5 h.

Figure 3 illustrates the function of exemplary TACI TD Fc fusion molecules in blocking APRIL (top of the figure) or BAFF (bottom of the figure).

Figure 4 shows the human TACI TDFc fusion molecule used to block APRIL (left) and BAFF (right) mediated signal transduction in mice.

Figure 5 shows the human TACI TD Fc fusion molecules used to block human APRIL (top) and BAFF (bottom) mediated signal transduction, relative to TACI 13-118-Fc, TACI 30-110-Fc, and belimumab.

Figures 6A-6I show the analysis of parameters assessed in a human SLE NZB/NZW mouse model. Proteinuria score (Figure 6A), mean percentage change in body weight (Figure 6B), and survival percentage (Figure 6C) were assessed starting at 20 weeks of age. Serum was analyzed for anti-double-stranded DNA IgG titer (Figure 6D) and blood urea nitrogen (BUN) (Figure 6E) (**** compared to Fc, p < 0.0001 for anti-dsDNA IgG via Student's t-test; *** compared to Fc, p = 0.0008 for BUN-4 via Student's t-test). Kidneys were processed and histologically analyzed in repeated periodic acid Schiff (PAS) stained sections, and individual components and total histological scores are depicted in Figure 6F. Frozen kidney sections were also stained for immunohistochemical analysis of mouse IgG and complement C3 glomerular deposits, as shown in Figures 6G and 6H, respectively. Figure 6I shows histological scores ± SEM.

Figure 7 shows the ability of the TACI mutation (K77E/F78Y/Y102D) to inhibit APRIL (left) and BAFF (right) mediated signal transduction, quantified by luciferase production in Jurkat/NF-κB/TACI cells.

Figures 8A and 8B depict schematic representations of exemplary TACI-Fc fusion proteins. Figure 8A depicts an exemplary TACI-Fc fusion protein containing two cysteine-rich pseudo-repetitive sequences (CRDs). Figure 8B depicts an exemplary TACI-Fc fusion protein containing one cysteine-rich pseudo-repetitive sequence (CRD, such as CRD2).

Figure 9 depicts an exemplary sequence alignment used 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 of amino acid substitutions used herein are indicated in bold text. Based on the alignment of two similar sequences having common identical residues, a person skilled in the art can identify “corresponding” positions in a sequence by comparing it to a reference sequence using conserved and identical amino acid residues as guidelines. Figure 9 provides an exemplary alignment of the reference TACI extracellular domain sequence shown in SEQ ID NO:122 (containing an intact extracellular domain with CRD1 and CRD2 and an initiating methionine residue) with the TACI extracellular domain sequence shown in SEQ ID NO:13 (containing only a single CRD, namely CRD2). The alignment of the same residues shows that, for example, amino acid residue E7 in SEQ ID NO:13 corresponds to residue E74 in SEQ ID NO:122, amino acid residue K10 in SEQ ID NO:13 corresponds to residue K77 in SEQ ID NO:122, amino acid residue Y12 in SEQ ID NO:13 corresponds to Y79 in SEQ ID NO:122, amino acid residue L15 in SEQ ID NO:13 corresponds to L82 in SEQ ID NO:122, amino acid residue R17 in SEQ ID NO:13 corresponds to R84 in SEQ ID NO:122, and amino acid residue D16 in SEQ ID NO:13 corresponds to D85 in SEQ ID NO:122. Technicians can skillfully perform similar comparisons between two similar protein sequences to identify corresponding residues, including based on the examples and descriptions in this article.

Figures 10A-10D show the analysis of parameters assessed in the mouse keyhole hemocyanin (KLH) model. Serum -KLH IgG1 OD levels were assessed as primary response (Figure 10A) and secondary response (Figure 10B). Similarly, serum anti-KLH IgG1 OD levels were assessed as both primary response (Figure 10C) and secondary response (Figure 10D).

Figures 11A-11B show the analysis of harvested spleens assessed using a mouse keyhole hemocyanin (KLH) immunization model. The spleens were processed and analyzed by weight (Figure 11A) and total cell count (Figure 11B).

Figure 12 depicts the analysis of the spleen based on the assessment of cell subtype population composition using the mouse keyhole hemocyanin (KLH) model, and shows the results of the number of B cell subsets relative to the group mean.

Figure 13 depicts the analysis of the spleen based on the assessment of cell subtype phenotypic composition from the mouse keyhole hemocyanin (KLH) model, and shows the results of the number of germinal center B cells and plasma cells (Figure 13).

Figures 14A-14D depict the number of T cells in a mouse keyhole hemocyanin (KLH) model. Splenic CD3+, CD8+, CD4+, and follicular helper T cells are depicted in Figures 14A, 14B, 14C, and 14D, respectively.

Figure 15 depicts the Tcm and Tem cell populations in a mouse keyhole hemocyanin (KLH) model.

Figures 16A-16B and 17A-17B depict the overall incidence and severity of sialorrhea (Figure 16A-16B) and pancreatitis (Figure 17A-17B) in diabetic mice after treatment with the tested molecules.

Detailed Implementation

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

The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and to protect tissues from excessive damage during immune responses, such as during an attack against a pathogen infection. However, in some situations, the immune system can become dysregulated and may launch abnormal immune responses against normal body parts or tissues, leading to autoimmune diseases or conditions or symptoms. In other situations, an undesirable immune response may be launched against foreign tissues such as grafts, resulting in transplant rejection.

In some respects, immunotherapies that alter the activity of immune cells (such as B cell activity) can treat certain diseases, disorders, and conditions with dysregulated immune responses. Specifically, suppression or attenuation of immune responses (such as B cell responses) may be desirable for reducing or preventing unwanted inflammation, autoimmune symptoms, and/or transplant rejection. However, therapies that seek to modulate the interactions between ligands and their receptors that mediate immune responses are not entirely satisfactory. In some cases, therapies for intervening in and altering the immunomodulatory effects of immune cell (e.g., B cell) activation are limited by spatial orientation requirements and size constraints imposed by immune synaptic boundaries. In some respects, 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 typically bind competitively (e.g., binding to no more than one target species at a time) and therefore lack the ability to bind multiple targets simultaneously. Additionally, pharmacokinetic differences between drugs that independently target one of these receptors can pose difficulties in maintaining the required blood concentrations of drug combinations targeting two different targets throughout treatment.

BAFF and APRIL are members of the TNF superfamily that bind to both TACI and BCMA on B cells; BAFF also binds to a third receptor, BAFF-R. Together, BAFF and APRIL support B cell development, differentiation, and survival, particularly for plasmablasts and plasma cells, and play a role in the pathogenesis of B cell-related autoimmune diseases. Their co-neutralization significantly reduces B cell function, including antibody production, while the inhibitory effects mediated by BAFF or APRIL alone are relatively small. Fc fusions of wild-type (WT) TACI (e.g., acetaxel and telitacicept) target both BAFF and APRIL and have shown promising clinical potential in diseases such as systemic lupus erythematosus (SLE) and IgA nephropathy, but long-term and/or complete disease remission have not yet been clearly demonstrated. Although B cell-targeted therapies have shown promising therapeutic potential, they are not entirely satisfactory. For example, soluble recombinant TACI shows great promise as a therapeutic agent, but its usefulness appears to be hampered by its low to medium affinity for APRIL.

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

The provided embodiments relate to the identification of variant TACI peptides engineered to possess improved affinity for APRIL and/or BAFF following random mutagenesis and directed evolution of the second cysteine-rich domain (CRD2) of TACI (spanning residues 68-110). As shown herein, affinity maturation involves five selections alternating between APRIL and BAFF, wherein the concentration of the selecting agent is decreased in parallel to maintain selection pressure. Results show that the variant TACI peptides exhibit significantly enhanced affinity for BAFF and APRIL compared to wild-type TACI. For example, variant TACI peptides containing one or more amino acid substitutions (substitutions or mutations) are provided herein, which confer improved binding affinity for BAFF and/or APRIL to the protein. Specifically, the provided embodiments include those providing improved combined BAFF and APRIL inhibition. Thus, the provided immunomodulatory proteins provide effective and durable disease suppression in the treatment of autoimmune or inflammatory diseases, including severe B-cell-related autoimmune diseases such as SLE.

For example, the provided implementation is based on the finding that directed evolution through affinity modification of the TNFR domain (TD) of the extracellular domain of TACI facilitates the development of molecules with improved affinity for APRIL and/or BAFF. Therefore, this affinity modification yields a variant TACI containing a variant TNFR domain (vTD). Fusion of such a molecule with immunoglobulin Fc yields an immunomodulatory protein that inhibits B cell activity and response. For example, reformatted into a soluble Fc fusion protein, the affinity-matured TACI variant output exhibits inhibition of APRIL and BAFF, as shown in this paper in the TACI-dependent reporter assay, and its _IC50 value is lower than that of wild-type TACI-Fc and belimumab comparisons. Furthermore, results in the evaluated animal models showed a rapid and significant reduction in key lymphocyte subsets, including plasma cells, germinal center B cells, and follicular T helper cells. Furthermore, the tested variant molecules exhibited improved activity in mouse models, including significantly reduced autoantibodies and sialadenitis in a spontaneous SjS model, suppressed glomerular IgG deposition in a bm12-induced lupus model, and effective inhibition of anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, sialadenitis, nephropathy, and renal immune complex deposition in an NZB/W lupus model. Additionally, the tested TACI-Fc fusions showed significantly and persistently reduced serum IgM, IgG, and IgA antibody titers in mice compared to wild-type TACI-Fc. These findings confirm that these immunomodulatory proteins consistently exhibit potent immunosuppressive activity and efficacy both in vitro and in vivo, outperforming existing and/or approved immunomodulators such as belimumab, abatacept, acecept, or teltacept. Such biologics could therefore be attractive development candidates for the treatment of severe autoimmune and/or inflammatory diseases, including B-cell-related diseases such as SLE, Sjögren's syndrome, and other connective tissue diseases.

All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated individually by reference. Where the definitions described herein contradict or otherwise are inconsistent with those set forth in patents, applications, publications, and other publications incorporated herein by reference, the definitions described herein shall prevail over those incorporated herein by reference.

The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

I. Definition

Unless otherwise defined, all field terms, symbols, and other technical and scientific terms or names used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or convenience of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from the meaning commonly understood in the art.

Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural indicators.

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

As used in the context of protein domains, the term "affinity-modified" refers to mammalian proteins that have an altered amino acid sequence (relative to the corresponding wild-type parent or unmodified domain) in their extracellular domain or their specific binding moiety, such that they have increased or decreased binding activity (e.g., binding affinity) with at least one of their binding partners (or alternatively, "anti-structure") compared to the parental wild-type or unmodified (i.e., non-affinity-modified domain) proteins. In some embodiments, the affinity-modified domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences (e.g., amino acid substitutions) in the wild-type or unmodified domain. The increase or decrease in binding activity (e.g., binding affinity) can be determined using well-known binding assays (including flow cytometry). Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005). See also Linsley et al., Immunity, 1:7930801 (1994). An increase in the binding activity (e.g., affinity) of the protein with one or more binding partners is an increase to values at least 10% greater than the wild-type control, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type control. A decrease in the binding activity (e.g., affinity) of the protein with at least one of its binding partners is a decrease to values not greater than 90% of the control but not less than 10% of the wild-type control value, and in some embodiments, not greater than 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the wild-type control value but not less than 10%. Affinity-modified proteins are altered in the primary amino acid sequence of their extracellular domains or their specific binding moieties through substitution, addition, or deletion of amino acid residues. The term "affinity-modified" is not intended to impose any particular starting composition or method on which the affinity-modified protein is produced. Therefore, affinity-modified proteins are not limited to wild-type protein domains subsequently converted to affinity-modified domains through any particular affinity-modification process. Affinity-modified domain peptides can, for example, be generated starting from wild-type mammalian domain sequence information, then modeled in a computer for binding to their binding partner, and finally recombined or chemically synthesized to produce an affinity-modified domain composition of the substance. In only one alternative example, the affinity-modified domain can be produced by site-directed mutagenesis of the wild-type domain. Therefore, an affinity-modified TD domain represents a product and is not necessarily a product produced by any given method. Various techniques can be employed, including recombinant methods, chemical synthesis, or combinations thereof.

The term "affinity-modified TD domain" refers to an affinity-modified domain of a member of the tumor necrosis receptor superfamily (TNFRSF) protein or its TNF ligand, having an altered amino acid sequence of either a TNFR domain or a TNF domain. For example, an affinity-modified TD domain of a TNFRSF protein has an altered amino acid sequence of the TNFR domain, the altered sequence consisting of at least one cysteine-rich domain (CRD) within the extracellular domain of the TNFRSF protein or its specific binding moiety (relative to the corresponding wild-type parent or unmodified domain), such that it has increased or decreased binding activity (e.g., binding affinity) with at least one of its binding pair (or alternatively, "anti-structure") compared to a parental wild-type or unmodified protein containing an unaffected or unmodified TD domain.

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

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

As used herein, “bind,” “bound,” or grammatical variations thereof refer to any attractive interaction between a molecule and another molecule, resulting in a stable association in which the two molecules are very close to each other. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as chemical compounds including pharmaceuticals.

As used herein, binding activity refers to a characteristic of a molecule (e.g., a polypeptide) in relation to whether and how it binds to one or more binding partners. Binding activity can include any measure of a molecule's binding to a binding partner. Binding activity includes the ability to bind to one or more binding partners, its affinity for binding to a binding partner (e.g., high affinity), the strength of its bond to a binding partner, and/or the specificity or selectivity of its binding to a binding partner.

As used herein, the term “binding affinity” refers to the specific binding affinity of a protein to its binding partner (i.e., its anti-structure) under specific binding conditions. Binding affinity refers to the strength of the interaction between two or more molecules (such as binding partners), typically the strength of the non-covalent interaction between the two binding partners. An increase or decrease in the binding affinity of an affinity-modified domain or an immunomodulatory protein containing an affinity-modified domain to its binding partner is determined relative to the binding affinity of an unmodified domain (e.g., a native or wild-type TD domain). Methods for determining binding affinity or relative binding affinity are known in the art, including solid-phase ELISA, 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 implementations, binding affinity can be measured by flow cytometry, such as based on mean fluorescence intensity (MFI) in flow binding assays.

As used herein, “binding affinity” refers to the specific binding affinity of a protein to its binding partner (i.e., its antistructure) under specific binding conditions. In biochemical kinetics, affinity refers to the cumulative strength of multiple affinities between individual non-covalent binding interactions (such as between a protein and its binding partner (i.e., its antistructure)). Therefore, affinity differs from affinity, which describes the strength of a single interaction.

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

As used herein, the term "cell surface antistructure" (or alternatively "cell surface binding partner") refers to an antistructure (or binding partner) expressed on mammalian cells. Typically, the cell surface binding partner is a transmembrane protein. In some embodiments, the cell surface binding partner is a receptor.

The term "binding partner" or "anti-structure" in relation to proteins (such as receptors, soluble ligands) or their extracellular domains or portions thereof, or affinity-modified variants thereof, refers to at least one molecule (typically a native mammalian protein) that specifically binds to a reference protein under specific binding conditions. In some aspects, affinity-modified domains or immunomodulatory proteins containing affinity-modified domains specifically bind to binding partners of the corresponding domains of native or wild-type proteins, but with increased or decreased affinity. "Cell surface binding partner" refers to a binding partner expressed on mammalian cells. Typically, said cell surface binding partner is a transmembrane protein. In some embodiments, said cell surface binding partner is a receptor or receptor ligand expressed on or by cells forming immune synapses (such as mammalian cells, e.g., immune cells).

The term "cis" refers to binding to two or more different cell surface molecules, each present on the surface of the same cell. In some embodiments, cis means that the two or more cell surface molecules are present on only one of the two mammalian cells that form IS, or on only the other (but not both).

As used herein, the term "conservative amino acid substitution" refers to 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 groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

The term "corresponds to" in relation to protein positions, such as the description of a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a published sequence, as described in the sequence listing, refers to the nucleotide or amino acid position identified after alignment with the published 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, for example, use conserved and identical amino acid residues as guidance to identify corresponding residues. Figure 9 illustrates the identification of corresponding residues by aligning two sequences.

As used herein, a “domain” (typically three or more, generally five or seven or more amino acids, e.g., 10 to 200 amino acid residues) refers to a portion of a molecule (e.g., a protein or encoding a nucleic acid) that is structurally and/or functionally distinct from other parts of the molecule and is identifiable. For example, a domain includes portions of a polypeptide chain that can form independently folded structures within a protein composed of one or more structural motifs and/or be identified by functional activity (e.g., binding activity). Proteins may have one or more unique domains. For example, domains can be identified, defined, or distinguished by the homology of their primary sequence or structure with related family members (e.g., homology with motifs). In another example, domains can be distinguished by their function, such as their ability to interact with biomolecules (e.g., homologous binding partners). A domain may independently exhibit a biological function or activity, such that the domain is active independently or after fusion with another molecule, e.g., binding. A domain can be a linear or non-linear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. Definitions are provided for the purposes of this article, but it should be understood that identifying a specific domain by name is within the scope of the art. If desired, appropriate software can be used to identify the domains. It should be understood that references to amino acids, including references to the specific sequence shown as SEQ ID NO used to describe the organization of the domains (e.g., the organization of the TD domain), are for illustrative purposes and are not intended to limit the scope of the embodiments provided. It should be understood that the description of peptides and their domains is theoretically derived based on homology analysis and alignment with similar molecules. Similarly, in some cases, adjacent N- and/or C-terminal amino acids of a given domain (e.g., TD) may also be included in the sequence, for example, to ensure proper folding of the domain during expression. Therefore, the exact locus can vary and is not necessarily the same for every protein. For example, a particular TD domain (e.g., a particular CRD domain) may be a few amino acids longer or shorter (1–10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

In this document, the terms “extracellular domain,” “extracellular structural domain,” or “ECD” are used interchangeably to refer to the region of a membrane protein located outside the vacuolar membrane (e.g., the space outside the cell) when expressing the full-length form of a membrane protein (such as a transmembrane protein) from a cell. For the purposes of this document, it should be understood that reference to an ECD refers to the sequence and domain constituting that region and does not require that the protein containing the ECD be a membrane protein or that the domain be located outside the cell. For example, soluble immunomodulatory proteins may contain the ECD sequence of a membrane protein fused to another portion (such as a polymerizing domain, e.g., the Fc region). Extracellular domains typically interact with specific ligands or specific cell surface receptors, for example, by specifically binding to binding domains of ligands or cell surface receptors. Examples of binding domains include cysteine-rich domains (CRDs). The extracellular domains of members of the TNFR superfamily contain TD domains (e.g., CRD domains). Therefore, reference to an ECD herein includes the full-length sequence of the ECD of a membrane protein, as well as the CRD-containing specific binding fragment that binds to a ligand or homologous binding partner.

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

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

The term "extended half-life fraction" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of a protein not so conjugated to the fraction. In some embodiments, the half-life is extended by more than or about 1.2 times, about 1.5 times, about 2.0 times, about 3.0 times, about 4.0 times, about 5.0 times, or about 6.0 times. In some embodiments, 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 the extended half-life fraction. Half-life refers to the length of time it takes for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extension portions include an Fc domain, a polymerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS) and polyglutamic acid (glutaminelated).

The Fc (crystallizable fragment) region or domain (also known as Fc polypeptide) of an immunoglobulin molecule primarily corresponds to the constant region of the immunoglobulin heavy chain and is responsible for multiple functions in some cases, including one or more effector functions of an antibody. The Fc domain contains part or all of the hinge domain of the immunoglobulin molecule plus CH2 and CH3 domains. In some cases used for inclusion in the provided fusion protein, all or part of the Fc hinge sequence may be missing. The Fc domain can form a dimer of two polypeptide chains linked by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits reduced (e.g., reduced by more than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) activity to promote effector function. In some embodiments, references to amino acid substitutions in the Fc region are made according to the EU numbering system unless described with respect to a specific SEQ ID NO. The EU number is known and conforms to the latest updated IMGTS Scientific Chart (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 as reported in the following literature: Kabat, E.A. et al. Sequences of Proteins of Immunological Interest. 5th edition. US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

An immunoglobulin Fc fusion (“Fc fusion”) (such as an immunomodulatory Fc fusion protein) is a molecule comprising one or more polypeptides operatively linked to the Fc region of an immunoglobulin. An Fc fusion may comprise, for example, an Fc region operatively linked to the TACI extracellular domain or its CRD-containing portion (including any variants with provided affinity modifications). The immunoglobulin Fc region may be linked indirectly or directly to one or more polypeptides. Various linkers are known in the art and may optionally be used to link Fc to a fusion partner to generate an Fc fusion. Fc fusions of the same kind may dimerize to form a homodimer of Fc fusions. Fc fusions of different kinds (e.g., pestle-and-mortar engineered structures) may be used to form heterodimers of Fc fusions. In some embodiments, the Fc is a mammalian Fc, such as a mouse or human Fc.

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 single-celled eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridomas. Examples of host cells include Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium, or the DHFR-deficient CHO strain DX-B11.

As used in this article, the term "immunological synapse" or "immunesynapse" refers to the interface between mammalian cells (such as antigen-presenting cells or tumor cells) that express MHC I (major histocompatibility complex) or MHC II and mammalian lymphocytes (such as effector T cells or natural killer (NK) cells).

As used herein, the term "immunoglobulin" (abbreviated as "Ig") is synonymous with the term "antibody" (abbreviated as "Ab") and refers to mammalian immunoglobulins, including any of the five human classes: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also includes immunoglobulins shorter than full length, whether wholly or partially synthetic (e.g., recombinant or chemically synthesized) or naturally occurring, including any fragment containing at least a portion of the variable heavy (VH) chain and/or variable light (VL) chain region of the immunoglobulin molecule, said fragment being sufficient to form an antigen-binding site and sufficient to specifically bind an antigen upon assembly. Antibodies may also include all or a portion of the constant region. Such fragments include antigen-binding fragments (Fab), variable fragments containing VH and VL (Fv), single-chain variable fragments containing VH and VL linked in one chain (scFv), and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv biantibodies, Fc, and Fd polypeptide fragments. Therefore, it should be understood that references to antibodies herein include both full-length antibodies and antigen-binding fragments. The term antibody also includes antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies, and single-chain molecules. Bispecific antibodies with and without bispecificity are included within the meaning of the term. Antibodies include polyclonal antibodies or monoclonal antibodies. Antibodies also include synthetic antibodies or recombinant antibodies. For the structure and properties of different antibody classes, 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.

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

"Antibody fragment" comprises a portion of a complete antibody, the antigen-binding region and/or variable region of the complete antibody. Antibody fragments (including but not limited to Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, disulfide-linked Fv(dsFv), Fd fragments, Fd'fragments;biantibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062[1995]); single-chain antibody molecules, including single-chain Fv(scFv) or single-chain Fab(scFab); antigen-binding fragments of any of the above and multispecific antibodies derived from antibody fragments.

The “Fv” consists of a heavy chain variable region domain and a light chain variable region domain linked by non-covalent association. Six complementarity-determining regions (CDRs) radiate from the folds of these two domains (three from the heavy chain and three from the light chain), contributing amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even if a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, in some cases its affinity is lower than that of the entire binding site.

“dsFv” refers to an engineered intermolecular disulfide bond with a stable V _H - V _L pair.

The “Fd fragment” is an antibody fragment containing a variable domain ( _VH ) and a constant domain ( _CH1 ) of the antibody heavy chain.

"Fab fragments" are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments with the same structure produced synthetically (e.g., by recombinant methods). Fab fragments contain a light chain (containing _VL and _CL ) and another chain containing a variable domain ( _VH ) of the heavy chain and a constant region domain ( _CH1 ) of the heavy chain.

The “F(ab’) ₂ fragment” is an antibody fragment obtained by digesting immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure produced synthetically (e.g., by recombinant methods). The F(ab’) ₂ fragment essentially contains two Fab fragments, each heavy chain portion containing several additional amino acids, including cysteine residues that form the disulfide bonds that connect the two fragments.

A “Fab’ fragment” is a fragment containing half of an F(ab’) ₂ fragment (one heavy chain and one light chain).

The “Fd’ fragment” is an antibody fragment containing a heavy chain portion of the F(ab’) ₂ fragment.

The “Fv” fragment is a fragment containing only the _VH and _VL domains of the antibody molecule.

"scFv fragment" refers to an antibody fragment containing a variable light chain ( _VL ) and a variable heavy chain ( _VH ) covalently linked in any order via a peptide linker. The length of the linker allows the two variable domains to be bridged without significant interference. An exemplary linker is (Gly-Ser) _n residues, some of which contain Glu or Lys residues distributed throughout to increase solubility.

"Biantibody" is a dimerized scFv; biantibodies typically have peptide linkers shorter than scFv and preferentially undergo dimerization.

As used herein, “immunoactivity” refers to one or more activities of immune cells (such as T cells or B cells), including, for example, activation, cell survival, cell proliferation, cytokine production (e.g., interferon-γ), cytotoxic activity, or the ability to activate the NF-κB pathway or other signaling cascades that lead to activation of transcription factors in immune cells. Assays used to assess the immunomodulatory activity of proteins can be compared to control proteins with inhibitory activity.

"Immunomotor proteins" or "immunomotor polypeptides" are proteins that regulate immune activity. "Modulation" or "modulating" an immune response means enhancing or suppressing immune activity. Such regulation includes any induction, alteration of degree or extent, or inhibition of the immune activity of immune cells (such as B cells or T cells). For example, the soluble Fc fusion protein described herein can suppress the immune activity of B cells. Immunomodulatory proteins can be single polypeptide chains or polymers (dimers or higher-order polymers) of at least two polypeptide chains covalently linked together by, for example, interchain disulfide bonds. Therefore, monomers, dimers, and higher-order polymers are within the scope of the defined terminology. Polymers can be homopolymers (having the same polypeptide chain) or heteropolymers (having different polypeptide chains).

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

The term "multimerizing domain" refers to an amino acid sequence that promotes the formation of multimers of two or more polypeptides. A multimerizing domain includes a sequence that promotes stable interactions between a polypeptide molecule and one or more other polypeptide molecules, each containing a complementary multimerizing domain (e.g., a first multimerizing domain and a second multimerizing domain), which may be the same or different. Interactions between complementary multimerizing domains (e.g., interactions between a first and a second multimerizing domain) form stable protein-protein interactions to produce multimers of the polypeptide molecule with other polypeptide molecules. In some cases, the multimerizing domains are identical and interact with themselves to form stable protein-protein interactions between two polypeptide chains. Typically, the polypeptide binds directly or indirectly to the multimerizing domain. Exemplary multimerizing domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. For example, the multiplying domain can be an immunoglobulin constant region or domain, such as the Fc domain or a portion thereof from IgG (including IgG1, IgG2, IgG3 or IgG4 subtypes), IgA, IgE, IgD and IgM and their modified forms.

The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to polymers of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form. Unless explicitly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to natural nucleotides and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implies coverage of variants with conserved modifications (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 mixture of bases and/or deoxyinosine residues. The terms nucleic acid or polynucleotide encompass cDNA or mRNA encoded by genes.

As used herein, the terms "operably combined," "in an operational sequence," and "operably linked" refer to the manner or orientation in which nucleic acid sequences are linked such that the segments are arranged to work synergistically for their intended purpose. In some embodiments, these terms refer to nucleic acid linkage to produce nucleic acid molecules capable of directing transcription of a given gene and/or producing desired protein molecules with functional properties. For example, segments of a DNA sequence (e.g., a coding sequence and one or more regulatory sequences) are linked in such a manner that gene expression is permitted when an appropriate molecule (e.g., a transcriptional activating protein) binds to the regulatory sequence.

The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammalian subjects (typically humans). Pharmaceutical compositions typically contain an effective amount of an active agent (e.g., an immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

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

The term "purified," when applied to nucleic acids (such as nucleic acids encoding immunomodulatory proteins or proteins such as immunomodulatory proteins), generally means nucleic acids or peptides that are substantially free of other components as determined by analytical techniques well known in the art (e.g., purified peptides or polynucleotides form discrete bands in electrophoresis gels, chromatographic eluents, and/or media subjected to density gradient centrifugation). For example, nucleic acids or peptides that produce substantially one band in an electrophoresis gel are "purified." Purified nucleic acids or proteins are at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99%, or higher (e.g., weight percentage or moles).

The term “recombinant” indicates that a material (e.g., a nucleic acid or polypeptide) has undergone artificial (i.e., non-natural) alteration through human intervention. This alteration can be performed on material within its natural environment or state, or on material taken from its natural environment or state. For example, a “recombinant nucleic acid” is a nucleic acid prepared, for example, during cloning, affinity modification, DNA shuffling, or other well-known molecular biology procedures. A “recombinant DNA molecule” consists of DNA segments joined together using such molecular biology techniques. As used herein, the terms “recombinant protein” or “recombinant polypeptide” refer to protein molecules (e.g., immunomodulatory proteins) expressed using recombinant DNA molecules. A “recombinant host cell” is a cell containing and/or expressing recombinant nucleic acids or otherwise altered through genetic engineering, for example, by introducing nucleic acid molecules encoding recombinant proteins (e.g., immunomodulatory proteins as presented herein). Transcriptional control signals in eukaryotes include “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (similar control elements, i.e., promoters, have also been found in prokaryotes). The choice of specific promoters and enhancers depends on the cell type to be used to express the target protein.

As used herein, the term "recombinant expression vector" refers to a DNA molecule containing a desired coding sequence (e.g., encoding an immunomodulatory protein) and an appropriate nucleic acid sequence required for expression in a specific cell. The required nucleic acid sequence for expression in prokaryotes includes a promoter, optionally an operon sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Secretory signal peptide sequences may also optionally be encoded by a recombinant expression vector operatively linked to a coding sequence such that the expressed protein can be secreted by a recombinant host cell, such as for its expression as a secretible protein or for easier isolation or purification of immunomodulatory proteins from cells (if desired). The term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the host cell genome. These vectors include viral vectors, such as lentiviral vectors.

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

As used herein with respect to the term "soluble," the protein is not a membrane protein or is not anchored to a cell membrane. Proteins can be constructed as soluble proteins by including only extracellular domains or portions thereof and without transmembrane domains. In some cases, protein solubility can be improved by directly or indirectly attaching to or via a linker to an Fc domain or other half-life-extending molecule, which in some cases may also improve the protein's stability and/or half-life. In some aspects, soluble proteins are Fc fusion proteins.

As used herein, the term "specific binding" means the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or co-affinity is at least 10 times, but optionally 50, 100, 250, or 500 times, or even at least 1000 times, greater than the average affinity or co-affinity of the same protein with a sufficiently statistically large set of random peptides or polypeptides. Specific binding proteins need not bind to only a single target molecule, but can bind specifically to more than one target molecule. In some cases, specific binding proteins can bind to proteins with structural conformations similar to the target protein (e.g., paralogs or orthologs). Those skilled in the art will recognize that specific binding to molecules that have the same function in different species of animals (i.e., orthologs) or molecules with substantially similar epitopes to the target molecule (e.g., paralogs) is possible without diminishing the binding specificity determined relative to a statistically effective set of unique non-targets (e.g., random polypeptides). Thus, the immunomodulatory proteins of the present invention can specifically bind to more than one different class of target molecules due to cross-reactivity. Solid-phase ELISA, ForteBio Octet, or Biacore measurements can be used to determine the specific binding between two proteins. Typically, the dissociation constant (Kd) of the interaction between the two binding proteins is less than about 1 x ^10⁻⁵ M, and usually as low as about 1 x ^10⁻¹² M. In some aspects of this disclosure, the dissociation constant of the interaction between the two binding proteins is less than about 1 x ^10⁻⁶ M, 1 x ^10⁻⁷ M, 1 x ^10⁻⁸ M, 1 x ^10⁻⁹ M, 1 x ^10⁻¹⁰ M, or 1 x ^10⁻¹¹ M or less.

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

As used in this article, “subject” is a mammal, such as a human or other animal, and is usually a human. Subjects can be male or female and can be of any suitable age, including infants, young children, adolescents, adults, and elderly subjects.

As used herein, “synthetic” in the context of, for example, synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, refers to nucleic acid or polypeptide molecules produced by recombination and/or by chemical synthesis.

As used herein, the term "TNF receptor superfamily" or "TNFRSF" refers to a group of cell surface cytokine receptors that are type I (N-terminal extracellular) transmembrane glycoproteins containing one to six cysteine-rich domains (CRDs) in their extracellular domain. Molecules are classified as members of this superfamily based on shared structural features, including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular region, which typically function in the binding of the protein to its homologous binding partner or ligand. TNFRSF proteins may have only one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or extracellular domain of TNFRSF members contains between one and six CRD pseudo-repetitive sequences. For example, the BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are typically trimeric or multimeric complexes, stabilized by intracysteine disulfide bonds. The binding of TNFRSF protein to its ligands promotes a variety of biological activities in cells, such as the induction of apoptotic cell death or cell survival and proliferation.

The term "TD" refers to one or more structural domains of a TNFRSF protein or a TNF family ligand. For example, the TD of the TNFRSF protein is a cysteine-rich domain (CRD) module of about 40 amino acids containing six (6) conserved cysteine residues. Therefore, the term TD can also be used interchangeably with the term TD concerning the TD of the TNFRSF protein. The six cysteine residues are involved in the formation of intrachain disulfide bonds. The extracellular domain (ECD) of TNFRSF members contains one or more CRD domains; therefore, the term TD is also used concerning the ECD of such protein molecules. When referring to a variant TD (vTD), it means a variant or modified sequence of the TD.

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

As used herein, the term "transmembrane protein" refers to a membrane protein that spans substantially or completely across a lipid bilayer, such as those found in biological membranes (e.g., mammalian cells) or in artificial constructs (e.g., liposomes). A transmembrane protein comprises a transmembrane domain ("transmembrane domain") through which the protein is integrated into the lipid bilayer, and this integration is thermodynamically stable under physiological conditions via the transmembrane domain. Transmembrane domains can generally be predicted from their amino acid sequences based on their increased hydrophobicity relative to protein regions interacting with aqueous environments (e.g., cytosol, extracellular fluid) using a variety of commercially available bioinformatics software applications. Transmembrane domains are typically hydrophobic α-helices that span the membrane. Transmembrane proteins may cross both layers of the lipid bilayer once or multiple times.

As used herein, the terms “treating,” “treatment,” or “therapeutic” for a disease or disorder mean to slow, stop, or reverse the progression of a disease, condition, or disorder, as demonstrated by a reduction, cessation, or elimination of clinical or diagnostic symptoms, achieved by the application of the immunomodulatory protein of the present invention, alone or in combination with another compound as described herein, or engineered cells. “Treating,” “treatment,” or “therapeutic” also means a reduction in the severity of symptoms of an acute or chronic disease, condition, or disorder, or a reduction in the relapse rate, such as in the case of a relapsing or remission course of an autoimmune disease or inflammatory condition, or a reduction in inflammation in the case of an autoimmune disease or inflammatory condition. As used in the context of the present invention, “preventing,” “prophylaxis,” or “prevention” for a disease, condition, or disorder means the application of the immunomodulatory protein of the present invention, alone or in combination with another compound, to prevent the occurrence or onset of the disease, condition, or disorder, or some or all of its symptoms, or to reduce the likelihood of its onset.

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

As used interchangeably in this article, the terms “wild-type”, “natural”, or “native” are used in conjunction with biological materials (such as nucleic acid molecules, proteins, host cells, etc.) found in nature and not modified by human intervention.

II. TACI Immunomodulatory Proteins and Variant TACI Peptides

This document provides TACI immunomodulatory proteins containing an extracellular domain (ECD) of the TACI receptor or a variant thereof that binds to at least one TACI homologous binding partner. This document also provides variant TACI peptides exhibiting altered (e.g., increased) binding activity or affinity for one or more TACI homologous binding partners. In some embodiments, the TACI homologous binding partner is one or more of BAFF or APRIL, or a BAFF/APRIL heterotrimer. The provided TACI immunomodulatory proteins and peptides comprise soluble fusion proteins thereof, wherein a TACI portion of the extracellular domain or a portion thereof is linked to another portion (such as immunoglobulin Fc or other polymerized domains or extended half-life portions). 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 peptide consisting of an extracellular domain of the TACI receptor or a portion thereof that binds to at least one TACI homologous binding partner or a variant of the TACI peptide, and (2) an Fc domain. The TACI polypeptide or its variants can be linked to the Fc domain directly or indirectly (e.g., via a peptide linker).

TACI is a member of the tumor necrosis factor receptor family, characterized by an extracellular domain (ECD) containing a cysteine-rich pseudorepetitive sequence domain (CRD). TACI is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudorepetitive sequences (CRD1 and CRD2), a transmembrane domain, and a cytoplasmic domain that interacts with CAML (calcium regulator and cyclic ligand), an integrated membrane protein located in intracellular vesicles that is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with subsets of B cells and T cells. The TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family. One ligand was named BAFF (a B-cell activating factor of the TNF family), and it was also named ZTNF4, "neutrokine-α", "BLyS", "TALL-1" and "THANK" in different ways (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)). Another ligand has been named APRIL, and has also been referred to in different ways 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 to the B cell maturation receptor (BCMA) (Gross et al., Nature 404:995 (2000)). The binding of the TACI receptor to its ligands BAFF or APRIL stimulates B cell responses, including non-T cell-dependent B cell antibody responses, allotype switching, and B cell homeostasis.

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

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

In some embodiments, the reference (e.g., unmodified) TACI sequence is a wild-type TACI sequence or a portion thereof containing one or two CRDs. In some embodiments, the reference (e.g., unmodified) TACI is or contains the extracellular domain (ECD) of TACI or a portion thereof containing one or two CRD domains. In some embodiments, the extracellular domain of the reference (e.g., unmodified) TACI peptide contains both CRD1 and CRD2. However, the variant TACI peptide need not contain both CRD1 and CRD2. In some embodiments, the variant TACI peptide contains CRD1 or a specific binding fragment thereof or is substantially composed of it. In some embodiments, the variant TACI peptide contains CRD2 or a specific binding fragment thereof or is substantially composed of it. In some embodiments, the variant TACI is a soluble peptide and lacks a transmembrane domain. In some embodiments, the variant TACI peptide also contains a transmembrane domain, and in some cases also contains a cytoplasmic domain.

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 TACI. In some embodiments, the reference (e.g., unmodified) TACI sequence is human. An exemplary human TACI sequence with extracellular domains is shown in SEQ ID NO:122.

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

TACI extracellular domain (ECD): SEQ ID NO:122

MSGLGRSRRGGRSRVDQEERFPQGLWTGVAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELRRQRSGEVENNSDNSGRYQGLEHRGSEASPALPGLKLSADQVALVYST

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

In some of the provided embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion lacking one or more residues of the stem portion containing the TACI extracellular domain. In some embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion lacking one or more consecutive C-terminal amino acid residues, said consecutive C-terminal amino acid residues starting at residue 105 and extending up to or including amino acid residue 166, said residues corresponding to residues of the ECD sequence shown in SEQ ID NO:122. In some implementations, the ECD sequence is missing numbers 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.

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

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

In some implementations, the reference TACI sequence is the TACI ECD sequence shown in international PCT publications WO 2000/067034, WO 2002/094852 or WO 2008/154814.

In some embodiments, the reference TACI sequence is a TACI ECD sequence that has or consists of the sequence shown in SEQ ID NO:131.

TACI ECD(CRD1/CRD2)：SEQ ID NO:131

SRVDQEER FPQGLWTGVA MRSCPEEQYW DPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYD HLLRDCISCA SICGQHPKQCAYFCENKLRSPVNLPPEL

In some implementations, the reference TACI sequence is a TACI ECD sequence that has or consists of the sequence shown in SEQ ID NO:130.

TACI ECD(CRD1/CRD2)：SEQ ID NO:130

AMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

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

TACI ECD(CRD1/CRD2)：SEQ ID NO:1

VAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

In some implementations, the reference TACI sequence is the extracellular domain region of TACI, which consists essentially of only the CRD2 sequence and lacks or omits the entire CRD1 sequence and essentially the entire stem region. Although previous studies have shown that residues in the stem region may contain proteolytic cleavage sites, it is believed that adequate expression of TACI and/or binding activity to its homologous ligands requires at least CRD1 and CRD2. For example, International PCT Publication WO 2002/094852 confirms that TACI molecules containing CRD1 and CRD2, but with the entire N-terminal region and a portion of the stem region sequence missing, exhibit reduced protein degradation upon expression. Other studies have shown that at least a portion of the N-terminal region preceding CRD1 is required for adequate binding activity of TACI to its homologous ligands, see, for example, International Publication WO 2008/154814, which identifies residues 13-118 or 13-108 in the extracellular region of TACI as being required for biological activity while minimizing TACI degradation during expression. Surprisingly, in this paper (e.g., Example 3), it was found that the extracellular region of TACI, which consists essentially of CRD2 with a small portion of the stem region, exhibits significantly improved homology-binding activity compared to a longer TACI molecule containing both CRD1 and CRD2.

This document provides an immunomodulatory protein (e.g., a TACI-Fc fusion protein) containing a TACI polypeptide, the TACI polypeptide being a CRD2-containing portion of the TACI extracellular domain (ECD) region, lacking the N-terminal region and CRD1, and containing one or more residues of the stem portion of the TACI extracellular domain, for example, corresponding to the amino acid sequence shown in SEQ ID NO: 122. In some embodiments, the CRD2-containing portion of the TACI extracellular domain includes amino acid residues 71-104, which correspond to the residues shown in SEQ ID NO: 122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein contains the deletion of N-terminal amino acid residues 1-66 corresponding to the residues shown in SEQ ID NO: 122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein contains the deletion of N-terminal amino acid residues 1-67 corresponding to the residues shown in SEQ ID NO: 122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein contains the deletion of N-terminal amino acid residues 1-68 corresponding to the residues shown in SEQ ID NO: 122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein contains the deletion of N-terminal amino acid residues 1-69 corresponding to the residues shown in SEQ ID NO:122. In the provided embodiments, the TACI polypeptide of the immunomodulatory protein contains the deletion of N-terminal amino acid residues 1-70 corresponding to the residues shown in SEQ ID NO:122. In some such embodiments, the TACI polypeptide of the immunomodulatory protein lacks one or more consecutive C-terminal amino acid residues, said consecutive C-terminal amino acid residues starting at residue 105 and continuing up to or including amino acid residue 166, said residues corresponding to residues of the ECD sequence shown in SEQ ID NO:122. In some implementations, the ECD sequence is missing numbers 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.

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

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

TACI ECD sequence (CRD2): SEQ ID NO:13

SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

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

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

In some embodiments, the equilibrium dissociation constant ( _Kd ) of any of the aforementioned embodiments with the BAFF can be less than 1 x ^10⁻⁵ M, 1 x ^10⁻⁶ M, 1 x ^10⁻⁷ M, 1 x ^10⁻⁸ M, 1 x ^10⁻⁹ M, 1 x ^10⁻¹⁰ M, 1 x ^10⁻¹¹ M, or 1 x ^10⁻¹² M. In some embodiments, the _Kd of any of the aforementioned embodiments with the BAFF is less than or less than about 1 x ^10⁻⁹ M, 1 x ^10⁻¹⁰ M, 1 x ^10⁻¹¹ M, or 1 x ^10⁻¹² M. In some embodiments, the _Kd of any of the aforementioned embodiments with the BAFF is between 1 x ^10⁻⁹ M and about 1 x ^10⁻¹² M. In some implementations, any of the foregoing implementations and BAFF's _Kd is or about ^1x10⁻⁹ M, or about ^2x10⁻⁹ M, or about ^4x10⁻⁹ M, or about ^6x10⁻⁹ M, or about ^8x10⁻⁹ M, or about ^1x10⁻¹⁰ M, or about ^2x10⁻¹⁰ M, or about ^4x10⁻¹⁰ M, or about ^6x10⁻¹⁰ M, or about ^8x10⁻¹⁰ M, or about ^1x10⁻¹¹ M, or about ^2x10⁻¹¹ M, or about ^{4x10⁻¹¹ M} , or about ^6x10⁻¹¹ M, or about ^8x10⁻¹¹ M, or about ^1x10⁻¹² M, or any value between ^the foregoing. In some embodiments, the provided embodiments include the variant TACI peptide as described above, and the K _d reduction (higher binding affinity) with BAFF is greater than or greater than about 1.5 times, such as greater than or about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

In some embodiments, the equilibrium dissociation constant (Kd) of any of the aforementioned embodiments with APRIL can be less than 1 x ^10⁻⁵ M, 1 x ^10⁻⁶ M, 1 x ^10⁻⁷ M, 1 x ^10⁻⁸ M, 1 x ^10⁻⁹ M, 1 x ^10⁻¹⁰ M, 1 x ^10⁻¹¹ M, or 1 x ^10⁻¹² M. In some embodiments, the _Kd of any of the aforementioned embodiments with APRIL is less than or less than about 1 x ^10⁻⁹ M, 1 x ^10⁻¹⁰ M, 1 x ^10⁻¹¹ M, or 1 x ^10⁻¹² M. In some embodiments, the _Kd of any of the aforementioned embodiments with APRIL is between 1 x ^10⁻⁹ M and about 1 x ^10⁻¹² M. In some implementations, any of the foregoing implementations and _Kd of APRIL are values between or about ^1x10⁻⁹ M, about ^2x10⁻⁹ M, about ^4x10⁻⁹ M, about ^6x10⁻⁹ M, about ^8x10⁻⁹ M, about ^1x10⁻¹⁰ M, about ^2x10⁻¹⁰ M, about ^4x10⁻¹⁰ M, about ^6x10⁻¹⁰ M, about 8x10⁻¹⁰ M, about ^1x10⁻¹¹ M, about ^2x10⁻¹¹ M, about ^4x10⁻¹¹ M, about ^6x10⁻¹¹ M, about ^8x10⁻¹¹ M, or about ^1x10⁻¹² M, or any of ^the foregoing. In some embodiments, the provided embodiments include the variant TACI peptide as described above, and the K _d reduction (higher binding affinity) with APRIL is greater than or greater than about 1.5 times, such as greater than or about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

A reference (e.g., unmodified or wild-type) TACI sequence does not necessarily have to be used as a starting composition to generate the variant TACI peptides described herein. Therefore, the use of the term “modification” (e.g., “substitution”) does not imply that embodiments of the invention are limited to a specific method for preparing variant TACI peptides or immunomodulatory proteins containing variant TACI peptides. Variant TACI peptides can be prepared, for example, by de novo peptide synthesis, and therefore modification (e.g., “substitution”) is not necessarily required in the sense of changing codons to encode a modification. This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific preparation method. Methods for designing or generating variant TACI peptides are not limited to any particular method. In some embodiments, however, the reference (e.g., unmodified or wild-type) TACI-encoding nucleic acid is mutagenized from the reference (e.g., unmodified or wild-type) TACI genetic material and screened for desired specific binding affinity or other functional activities. In some embodiments, variant TACI peptides are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases, followed by screening. This information is provided by the National Center for Biotechnology Information, and its website is publicly accessible via the Internet, as is the UniProtKB database mentioned earlier.

Unless otherwise stated, as indicated throughout this disclosure, one or more amino acid modifications in the variant TACI polypeptide are named according to the amino acid position number corresponding to the position number of the reference ECD sequence shown in SEQ ID NO:122. Those skilled in the art can skillfully identify the corresponding position of a modification (e.g., amino acid substitution) in the TACI polypeptide (including its portion containing TDs (e.g., CRD1 and/or CRD2)) by comparing a reference sequence (e.g., SEQ ID NO:1 or 13) with SEQ ID NO:122. An example of identifying the corresponding residue is shown in Figure 9. In the list of modifications throughout this disclosure, the amino acid position is indicated in the middle, the corresponding reference (e.g., unmodified wild-type) amino acid is listed before the number, and the identified variant amino acid substitution is listed after the number. If the modification is a positional deletion, then “del” is indicated, and if the modification is a positional insertion, then “ins” is indicated. In some cases, the insertion is listed together with the amino acid position indicated in the middle, and the corresponding reference amino acid is listed before and after the number, and the identified variant amino acid insertion is listed after the unmodified (e.g., wild-type) amino acid.

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

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

In some embodiments, the variant TACI polypeptide with one or more amino acid modifications (e.g., amino acid substitutions) as described above 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 a specific binding fragment thereof containing CRD1 and/or CRD2 domains. In some embodiments, the specific binding fragment contains a CRD1 domain, for example, the specific binding fragment contains the sequence of amino acids 34-66 shown in SEQ ID NO:122. In some cases, the CRD1 domain is the only intact CRD domain in the specific binding fragment. In some embodiments, the specific binding fragment is or contains a CRD2 domain, for example, the specific binding fragment contains the sequence of amino acids 71-104 shown in SEQ ID NO:122. In some embodiments, the CRD2 domain is the only intact CRD domain in the specifically binding fragment. In some embodiments, the specifically binding fragment is or contains both the CRD1 and CRD2 domains, for example, the specifically binding fragment contains amino acids 34-104 of SEQ ID NO:122. In some embodiments, the specifically binding fragment contains a continuous portion of a stem domain, for example, the specifically binding fragment contains a continuous portion of amino acids 105-165 of SEQ ID NO:122. In some embodiments, the specifically binding fragment of SEQ ID NO:122 is smaller than the full-length ECD shown in SEQ ID NO:122. In some embodiments, the specifically binding fragment is shown in SEQ ID NO:1. In some embodiments, the specifically binding fragment is shown in SEQ ID NO:13. In some embodiments, the specifically binding fragment is shown in SEQ ID NO:130. In some embodiments, the specifically binding fragment is shown in SEQ ID NO:131.

In some embodiments, variant TACI polypeptides containing one or more amino acid modifications (e.g., amino acid substitutions) as described have 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 (e.g., with the amino acid sequence of SEQ ID NO: 1, 13, or 122).

In some embodiments, the variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described above 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.

In some embodiments, the variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described above 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.

In some embodiments, variant TACI polypeptides containing one or more amino acid modifications (e.g., amino acid substitutions) as described have 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.

In some embodiments, variant TACI polypeptides containing one or more amino acid modifications (e.g., amino acid substitutions) as described have 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.

In some embodiments, the variant TACI polypeptide containing one or more amino acid modifications (e.g., amino acid substitutions) as described above 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.

In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) in the reference TACI polypeptide or its specific binding fragment, wherein the amino acid modifications are numbered with respect to SEQ ID NO:122 corresponding to one or more positions 40, 59, 60, 61, 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) selected from the following: 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 conserved amino acid substitutions thereof. In some embodiments, the reference TACI polypeptide includes a CRD1 domain or a CRD2 domain, for example, the reference TACI polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:122.

In some embodiments, the amino acid substitution is located only in the CRD2 domain. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) in the reference TACI polypeptide or its specific binding fragment, wherein the amino acid modifications are numbered with respect to SEQ ID NO:122 corresponding to one or more positions 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103. In some embodiments, the variant TACI polypeptide has one or more amino acid modifications (e.g., substitutions) selected from the following: 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 conserved amino acid substitutions thereof. In some embodiments, in the CRD domain, the reference TACI polypeptide includes only the CRD2 domain but lacks the CRD1 domain, for example, the reference TACI polypeptide is shown in SEQ ID NO:13. Therefore, in some embodiments, the variant TACI polypeptide includes a portion of the TACI polypeptide's ECD sequence that includes the CRD2 domain but lacks the CRD1 domain.

Conservative amino acid modifications (e.g., substitutions) are any amino acid that falls into the same amino acid class as the substituted amino acid, except for a reference (e.g., unmodified) or wild-type amino acid. Amino acid classes are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic/amide (aspartic acid, glutamic acid, asparagine, and glutamine).

In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 75 of the number associated with SEQ ID NO:122. In some embodiments, the amino acid substitution at position 75 confers increased binding with BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide without said amino acid substitution. In some embodiments, the substituted amino acid is an acidic amino acid or amide, such as 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).

In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 77 of the number associated with 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 without said amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 77 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 77 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 77 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 77 is glutamine (Gln, Q).

In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 78 of the number associated with 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 without said amino acid substitution. In some embodiments, the substituted amino acid at position 78 is an aromatic amino acid, such as 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).

In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 84 of the number associated with 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 without said amino acid substitution. In some embodiments, the substituted amino acid at position 84 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 84 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 84 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 84 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 84 is glutamine (Gln, Q).

In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 101 of the number associated with 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 without said amino acid substitution. In some embodiments, the substituted amino acid at position 101 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 101 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 101 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 101 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 101 is glutamine (Gln, Q).

In some embodiments, the variant TACI polypeptide includes at least one amino acid substitution at position 102 of the number associated with 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 without said amino acid substitution. In some embodiments, the substituted amino acid at position 102 is an acidic amino acid or an amide. In some embodiments, the substituted amino acid at position 102 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 102 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 102 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 102 is glutamine (Gln, Q).

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

In some embodiments, the variant TACI polypeptide includes amino acid substitution E74V. In some embodiments, the variant TACI polypeptide includes amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide includes amino acid substitution K77E. In some embodiments, the variant TACI polypeptide includes amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide includes amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide includes amino acid substitution L82H. In some embodiments, the variant TACI polypeptide includes amino acid substitution L82P. In some embodiments, the variant TACI polypeptide includes amino acid substitution R84G. In some embodiments, the variant TACI polypeptide includes amino acid substitution R84L. In some embodiments, the variant TACI polypeptide includes amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide includes amino acid substitution D85V. In some embodiments, the variant TACI polypeptide includes amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide includes amino acid substitution A102D. In some embodiments, the variant TACI polypeptide includes amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide contains two or more amino acid substitutions from any two or more of the foregoing. In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions, said amino acid substitutions being conserved amino acid substitutions of any of the foregoing. In the provided embodiments, the variant TACI polypeptide comprises an amino acid substitution in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid 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.

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

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

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

In some embodiments, the variant TACI polypeptide comprises one or more amino acid substitutions for R84G, A101D, K77E/R84Q, K77E/A101D, K77E/F78Y, K77E/F78Y/Y102D, Q75E/R84Q, K77E/A101D/Y102D, R84Q, K77E, A101D, Q75E, K77E/F78Y/R84Q, F78Y, F78Y/R84Q, F78Y/A101D, F78Y/Y102D, or K77E/Y102D. In the provided embodiments, the variant TACI polypeptide comprises amino acid substitutions in any of the reference TACI polypeptide sequences described herein (such as those shown in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130, or SEQ ID NO:131).

In some embodiments, the variant TACI polypeptide includes amino acid substitutions for K77E and F78Y (K77E/F78Y). In the provided embodiments, the variant TACI polypeptide includes amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

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

In some embodiments, the variant TACI polypeptide contains amino acid substitutions for F78Y and Y102D (F78Y/Y012D). In the provided embodiments, the variant TACI polypeptide includes amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

In some embodiments, the variant TACI polypeptide contains amino acid substitutions for K77E, F78Y, and Y102D (K77E/F78Y/Y102D). In the provided embodiments, the variant TACI polypeptide includes amino acid substitutions in any of the reference TACI polypeptide sequences as described. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:1. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:13. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:130. In some embodiments, the amino acid substitution is located in the reference TACI sequence shown in SEQ ID NO:131.

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

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

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

The use of the term "modification" (such as "substitution" or "mutation") does not imply that embodiments of the invention are limited to a specific method for preparing immunomodulatory proteins. Variant TACI peptides can be prepared, for example, by de novo peptide synthesis, and therefore, in the sense of changing codons to encode a modification (e.g., substitution), such modification (e.g., substitution) is not necessarily required. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which similarly do not imply a specific preparation method. The means used to design or generate vTDs are not limited to any particular method. However, in some embodiments, the nucleic acid encoding the wild-type or unmodified TD is mutated from the wild-type or unmodified TD genetic material and screened for alterations to desired specific binding activity (e.g., binding affinity) and/or NF-κB regulation or other functional activities. In some embodiments, vTDs are synthesized de novo 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, and its website is publicly accessible via the Internet, as is the UniProtKB database.

In some embodiments, the variant TACI polypeptide comprises extracellular domain (ECD) sequences containing CRD1 and CRD2, such as the variant TACI polypeptide shown in any one of SEQ ID NO:2-12, 21, 22, 101-120. In some embodiments, the variant TACI polypeptide comprises a polypeptide sequence exhibiting at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, such as at least about 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:2-12, 21, 22, 101-120, and retaining one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide comprises a specific binding fragment of any one of SEQ ID NO: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds to BAFF, APRIL, or BAFF/APRIL heterotrimer and contains a sequential sequence therein containing one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI.

In some embodiments, the variant TACI polypeptide consists of or is substantially composed of the 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 substantially composed of a polypeptide sequence exhibiting at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, such as at least about 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:2-12, 21, 22, 101-120, and retains one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or is substantially composed of a specific binding fragment of any one of SEQ ID NO: 2-12, 21, 22, 101-120, wherein the specific binding fragment binds to BAFF, APRIL, or APRIL/BAFF heterotrimer and contains a sequential sequence therein containing one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI.

In some embodiments, the variant TACI polypeptide comprises an extracellular domain (ECD) sequence containing CRD2 but lacking CRD1 of a reference TACI polypeptide, such as the variant TACI polypeptide shown in any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192. In some embodiments, the variant TACI polypeptide comprises a polypeptide sequence exhibiting at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, such as at least about 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192, and retaining one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide comprises a specific binding fragment of any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or BAFF/APRIL heterotrimer and contains a sequential sequence therein containing one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI.

In some embodiments, the variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192. In some embodiments, the variant TACI polypeptide consists of or is substantially composed of polypeptide sequences exhibiting at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, such as at least about 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192, and retaining one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide consists of or is substantially composed of a specific binding fragment of any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192, wherein the specific binding fragment binds to BAFF, APRIL, or BAFF/APRIL heterotrimer and contains a sequential sequence therein containing one or more amino acid modifications (e.g., one or more substitutions) not present in a reference (e.g., unmodified or wild-type) TACI.

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

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

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

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

In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence shown in any one of SEQ ID NO:37-47, 56, or 57. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence exhibiting at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, such as at least about 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:37-47, 56, or 57, and retaining one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI. This document also provides a nucleic acid containing the sequence shown in any one of SEQ ID NO:37-47, 56 or 57, or a sequence exhibiting at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identity (e.g., at least 96%, 97%, 98% or 99% identity) with any one of SEQ ID NO:37-47, 56 or 57.

In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence shown in any one of SEQ ID NO:49-55 or 58-70. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, such as at least about 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:49-55 or 58-70, and retains one or more amino acid modifications (e.g., one or more substitutions) not present in the reference (e.g., unmodified or wild-type) TACI. This article also provides a nucleic acid containing the sequence shown in any one of SEQ ID NO:49-55 or 58-70, or a sequence showing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identity (e.g., at least 96%, 97%, 98%, or 99%) with any one of SEQ ID NO:549-55 or 58-70.

In some implementations, this document also provides TACI ECD fusion sequences, wherein any of the above TACI ECD sequences is connected to or fused with a multi-merging domain (as described herein).

The interaction of two or more polypeptides of an immunomodulatory protein can be facilitated by their direct or indirect linkage with any moiety or other polypeptide that itself can interact to form a stable structure. For example, individually encoded polypeptide chains can be joined by polymerization, thereby mediating polypeptide polymerization through a polymerization domain. Typically, the polymerization domain provides for the formation of stable protein-protein interactions between the first and second polypeptides.

In some embodiments, two or more individual polypeptides of an immunomodulatory protein can be conjugated via polymerization, such as conjugating into dimers, trimers, tetramers, or pentamers. In some cases, the individual polypeptides are identical. For example, a trimer can be formed from three copies of the same individual polypeptide. In other examples, a tetramer is generated from four copies of the same individual polypeptide. In other examples, a pentamer is generated from five copies of the same individual polypeptide. The polymerizing domain can be a domain that promotes the dimerization, trimerization, tetramerization, or pentamerization of the polypeptide chain.

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

In some embodiments, the multimerizing domain includes any domain capable of forming stable protein-protein interactions. The multimerizing domain can interact via: immunoglobulin sequences (e.g., Fc domains; see, for example, International Patent Publications WO 93/10151 and WO 2005/063816 US; US Publication 2006/0024298; US Patent No. 5,457,035); leucine zippers (e.g., from nuclear converting proteins fos and jun, or proto-oncogene c-myc, or from 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 free thiols forming intermolecular disulfide bonds between homopolymers or heteropolymers. Additionally, the polymerizing domain may include an amino acid sequence containing a protrusion that is complementary to the amino acid sequence containing the pore, as described, for example, in the following documents: U.S. Patent No. 5,731,168; International Patent Publications Nos. WO 98/50431 and WO 2005/063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such polymerizing regions can be engineered such that spatial interactions not only promote stabilizing interactions but also promote the formation of heterodimers from mixtures of chimeric monomers beyond homodimers. Typically, the protrusion is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Optionally, a compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine). Exemplary polymerizing domains are described below.

A TACI polypeptide sequence (e.g., a variant TACI polypeptide sequence) can be conjugated at any position (but typically via its N-terminus or C-terminus) to the N-terminus or C-terminus of a polymerizing domain to form a chimeric polypeptide. The conjugation can be direct or indirect via a linker. Similarly, the chimeric polypeptide can be a fusion protein or can be formed by chemical conjugation, such as through covalent or non-covalent interactions. For example, in preparing a chimeric polypeptide containing a polymerizing domain, a nucleic acid encoding all or part of the TACI polypeptide sequence (such as any of the described TACI ECDs, including variant TACI polypeptide sequences) can be operatively conjugated directly or indirectly or optionally via a linker domain to a nucleic acid encoding the sequence of said polymerizing domain. In some cases, the construct encodes a chimeric protein in which the C-terminus of the TACI polypeptide sequence conjugates to the N-terminus of the polymerizing domain. In some cases, the construct can encode a chimeric protein in which the N-terminus of the TACI polypeptide sequence conjugates to either the N-terminus or C-terminus of the polymerizing domain.

The polypeptide multimer contains two chimeric proteins, which are generated by directly or indirectly linking two identical or different TACI polypeptide sequences (e.g., two identical or different variant TACI polypeptide sequences) to a multimerizing domain. In some examples, where the multimerizing domain is a polypeptide, a fusion of a gene encoding a 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 in host cells transformed with a recombinant expression vector and is allowed to assemble into a multimer, wherein the multimerizing domains interact to form a multivalent polypeptide. The chemical linking of the multimerizing domain to the TACI polypeptide (e.g., a variant TACI polypeptide) can be achieved using a heterobifunctional linker.

The resulting chimeric polypeptides (such as fusion proteins) and the multimers formed therefrom can be purified by any suitable method, such as affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, homodimers and heterodimers will form. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

In some implementations, the polymerized domain is the Fc region of an immunoglobulin.

In some embodiments, the polymerizing domain is an immunoglobulin (e.g., IgG1) Fc region, wherein the fusion protein is a TACI-Fc region containing: (1) any of the provided TACI ECD sequences or TACI sequences composed thereof; and (2) an immunoglobulin Fc region. Therefore, the provided embodiments include TACI-Fc fusion proteins containing: (1) any of the above-described TACI ECD polypeptide sequences (e.g., variant TACI polypeptides) or TACI sequences composed thereof; and (2) an immunoglobulin Fc region.

In some embodiments, this document provides a TACI-Fc fusion sequence comprising (1) a TACI ECD sequence comprising the sequence shown in SEQ ID NO:13, and (2) an immunoglobulin Fc region. In some embodiments, this document provides a TACI-Fc fusion sequence comprising (1) a TACI ECD sequence consisting of or substantially consisting of the sequence shown in SEQ ID NO:13, and (2) an immunoglobulin Fc region.

In some embodiments, the TACI-Fc fusion is a variant TACI-Fc fusion containing any one of the aforementioned variant TACI polypeptides and an immunoglobulin Fc region, or being composed of such a region.

In some embodiments, this document provides a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence containing CRD1 and CRD2, such as a TACI sequence containing the sequence shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120, and (2) an immunoglobulin Fc region.

In some embodiments, this document provides a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence containing a CRD2 domain but lacking a CRD1 domain, such as a TACI sequence comprising the sequence shown in any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192, and (2) an immunoglobulin Fc region. In some embodiments, this document provides a variant TACI-Fc fusion sequence comprising (1) a TACI ECD sequence containing a CRD2 domain but lacking a CRD1 domain, such as a TACI sequence comprising or substantially comprising the sequence shown in any one of SEQ ID NO: 14-20, 23-35, 92-100, 177-192, and (2) an immunoglobulin Fc region.

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

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

In some embodiments, the Fc region is or comprises a 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 is derived from IgG1, as 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 is derived from IgG2, as shown in any one of SEQ ID NO: 138 or 219. In some embodiments, the Fc region is or is derived from IgG4, such as any of those shown in SEQ ID NO: 139, 140, or 220. In some embodiments, the Fc region in the Fc fusion protein provided herein may further include an Fc region exhibiting at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of any of the Fc regions described above.

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

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

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:81)

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

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

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

In some embodiments, the Fc region is a variant Fc region, wherein the wild-type Fc is modified by substitution of one or more amino acids to reduce effector activity or to render the Fc inert to Fc effector function. Exemplary effector-free or inert mutations include those described herein.

In some embodiments, the Fc region contains one or more modifications that alter (e.g., reduce) one or more of its normal functions. Typically, in addition to its primary function as an immunoglobulin—antigen-binding capacity—the Fc region is also responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC). Furthermore, the FcRn sequence present in the Fc region can regulate serum IgG levels by increasing the in vivo half-life through conjugation to an in vivo FcRn receptor. In some embodiments, such functions can be reduced or altered in the Fc region for use with the provided Fc fusion protein.

In some embodiments, one or more amino acid modifications may be introduced into the Fc region to generate Fc region variants. In some embodiments, the Fc region variants have reduced effector function. There are many examples of Fc sequence variations or mutations that can alter effector function. For example, WO 00/42072, WO 2006019447, WO 2012125850, WO 2015/107026, US 2016/0017041, and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to the FcR. The contents of those publications are expressly incorporated herein by reference.

In some embodiments, the provided immunomodulatory protein includes an Fc region exhibiting reduced effector function, making it a desirable candidate for certain applications where the in vivo half-life of the immunomodulatory protein is important, but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the immunomodulatory protein lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells, which mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc 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 ADCC activity of target molecules are described in the following literature: U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)); and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA); and the CytoTox 96 ^™ non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the target molecule can be assessed in vivo (e.g., in animal models), as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the immunomodulatory protein n cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (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 assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Immunomodulatory proteins with reduced effector function include those with substitutions of one or more of the following Fc region residues according to EU designations: 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327 according to EU designations, including the so-called “DANA” Fc mutant with residues 265 and 297 substituted with alanine (US Patent No. 7,332,581).

In some embodiments, the Fc region of the immunomodulatory protein has an Fc region in which any one or more amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are replaced by different amino acids compared to the native Fc region. Such changes in the Fc region include, for example, deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A/L235A, IgG1-L234A/L235E/G237A, IgG1-A325A/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L2 as described in Current Opinion in Biotechnology (2009) 20(6), 685-691. Changes in IgG1-E233P/L234V/L235A/G236del/S267K, IgG1-L234F/L235E/P331S, IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4-L235A/G237A/E318A, and IgG4-L236E; such as WO The changes to G236R/L328R, L235G/G236R, N325A/L328R and N325LL328R as described in 2008/092117; the amino acid insertions at positions 233, 234, 235 and 237 (as indicated by EU numbers); and the changes at the sites described in WO 2000/042072.

Certain Fc variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004/056312, WO 2006019447 and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

In some embodiments, an immunomodulatory protein is provided comprising a variant Fc region containing one or more amino acid substitutions, said substitutions prolonging the half-life and/or improving binding to nascent Fc receptors (FcRn). Antibodies having prolonged half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et al.) or WO 2015107026. Those antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the following Fc region residues according to EU numbers: 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, the substitution of Fc region residue 434 (US Patent No. 7,371,826).

In some embodiments, the Fc region of the immunomodulatory protein contains one or more amino acid substitutions for C220S, C226S, and/or C229S according to EU numbers. In some embodiments, the Fc region of the immunomodulatory protein contains one or more amino acid substitutions for R292C and V302C. Other examples of Fc region variants can be found in: Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351.

In some embodiments, alterations are made in the Fc region that result in reduced C1q binding and/or complement-dependent cytotoxicity (CDC), for example, as described in the following literature: U.S. Patent No. 6,194,551, WO 99/51642; and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

In some embodiments, the variant Fc region containing one or more amino acid modifications (e.g., amino acid substitutions) is derived from wild-type IgG1, such as wild-type human IgG1. In some embodiments, the wild-type IgG1 Fc may be the 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 numbers. 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, such as residues containing Asp(D) and Leu(L) at positions 356 and 358, for example as shown in SEQ ID NO:81. Therefore, in some cases, the variant Fc is derived from the amino acid sequence shown in SEQ ID NO:81.

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

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

In some embodiments, the Fc region is a variant Fc containing at least one amino acid substitution, the amino acid substitution being designated N82G according to SEQ ID NO:71 (corresponding to N297G according to EU designation). In some embodiments, the Fc also contains at least one amino acid substitution according to SEQ ID NO:71, namely R77C or V87C (corresponding to R292C or V302C according to EU designation). In some embodiments, the variant Fc region further contains a C5S amino acid modification according to SEQ ID NO:71 (corresponding to C220S according to EU designation). For example, in some embodiments, the variant Fc region contains the following amino acid modification: N297G according to EU designation and one or more of the following amino acid modifications C220S, R292C, or V302C (regarding SEQ ID NO:71, corresponding to N82G and one or more of the following amino acid modifications C5S, R77C, or V87C), for example, the Fc region contains the sequence shown in SEQ ID NO:82.

In some embodiments, according to EU designations, variant Fc contains amino acid substitutions of L234A/L235E/G237A. In some embodiments, according to EU designations, variant Fc contains amino acid substitutions of A330S/P331S. In some embodiments, variant Fc contains amino acid substitutions of 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.

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

In some implementations, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:73.

EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:73)

In some embodiments, the Fc region is an IgG1 Fc region, 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).

In some implementations, the Fc region is a variant Fc having the sequence shown in SEQ ID NO:221.

DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:221)

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

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

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

Examples of such Fc regions included in immunomodulatory peptides are shown in Table 2.

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

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

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

In some embodiments, one or both of the Fc domains of the first and second immunomodulatory Fc fusion peptides contain modifications (e.g., substitutions) such that the interface of the Fc molecule is modified to favor and/or promote heterodimerization. Methods for promoting Fc chain heterodimerization include mutagenesis of the Fc region, such as by including a set of "mortar and pestle" mutations or by including mutations to achieve electrostatic manipulation of the Fc to favor attractive interactions between different polypeptide chains. In some embodiments, the Fc region of the heterodimeric molecule may additionally contain one or more other Fc mutations, as described above. In some embodiments, the heterodimeric molecule contains an Fc region with a mutation that reduces effector function. In some embodiments, such an Fc region contains mutations C220S, L234A, L235E, and/or G237A according to EU numbers. In some embodiments, any of the above mutations in the Fc backbone can be carried out in an allotype containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbers. In other embodiments, any of the above mutations in the Fc backbone can be performed in an allotype containing residues Asp(D) and Leu(L) at positions 356 and 358 according to EU numbering.

In some embodiments, the modification includes introducing a protrusion (pestle) into a first Fc polypeptide and a cavity (pothole) into a second Fc polypeptide, such that the protrusion can be located in the cavity to promote the complexation of the first and second Fc-containing polypeptides. The amino acid targeted for substitution and/or modification to create the protrusion or cavity in the polypeptide is typically an interfacial amino acid that interacts with or contacts one or more amino acids at the interface of the second polypeptide.

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

In some embodiments, the second polypeptide modified to contain a cavity (pothole) is a polypeptide comprising replacing a natural or original amino acid with an amino acid having at least one side chain recessed from the interface of the second polypeptide, thus enabling the reception of a corresponding protrusion from the interface of the first polypeptide. Most commonly, the substituted amino acid is an amino acid having a side chain volume smaller than that of the original amino acid residue. Those skilled in the art understand how to determine and/or evaluate the properties of amino acid residues to identify ideal substituted residues for forming a cavity. Typically, the substituted residues used to form a cavity are naturally occurring amino acids and 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.

For example, the CH3 interface of human IgG1 involves sixteen residues on each domain located on four antiparallel β chains, which bury 1090 2 from each surface (see, for example, Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al. (1990) J Mol. Biol., 216, 965-973; Ridgway et al. (1996) Prot. Engin., 9:617-621; U.S. Patent No. 5,731,168). Modification of the CH3 domain to create protrusions or cavities is described in, for example, the following literature: U.S. Patent No. 5,731,168; International Patent Applications WO 98/50431 and WO 2005/063816; and Ridgway et al. (1996) Prot. Engin., 9:617-621. In some cases, modifying the CH3 domain to create protrusions or cavities typically targets residues located on two centrally antiparallel β chains. The aim is to minimize the risk that the resulting protrusions could be contained by protrusions into the surrounding solvent rather than by compensating cavities within the CH3 domain of the partner body.

In some embodiments, the heterodimer molecule contains the T366W mutation in the CH3 domain of the "jaw mortar" chain and the T366S, L368A, Y407V mutations in the CH3 domain of the "mortar" chain. In some cases, additional interchain disulfide bridges between the CH3 domains can also be used, for example, by introducing the Y349C mutation into the CH3 domain of the "jaw mortar" or "mortar" chain, and by introducing the E356C or S354C mutation into the CH3 domain of the other chain (Merchant, A.M. 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 mortar Fc can contain the sequence shown in SEQ ID NO:89, which contains S354C and T366W, and the mortar Fc shown in SEQ ID NO:90, which contains the mutations Y349C, T366S, L368A, and Y407V. In some embodiments, the heterodimer contains the E356C, T366W mutation in one of the two CH3 domains, and the Y349C, T366S, L368A, and Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimer contains the Y349C, T366W mutation in one of the two CH3 domains, and the E356C, T366S, L368A, and Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimer molecule comprises 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 pestle-and-mortar structure techniques are known in the art, for example, as described in EP 1 870 459 A1.

In some embodiments, Fc variants containing CH3 protrusion (pallet) or cavity (pothole) modifications can conjugate to multi-domain immunoregulatory peptides at any location, but typically via their N-terminus or C-terminus, to the N-terminus or C-terminus of one or more TACI peptide sequences (e.g., variant TACI peptide sequences), to form fusion peptides. The conjugation can be direct or indirect via a linker. Typically, palmetto and pothole molecules are generated by co-expression of a first immunoregulatory peptide conjugated to an Fc variant containing one or more CH3 protrusion modifications and a second immunoregulatory peptide conjugated to an Fc variant containing one or more CH3 cavity modifications.

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

In some embodiments, the individual polypeptides of a multi-domain polypeptide or a single polypeptide of a single-domain polypeptide are linked to a multimerizing domain that forms an immunomodulatory protein, and are trimers, tetramers, or pentamers. In some embodiments, the individual polypeptides of such molecules are identical. In some embodiments, such multimerizing domains are chondrocyte oligomeric matrix protein (COMP) assembly domains, vasodilator-stimulated phosphoprotein (VASP) tetramerizing domains, or ZymoZipper (ZZ) 12.6 domains.

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

In some embodiments, the multimerizing domain is a vasodilator-stimulated phosphoprotein (VASP) tetramerizing domain (Bachmann et al., J Biol Chem (1999) 274(33):23549-23557). In some embodiments, VASP is or contains an amino acid sequence as shown in SEQ ID NO:147 (e.g., amino acids 343-375 of full-length VASP; Uniprot accession number P50552) or has about 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the sequence identity with SEQ ID NO:147.

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

In some embodiments, the linker is (by single-letter amino acid code): GGGGS (“4GS”; SEQ ID NO:77) or a polymer of the 4GS linker, such as repeating sequences of 2, 3, 4, or 5 4GS linkers. In some embodiments, the peptide linker is (GGGGS) ₂ (SEQ ID NO:78), (GGGGS) ₃ (SEQ ID NO:79), (GGGGS) ₄ (SEQ ID NO:84), or (GGGGS) ₅ (SEQ ID NO:91). In some embodiments, the linker may also comprise a series of alanine residues alone or include another peptide linker (such as the 4GS linker or a polymer thereof). In some embodiments, the linker is (by single-letter amino acid code) GSGGGGS (SEQ ID NO:74) or GGGGSSA (SEQ ID NO:80). In some examples, the linker is 2xGGGGS followed by three alanine residues (GGGGSGGGGSAAA; SEQ ID NO:133). In some examples, the connector is shown in SEQ ID NO:194 or 195.

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

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

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

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

SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:171)

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

SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:172)

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.

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

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

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

SLSCRKEQGEYYDHLLRDCISCASICGQHPKQCADFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:167)

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

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

SLSCRKEQGEYYDHLLRDCISCASICGQHPKQCADFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:168)

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

SLSCRKEEGKFYDHLLQDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:169)

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

SLSCRKEEGKFYDHLLQDCISCASICGQHPKQCAYFCENKLRSGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:170)

In some embodiments, the TACI-Fc fusion protein contains multiple copies of the TACI polypeptide sequence (e.g., variant TACI-polypeptide sequences), such as 2, 3, or 4 TACI polypeptide sequences. In some embodiments, the TACI-Fc fusion protein contains two TACI polypeptide sequences (e.g., two variant TACI polypeptide sequences). In some cases, the TACI polypeptide sequences can be directly linked or indirectly linked via a linker (e.g., a peptide linker, including any of those described above). In such examples, one of the TACI polypeptide sequences binds to or is linked to the Fc region, such as to the N-terminus or C-terminus of the Fc region. In other cases, the TACI polypeptide sequences can be separated from each other by the Fc region and each binds individually to the N-terminus or C-terminus of the Fc region. The linking to the Fc region can be direct or indirect via a linker (e.g., a peptide linker, including any of those described above).

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

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

In some embodiments, the TACI polypeptide sequences (e.g., variant TACI polypeptide sequences) may be separated by Fc regions within the fusion protein, wherein the Fc regions are located between 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). In some embodiments, the linker 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, the interaction between the Fc regions of two identical polypeptide fusions forms a covalent disulfide bond to obtain a dimer containing four TACI polypeptides (e.g., four variant TACI polypeptides).

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

In some embodiments, a TACI-Fc fusion protein is provided as a dimer formed from two identical TACI polypeptides (e.g., variant TACI polypeptides) linked to an Fc domain as described. In some embodiments, any provided TACI-Fc fusion polypeptide (e.g., variant TACI-Fc fusion) of the same kind (also referred to as a copy) will dimerize to produce a homodimer. In some embodiments, the dimer is a homodimer in which the two TACI-Fc polypeptides (e.g., variant TACI-Fc polypeptides) are identical. For the generation of a homodimeric Fc molecule, the Fc region is an Fc region capable of forming a homodimer with a matching Fc region through co-expression in the cell of a single Fc region. In some embodiments, dimerization is mediated by one or more covalent disulfide bonds formed between the Fc regions of the polypeptide fusion.

Nucleic acid molecules encoding immunomodulatory proteins are also provided. In some embodiments, for the generation of immunomodulatory proteins, the nucleic acid molecule encoding said immunomodulatory protein is inserted into a suitable expression vector. The resulting immunomodulatory protein can be expressed in host cells transformed with expression, wherein assembly between Fc domains is performed via interchain disulfide bonds formed between Fc moieties to produce a dimer (e.g., divalent) immunomodulatory protein.

Nucleic acid molecules encoding TACI-Fc fusion proteins (e.g., variant TACI-Fc fusion proteins) are also provided. In some embodiments, for the generation of Fc fusion proteins, nucleic acid molecules encoding TACI-Fc fusion proteins (e.g., variant TACI-Fc fusion proteins) are inserted into a suitable expression vector. The resulting TACI-Fc fusion protein (e.g., variant TACI-Fc fusion protein) can be expressed in host cells transformed with expression, wherein assembly between Fc domains is performed via interchain disulfide bonds formed between Fc moieties to produce a dimer (e.g., divalent) TACI-Fc fusion protein. The resulting Fc fusion protein can be readily purified by affinity chromatography on a protein A or protein G column. To generate heterodimers, additional purification steps may be required. For example, in the case of transforming two nucleic acids encoding different immunomodulatory proteins into cells, heterodimer formation must be achieved biochemically because the immunomodulatory protein carrying the Fc domain will also be expressed as a disulfide-linked homodimer. Therefore, the homodimer can be reduced under conditions that favor the disruption of interchain disulfide bonds without affecting intrachain disulfide bonds. In some cases, different immunomodulatory protein monomers are mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, this type of heterodimer formation can be biased towards the genetic engineering and expression of immunomodulatory proteins containing Fc fusion molecules with one or more TACI variants using the mortar and pestle structure method as described.

In the implementation, the immunomodulatory protein (such as TACI-Fc (e.g., variant TACI-Fc)) provided when generated and expressed from cells is a homodimer containing two identical polypeptide chains. Figures 8A and 8B depict the structure of the exemplary TACI-Fc fusion protein provided herein.

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

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

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

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

In some embodiments, the provided TACI-Fc (e.g., variant TACI-Fc) fusion protein (such as its homodimer) exhibits an _IC50 of less than 400 pM for neutralizing BAFF. In some embodiments, the IC50 for neutralizing BAFF is between 1 pM and 400 pM, such as between 10 pM and 300 pM, between 10 pM and 200 pM, between 10 pM and 100 pM, between 10 pM and 50 pM, between 10 pM and 20 pM, between 20 pM and 400 pM, between 20 pM and 300 pM, between 20 pM and 200 pM, between 20 pM and 100 pM, etc. Between 20pM and 50pM, between 50pM and 400pM, between 50pM and 300pM, between 50pM and 200pM, between 50pM and 100pM, between 100pM and 400pM, between 100pM and 300pM, between 100pM and 200pM, between 200pM and 400pM, between 200pM and 300pM, or between 300pM and 400pM. In some implementations, the IC ₅₀ for neutralizing BAFF is or about 10pM, 15pM, 20pM, 25pM, 30pM, 35pM, 40pM, 45pM, 50pM, 55pM, 60pM, 65pM, 70pM, 75pM, 80pM, 85pM, 90pM, 95pM, or 100pM, or any value between the foregoing.

In some embodiments, the provided TACI-Fc (e.g., variant TACI-Fc) fusion protein (such as its homodimer) exhibits an _IC50 of less than 400 pM for neutralizing APRIL. In some embodiments, the IC50 for neutralizing APRIL is between 0.5 pM and 100 pM, such as between 0.5 pM and 50 pM, between 0.5 pM and 25 pM, between 0.5 pM and 10 pM, between 0.5 pM and 5 pM, between 0.5 pM and 1 pM, between 1 pM and 100 pM, between 1 pM and 50 pM, between 1 pM and 25 pM, between 1 pM and 1... Between 0pM, between 1pM and 5pM, between 5pM and 100pM, between 5pM and 50pM, between 5pM and 25pM, between 5pM and 10pM, between 10pM and 100pM, between 10pM and 50pM, between 10pM and 25pM, or between 25pM and 100pM, between 25pM and 50pM, or between 50pM and 100pM. In some implementations, the IC ₅₀ for neutralizing APRIL is or about 0.5pM, 0.75pM, 1pM, 2pM, 3pM, 4pM, 5pM, 6pM, 7pM, 8pM, 9pM, 10pM, 11pM, 12pM, 13pM, 14pM, 15pM, 20pM or 25pM, or any value between the foregoing.

III. Nucleic acids, vectors, and methods for producing the said polypeptide or cells

This document provides isolated or recombinant nucleic acids (collectively, “nucleic acids”) that encode any of the immunomodulatory proteins provided herein. In some embodiments, the nucleic acids provided herein (including all nucleic acids described below) can be used for the recombinant production (e.g., expression) of the immunomodulatory proteins provided herein. In some embodiments, the nucleic acids provided herein (including all nucleic acids described below) can be used for the expression of the immunomodulatory proteins provided herein (such as the TACI fusion protein provided herein). The nucleic acids provided herein may be in RNA or DNA form and include mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleic acids provided herein are generally DNA molecules and are generally double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleic acids or combinations thereof comprising any nucleotide sequence of the present invention are also provided.

In some cases, a heterologous (non-natural) signal peptide may be added to a nucleic acid encoding an immunomodulatory protein. This may be desirable, for example, in the case of expressing a TACI fusion protein that does not contain an N-terminal signal sequence. In some embodiments, the signal peptide is derived from: immunoglobulins (such as IgG heavy chain or IgG-κ light chain), cytokines (such as interleukin-2 (IL-2) or CD33), serum albumin proteins (such as HSA or albumin), human astaxanthin proprotein signal sequence, luciferase, trypsinogen (such as chymotrypsinogen or trypsinogen), or other signal peptides that can be efficiently expressed and secreted from the cell in some respects. Exemplary signal peptides include any of those described in Table 3.

In some implementations, the immunomodulatory protein contains a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the immunomodulatory protein upon secretion.

This article also provides recombinant expression vectors and recombinant host cells that can be used to generate immunomodulatory proteins, such as the TACI fusion protein provided herein.

In any of the embodiments provided above, recombinant DNA and cloning techniques can be used to introduce nucleic acids encoding the immunomodulatory peptides provided herein into cells. For this purpose, recombinant DNA molecules encoding immunomodulatory peptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the coding sequence of the peptide can be excised from DNA using a suitable restriction enzyme. Alternatively, chemical synthesis techniques (such as the phosphoramide process) can be used to synthesize DNA molecules. Similarly, combinations of these techniques can be used. In some cases, recombinant or synthetic nucleic acids can be generated by polymerase chain reaction (PCR). As known to those skilled in the art, DNA inserts encoding immunomodulatory proteins can be cloned into suitable transduction/transfection vectors. Expression vectors containing said nucleic acid molecules are also provided.

In some embodiments, the expression vector can express an immunomodulatory protein in suitable cells under conditions appropriate for protein expression. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding an immunomodulatory protein, which is operatively linked to a suitable expression control sequence. Methods for achieving this operative linking before or after inserting the DNA molecule into the vector are well known. Expression control sequences include promoters, activators, enhancers, operons, ribosome binding sites, initiation signals, termination signals, capping signals, polyadenylation signals, and other signals involved in transcriptional or translational control.

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

The resulting recombinant expression vector, containing DNA molecules, is used to transform a suitable host. This transformation can be performed using methods well known in the art. In some embodiments, the nucleic acid provided herein further comprises a nucleotide sequence encoding a secretory or signal peptide, said nucleotide sequence being operatively linked to a nucleic acid encoding an immunomodulatory polypeptide, such that a soluble immunomodulatory polypeptide is recovered from a culture medium, host cell, or host cell periplasm. In other embodiments, an appropriate expression control signal is selected to allow membrane expression of the immunomodulatory polypeptide. Furthermore, commercially available kits and contract manufacturing companies can also be used to prepare the engineered cells or recombinant host cells provided herein.

In some embodiments, the resulting expression vector, having a DNA molecule thereon, is used to transform (e.g., transduce) suitable cells. Introduction can be performed using methods well known in the art. Exemplary methods include those for transferring nucleic acids encoding receptors, including transduction via viruses (e.g., retroviruses or lentiviruses), transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred into cells via lentiviral or retroviral transduction methods.

Any of a large number of publicly available and well-known mammalian host cells (including mammalian T cells or APCs) can be used to prepare peptides or engineered cells. Cell selection depends on a number of factors recognized in the art. These factors include, for example, compatibility with the chosen expression vector, the toxicity of the peptide encoded by the DNA molecule, conversion efficiency, ease of peptide recovery, expression characteristics, biosafety, and cost. To balance these factors, it is essential to understand that not all cells are equally effective for the expression of a particular DNA sequence.

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

In some embodiments, the host cell can be a variety of eukaryotic cells, such as yeast cells, or mammalian cells, such as Chinese hamster ovary (CHO) cells or HEK293 cells. In some embodiments, the host cell is a suspension cell, and the peptide is engineered or generated in a culture suspension, such as 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 deficient in glutamine synthase (GS), for example, CHO-S cells, CHOK1 SV cells, and CHOZN((R))GS-/- cells. In some embodiments, the CHO cells (e.g., suspension CHO cells) can be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

In some embodiments, the host cell can also be a prokaryotic cell, such as *Escherichia coli*. The transformed recombinant host cell is cultured under peptide expression conditions and then purified to obtain a soluble protein. The recombinant host cell can be cultured under conventional fermentation conditions to express the desired peptide. Such fermentation conditions are well known in the art. Finally, the peptide provided herein can be recovered and purified from the recombinant cell culture by any of a variety of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, and affinity chromatography. A protein refolding step can be used as needed to complete the conformation of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be used in the final purification step.

In some embodiments, the recombinant vector is a viral vector. Exemplary recombinant viral vectors include lentiviral vector genomes, poxvirus vector genomes, vaccinia virus vector genomes, adenovirus vector genomes, adenovirus-associated virus vector genomes, herpesvirus vector genomes, and alphavirus vector genomes. The viral vector can be a live, attenuated, replication-conditioning, or replication-defective, non-pathogenic (defective), reproducible viral vector, and/or modified to express a heterologous gene product, such as the variant immunomodulatory peptides provided herein. The vector used to generate the virus can also be modified to alter viral attenuation, including any method of increasing or decreasing transcriptional or translational load.

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

In some respects, the nucleic acid or expression vector contains a nucleic acid sequence encoding an immunomodulatory protein that is operatively linked to a suitable expression control sequence. Methods for achieving this operative linking before or after the insertion of the nucleic acid sequence encoding the immunomodulatory protein into the vector are well known. Expression control sequences include promoters, activators, enhancers, operons, ribosome binding sites, initiation signals, termination signals, capping signals, polyadenylation signals, and other signals involved in transcriptional or translational control. The promoter may be operatively linked to a portion of the nucleic acid sequence encoding the immunomodulatory protein.

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

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

Expression vectors can be introduced into host cells using a variety of standard techniques, including calcium phosphate transfection, liposome-mediated transfection, micro-projectile-mediated delivery, electroporation, etc. Transfected cells can be selected and propagated 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 by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).

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

In some embodiments, the peptides provided herein can 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 preparing small peptides. Well-known solid-phase synthesis techniques include, for example, 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 peptides as provided herein.

IV. Pharmaceutical Compositions

This document provides compositions containing any of the immunomodulatory proteins described herein. The pharmaceutical compositions may also contain pharmaceutically acceptable excipients. For example, a pharmaceutical composition may contain one or more excipients for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. Such compositions may contain buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

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

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

In some embodiments, the pharmaceutical composition comprises 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 gluten, zein, or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethyl cellulose, or sodium glycolate starch. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, or sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

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

In some embodiments, the pharmaceutical composition is sterile. Sterilization can be accomplished by filtration through a sterile filter membrane or by radiation. In the case of lyophilized compositions, sterilization can be performed using this method before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized or solution form. Additionally, parenteral compositions are typically placed in containers with sterile access ports, such as intravenous infusion bags or vials with stoppers that can be punctured by hypodermic needles.

A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or medium. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of a carrier must be "pharmaceutically acceptable" because it must be compatible with the other components in the formulation. It must also be suitable for contact with any tissue, organ, or part of the body it may encounter, meaning it must not carry the risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that outweigh its therapeutic benefit.

In some implementations, the pharmaceutical composition is administered to a subject. Typically, the dosage and route of administration of the pharmaceutical composition are determined according to standard pharmaceutical practice, based on the subject's body size and condition. For example, the therapeutically effective dose may initially be estimated in cell culture assays or in animal models (such as mice, rats, rabbits, dogs, pigs, or monkeys). Animal models may also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine the available dosage and route of administration in humans. The exact dosage will be determined based on factors relevant to the subject requiring treatment. The dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's overall health condition, the subject's age, weight, and sex, the time and frequency of administration, one or more drug combinations, sensitivity to response, and response to therapy.

Long-acting pharmaceutical compositions can be administered every 3 to 4 days, weekly, or bi-weekly, depending on the half-life and clearance rate of the specific formulation. The dosing frequency will depend on the pharmacokinetic parameters of the molecules in the formulation used. Typically, the composition is administered until the desired effect is achieved. Therefore, the composition can be administered as a single dose, or over time in multiple doses (at the same or different concentrations/dosages), or via continuous infusion. Further refinement of the appropriate dosage is routinely made. The appropriate dosage can be determined by using suitable dose-response data.

In some implementations, the pharmaceutical composition is administered to the subject via any route, including oral, transdermal, inhalation, intravenous, intra-arterial, intramuscular, direct application to a wound site, application to a surgical site, intraperitoneal, via suppository, subcutaneous, intradermal, percutaneous, via nebulization, intrapleural, intracardiac, intra-articular, intraocular, or intraspinal.

The provided drug formulation may be, for example, in a form suitable for intravenous infusion.

In some embodiments, the dosage of the pharmaceutical composition is a single dose or repeated doses. In some embodiments, the dose is given to the subject once daily, twice daily, three times daily, or four or more times daily. In some embodiments, about one or more doses (e.g., about two or more, about three or more, about four or more, about five or more, about six or more, or about seven or more) are given over a week. In some embodiments, multiple doses are given over a period of days, weeks, months, or years. In some embodiments, the treatment course is about one or more doses (e.g., about two or more doses, about three or more doses, about four or more doses, about five or more doses, about seven or more doses, about ten or more doses, about fifteen or more doses, about twenty-five or more doses, about forty or forty, about fifty or forty, or about one hundred or more doses).

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

V. Methods for assessing the activity and immunomodulation of immunomodulatory proteins

In some implementations, the provided immunomodulatory proteins (such as the TACI fusion protein provided herein) exhibit immunomodulatory activity. The provided immunomodulatory proteins (such as the TACI fusion protein) can regulate one or more of B cell activities, such as B cell proliferation, differentiation, or survival.

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

The provided immunomodulatory proteins can also be evaluated in any of a variety of assessments to assess their regulation of B cell activity. One such assay is a cell proliferation assay. Cells are cultured with or without the test compound (e.g., an immunomodulatory protein), and cell proliferation is detected by colorimetric assay, for example, by measuring the incorporation of tritylthymidine or by metabolic degradation based on 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazole bromide (MTT) (Mosman, J. Immunol. Meth. 65:55-63, 1983). Alternative assay formats use cells further engineered to express a reporter gene. The reporter gene is linked to a promoter element that responds to a receptor-linked pathway, and the assay detects activation of transcription of the reporter gene. A variety of reporter genes that are readily measurable in cell extracts are known in the art, for example, E. coli lacZ, chloramphenicol acetyltransferase (CAT), and serum response elements (SRE) (see, for example, Shaw et al., Cell 56:563-72, 1989). An exemplary reporter gene is the luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:29094-101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase activity assay kits are commercially available from, for example, Promega Corp. (Madison, Wisconsin).

The provided immunomodulatory protein may be characterized by its ability to inhibit the stimulation of human B cells by soluble APRIL or BAFF, as described by Gross et al. in International Publication No. WO00/40716. In short, human B cells are isolated from peripheral blood mononuclear cells, such as using CD19 magnetic beads (e.g., Miltenyi Biotec Auburn, CA). The purified B cells can be incubated under stimulating conditions (e.g., in the presence of soluble APRIL) and further incubated in the presence of increasing concentrations of the immunomodulatory protein. The B cells can be labeled with a proliferation dye, or with 1 μCi ³ H-thymidine, to measure proliferation. The number of B cells can be determined over time.

Reporter cell lines expressing reporter genes under the operative control of transcription factors such as NF-κB, NFAT-1, and AP-1, expressing TACI or BCMA, can be prepared. For example, reporter cells can include Jurkat and other B-cell lymphoma cell lines. These cells, incubated with soluble BAFF or APRIL ligands, signal via the reporter gene in these constructs. The role of the provided immunomodulatory proteins in regulating this signal transduction can be evaluated.

Well-established animal models are available to test the in vivo efficacy of the provided immunomodulatory proteins in certain disease states, including those involving autoimmune or inflammatory conditions. For example, animal models of autoimmune diseases include, for instance, the MRL-lpr/lpr or NZB×NZW F1 homologous mouse strains, which are used as models of SLE (systemic lupus erythematosus). Such animal models are known in the art; see, for example, *Autoimmune Disease Models A Guidebook*, edited by Cohen and Miller, Academic Press. The offspring of a cross between New Zealand Black (NZB) and New Zealand White (NZW) mice develop a spontaneous form of SLE that is very similar to human SLE. Progeny mice, designated NZBW, begin producing IgM autoantibodies against T cells at 1 month of age, and by 5–7 months of age, Ig anti-DNA autoantibodies are dominant immunoglobulins. Overactivity of polyclonal B cells leads to excessive production of autoantibodies. The deposition of these autoantibodies, particularly those against single-stranded DNA, is associated with the development of glomerulonephritis, clinically manifested as proteinuria, azotemia, and death from renal failure. Renal failure is the leading cause of death in mice affected by spontaneous SLE, and in the NZBW strain, the process is chronic and closed. The disease progresses more rapidly and severely in females than in males, with a mean survival of only 245 days, compared to 406 days in males. Although many female mice will develop symptoms (proteinuria) by 7–9 months of age, some female mice may appear significantly younger or older than normal. The fatal immune nephritis observed in NZBW mice is very similar to glomerulonephritis seen in human SLE, making this spontaneous 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 the provided immunomodulatory proteins to these mice could be evaluated to assess their efficacy in improving symptoms and altering disease course.

Another mouse model of inflammation and lupus-like disease is the bm12-induced mouse SLE model (Klarquist and Janssen, 2015. J. Vis. Exp. (105), e53319). A suspension of spleen cells from female IA ^bm12 B6(C)-H2-Ab1 ^bm12 /KhEgJ (“bm12”) mice was adoptively transferred to female C57BL/6NJ recipient mice. H2-Ab1 ^bm12 differs from H2-Ab1 ^b by 3 nucleotides, resulting in a 3-amino acid alteration in the β-chain of the MHC class II IA molecule. Homologous activation of donor bm12 CD4+ T cells by receptor antigen-presenting cells leads to chronic GVHD, with symptoms very similar to SLE, including autoantibody production, changes in immune cell subsets, and mild nephropathy. Glomerulonephritis with immune complex deposition appears later in this model and is primarily composed of autoantigens that bind to antibodies against IgG1, IgG2b, IgG2c, and IgG3. The model endpoints may include the concentration of anti-dsDNA antibodies, selection of IgG isotypes, blood urea nitrogen (BUN), serum creatinine, subset composition of immune cells in the spleen and cervical lymph nodes, and renal histology.

In some implementations, a mouse model of Sjögren's syndrome (SjS) can be used. SjS disease and accelerated onset of diabetes can be induced in female, diabetic, non-obese diabetic (NOD) mice using repeated administration of an anti-mice (m)PD-L1 antibody, based on a modified version of the protocol disclosed by Zhou et al., 2016 Sci. Rep. 6, 39105. Starting at 6 weeks of age, mice were injected intraperitoneally (IP) with 100 μg of the anti-PD-L1 antibody on days 0, 2, 4, and 6 of the study, and the mice were treated with the provided immunomodulatory protein at different days. Initially tested mice were included as controls for the endpoint analysis. All mice were typically sacrificed on day 10 of the study, and the submandibular gland (SMG) and pancreas were collected from each mouse for histopathological evaluation to assess the signs and severity of sialodenitis and insulitis. Blood glucose levels can be measured at different days.

In some implementations, a mouse model of experimental allergic encephalomyelitis (EAE) can be used. This model is similar to human multiple sclerosis and involves demyelination due to T-cell activation of neuronal proteins such as myelin basic protein (MBP) or protein lipoprotein protein (PLP). Inoculation with the antigen induces CD4+ class II MHC-restricted T cells (Th1). Variations in the protocols used for EAE can produce acute, chronic-relapsing, or passively metastatic variants of the model (Weinberg et al., J. Immunol. 162:1818-26, 1999; Mijaba et al., Cell. Immunol. 186:94-102, 1999; and Glabinski, Meth. Enzym. 288:182-90, 1997). Administration of the provided immunomodulatory proteins can be evaluated for improvement in symptoms and alterations in disease course.

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

In some implementations, a model of bronchial infection (such as asthma) can be created in mice by injecting ovalbumin and then restimulating them nasally with an antigen (which produces an asthmatic response in the bronchi similar to asthma). The administration of the provided immunomodulatory protein can be evaluated to improve symptoms and alter the course of the disease.

In some implementations, myasthenia gravis (MG) is another autoimmune disease for which a mouse model can be obtained. MG is a neuromuscular transmission disorder involving the production of autoantibodies against the nicotinic acetylcholine receptor (AChR). MG is acquired or hereditary, and its clinical features include abnormal weakness and fatigue during physical activity. Mouse models of MG have been established. (Christadoss et al., Establishment of a Mouse Model of Myasthenia Gravis Which Mimics Human Myasthenia Gravis Pathogenesis for Immune Intervention, Immunobiology of Proteins and Peptides VIII, Atassi and Bixler eds., 1995, pp. 195-99.) Experimental autoimmune myasthenia gravis (EAMG) is an antibody-mediated disease characterized by the presence of antibodies against the AChR. These antibodies disrupt the receptor, leading to a deficiency in neuromuscular electrical impulses, resulting in muscle weakness. In the EAMG model, mice are immunized with nicotinic acetylcholine receptors. Clinical signs of MG become apparent several weeks after the second immunization. EAMG was evaluated using several methods, including measuring 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), measuring muscle AChR, or electromyography (Wu et al. Protocols in Immunology. Vol. 3, Coligen, Kruisbeak, Margulies, Shevach and Strober eds. John Wiley and Sons, New York, 15.8.1, 1997).

Another use of in vivo models involves delivering antigen challenge to animals, followed by administration of immunomodulatory proteins and measurement of T-cell and B-cell responses. T-cell-dependent and non-T-cell-dependent immune responses can be measured, as described in Perez-Melgosa et al., J. Immunol. 163:1123-7, 1999. The effect on B-cell responses can be measured by administering the provided immunomodulatory protein to animals that have undergone routine antigen challenge (e.g., keyhole cyanin (KLH), sheep erythrocytes (SRBC), ovalbumin, or collagen).

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

VI. Therapeutic applications

The pharmaceutical compositions described herein (including those comprising the immunomodulatory proteins described herein, such as TACI-Fc) can be used in a variety of therapeutic applications, such as the treatment of diseases. For example, in some embodiments, the pharmaceutical compositions are used to treat inflammatory or autoimmune disorders in mammals, cancer, organ transplantation, viral infections, and/or bacterial infections. The pharmaceutical compositions can modulate (e.g., reduce) the immune response to treat the disease.

Such methods and uses include therapeutic methods and uses, for example, those designed to administer a molecule or a composition containing said molecule to a subject suffering from a disease, condition, or disorder. In some cases, as described, said disease, condition, or disorder is an autoimmune or inflammatory disease or disorder. In some embodiments, said molecule or engineered cells are administered in an effective amount to achieve treatment of said disease or disorder. Uses include the use of molecules containing immunomodulatory proteins, and their use in the preparation of pharmaceuticals for carrying out such therapeutic methods. In some embodiments, said methods are carried out by administering the provided immunomodulatory protein or a composition containing it to a subject suffering from or suspected of suffering from a disease or condition. In some embodiments, said methods thereby treat the subject's disease, disorder, or condition or disorder.

Illustrative subjects include mammalian subjects, such as draft animals, livestock, and human patients. In a particular embodiment, the subjects are human subjects.

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

The provided methods are believed to be applicable in a variety of applications, including, but not limited to, preventative or therapeutic methods for treating various immune system diseases or conditions in mammals, in which the regulation or control of the immune system and immune system responses is beneficial. For example, suppressing an immune response can be beneficial in preventative and/or therapeutic methods to inhibit the recipient's rejection of tissue, cell, or organ transplants from a donor. In therapeutic cases, the mammalian subject is typically a subject suffering from an immune system disease or condition, and administration is performed to prevent further progression of the disease or condition.

The provided immunomodulatory proteins (including TACI fusion proteins) can be used to treat the following diseases: autoimmune diseases, B-cell carcinoma, immunomodulation, EBD and any antibody-mediated conditions (e.g., ITCP, myasthenia gravis, etc.), nephropathy, indirect T-cell immune responses, graft rejection, and graft-versus-host disease. Administration of immunomodulatory proteins (e.g., TACI-Fc) can specifically regulate B-cell responses during immune responses. Additionally, administration of the provided immunomodulatory proteins can regulate B-cell development, the development of other cells, antibody production, and cytokine production. Administration or use of the provided immunomodulatory proteins can also modulate B-cell communication, such as by neutralizing the proliferative effects of BAFF or APRIL.

In some embodiments, the pharmaceutical composition suppresses the immune response and can be used in the treatment of inflammatory or autoimmune disorders or organ transplantation. In some embodiments, the pharmaceutical composition contains an immunomodulatory protein exhibiting antagonistic activity against B-cell stimulating receptors, thereby reducing or decreasing the immune response.

In some embodiments, the composition can be used to treat autoimmune diseases. In some embodiments, administration of a therapeutic composition containing the immunomodulatory protein provided herein to a subject suffering from an immune system disease (e.g., an autoimmune disease) can result in the suppression or inhibition of such immune system attack or associated biological responses. By suppressing such immune system attack on healthy body tissues, resulting physical symptoms (e.g., pain, joint inflammation, joint swelling, or tenderness) caused by or associated with said attack on healthy tissues can be reduced or alleviated, and biological and physical damage caused by or associated with said immune system attack can be reduced, slowed, or stopped. In prophylactic cases, the subject may be a subject suffering from, susceptible to, or believed to present with an immune system disease, disorder, or condition, and administration is typically performed to prevent the progression of the disease, disorder, or condition, to suppress or alleviate associated symptoms, signs, or biological responses, to prevent potential bodily damage, and/or to maintain or improve the subject's physical function.

In some embodiments, the diseases or conditions that can be treated by the pharmaceutical compositions described herein are any diseases mediated by: immune complex deposition (e.g., lupus nephritis, vasculitis); direct interference with pathways (e.g., catastrophic antiphospholipid syndrome, myasthenic crisis; anti-Jo-1 disease); opsonization or direct damage to cells (e.g., idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia); antibody-mediated rejection of allogeneic grafts (e.g., highly sensitized kidney transplant patients); or anti-drug antibodies against biological substitute factors or carriers (e.g., anti-factor 8).

In some implementations, the inflammatory and autoimmune disorders, conditions, or diseases that can be treated with the pharmaceutical compositions described herein are systemic lupus erythematosus (SLE), including blast prophylaxis without the use of glucocorticoids; Sjögren's syndrome; primary biliary cirrhosis (PBC); systemic scleroderma; polymyositis; diabetes prophylaxis; IgA nephropathy; IgA vasculitis; B-cell carcinomas, such as myeloma; multiple sclerosis; or optic neuritis.

In some embodiments, the provided immunomodulatory protein can be used to treat pre-B-cell leukemia or B-cell leukemia (such as plasma cell leukemia, chronic or acute lymphoblastic leukemia), myeloma (such as multiple myeloma, plasma cell myeloma, endothelial myeloma, and giant cell myeloma), and lymphoma (such as non-Hodgkin lymphoma). In any embodiment, the type of myeloma includes multiple myeloma, plasmacytoma, multiple solitary plasmacytoma, and/or extramedullary myeloma. In any embodiment, the type of myeloma includes light chain myeloma, non-secreting myeloma, and/or IgD or IgE myeloma.

In some implementations, the provided immunomodulatory proteins can be used as immunosuppressants to selectively block the action of B lymphocytes for the treatment of diseases. For example, certain autoimmune diseases are characterized by the production of autoantibodies that promote tissue destruction and disease exacerbation. Autoantibodies can also lead to complications of immune complex deposition and contribute to many symptoms of systemic lupus erythematosus (including kidney failure, neuropathic symptoms, and death). Regulating antibody production independent of cellular responses can also be beneficial in many disease states. B cells have also been shown to play a role in the secretion of arthrogenic immunoglobulins in rheumatoid arthritis. The methods and uses of the provided immunomodulatory proteins to inhibit, block, or neutralize the action of B cells, thereby suppressing antibody production, would be beneficial in the treatment of autoimmune diseases such as myasthenia gravis, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis.

In some implementations, the provided immunomodulatory proteins can be used to block or neutralize B-cell activity associated with end-stage renal disease, which may or may not be related to autoimmune diseases. Such methods can also be used to treat immune-mediated nephropathy. These methods can also be used to treat glomerulonephritis associated with diseases such as: membranous nephropathy, IgA nephropathy or Berger's disease, IgM nephropathy, IgA vasculitis, Goodpasseur's disease, post-infectious glomerulonephritis, mesangial proliferative disorders, chronic lymphocytic leukemia, and minimal change disease syndrome. Such methods will also be used as a therapeutic application for treating secondary glomerulonephritis or vasculitis associated with diseases such as: lupus, Takayasu arteritis, allergic purpura, scleroderma, HTV-related diseases, amyloidosis, or hemolytic uremic syndrome. The provided methods can also be used as part of a therapeutic application for treating interstitial nephritis or pyelonephritis associated with: chronic pyelonephritis, analgesic abuse, nephrocalcification, nephropathy caused by other factors, kidney stones, or chronic or acute interstitial nephritis. The methods provided also include the use of the provided immunomodulatory protein in the treatment of hypertension or macrovascular diseases, including renal artery stenosis or occlusion, cholesterol embolism, or renal embolism. The provided methods and uses can also be used to treat renal or urinary tract tumors, multiple myeloma, lymphoma, light chain neuropathy, or amyloidosis.

In some implementations, the provided immunomodulatory protein can also be used to treat asthma and other chronic airway diseases, such as bronchitis and emphysema. The provided immunomodulatory protein can also be used to treat Sjögren's syndrome.

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

In some embodiments, inflammatory and autoimmune disorders that can be treated with pharmaceutical compositions containing the immunomodulatory proteins described herein include, but are not limited to, achalasia; Addison's disease; adult-onset Still's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM/anti-TBM nephritis; antiphospholipid syndrome; autoimmune adrenalitis (Addison's disease); autoimmune angioedema; autoimmune autonomic dysfunction; autoimmune encephalomyelitis; autoimmune hepatitis; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune orchitis; autoimmune pancreatitis; and autoimmune polygonatum syndrome type II (APS). II); Autoimmune retinopathy; Autoimmune thyroid disease (AITD), i.e., Hashimoto's thyroiditis; Autoimmune urticaria; Axonal and neuronal neuropathy (AMAN); Barlow's disease; Ocular-oral-genital syndrome; Benign mucosal pemphigoid; Bullous pemphigoid; Kaselman's disease (CD); Celiac disease; Chagas disease; Chronic inflammatory demyelinating polyneuropathy (CIDP); Chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA); Cicatricial pemphigoid; Kogan syndrome Combination syndromes; cold agglutinin disease; congenital heart block; Coxsackievirus myocarditis; CREST syndrome; Crohn's disease; herpetic dermatitis; dermatomyositis; Dermatomyositis; Dervec disease (neuromyelitis optica); discoid lupus; post-myocardial infarction syndrome; endometriosis; eosinophilic esophagitis (EoE); eosinophilic fasciitis; erythema nodosum; severe mixed cryoglobulinemia; Evans syndrome; fibromyalgia; fibrotic alveolitis; giant cell arteritis (temporal arteritis); giant cell myocarditis; glomerulonephritis; Goodpassuu syndrome; granulomatous polyangiitis; Graves' disease; Guillain-Barré syndrome Syndrome; Hashimoto's thyroiditis; Hemolytic anemia; Allergic purpura (HSP); Herpes gestationis or pemphigoid of pregnancy (PG); Hidradenitis suppurativa (HS) (acne paradox); Hypogammaglobulinemia; IgA nephropathy; IgA vasculitis; IgG4-related sclerotic disease; Immune thrombocytopenic purpura (ITP); Inclusion body myositis (IBM); Interstitial cystitis (IC); Juvenile arthritis; Juvenile diabetes mellitus (type 1 diabetes); Juvenile myositis (JM); Kawasaki disease; Lambert-Eton syndrome; Leukocytic clotting vasculitis; Purpura; Lichen sclerosus; Phoebe conjunctivitis Inflammation; Linear IgA disease (LAD); Lupus; Chronic Lyme disease; Meniere's disease; Microscopic polyangiitis (MPA); Mixed connective tissue disease (MCTD); Erosive corneal ulcer; Muhammad-Haas disease; Multifocal motor neuropathy (MMN) or MMNCB; Multiple sclerosis; Myasthenia gravis; Myositis; Narcolepsy; Neonatal lupus; Neuromyelitis optica; Neutropenia; Ocular cicatricial pemphigoid; Optic neuritis; Relapsing rheumatism (PR); Pandas; Paraneoplastic cerebellar degeneration (PCD); Paroxysmal nocturnal hemoglobinuria (PNH); Parry Romberg syndrome; ciliary body plaque inflammation (intermediate uveitis); Parsonage-Turner syndrome; pemphigus, pemphigus vulgaris; peripheral neuropathy; perivenous encephalomyelitis; pernicious anemia (PA); POEMS syndrome; polyarteritis nodosa; polyglandular syndrome type I, II, III; polymyalgia rheumatica; polymyositis; post-myocardial infarction syndrome; post-pericardiotomy syndrome; primary biliary cirrhosis; primary sclerosing cholangitis; progesterone dermatitis; psoriasis; psoriatic arthritis; pure red cell aplasia (PRCA); pyoderma gangrenosum; Raynaud's phenomenon; reactive arthritis; recurrent polydipsia; Chondritis; Restless Legs Syndrome (RLS); Retroperitoneal fibrosis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidt syndrome; Scleritis; Scleroderma; Sjögren's syndrome; Sperm and testis autoimmunity; Stiff-person syndrome (SPS); Subacute bacterial endocarditis (SBE); Susac syndrome; Sympathetic ophthalmia (SO); Takayasu arteritis; Temporal arteritis/giant cell arteritis; Thrombocytopenic purpura (TTP); Tolosa-Hunter syndrome (THS); Transverse myelitis; Type 1 diabetes; Ulcerative colitis (UC); Undifferentiated connective tissue disease (UCTD); Uveitis; Vasculitis; Vitiligo or Vogt-Koyanagi-Harada disease.

In some implementations, the provided immunomodulatory proteins (e.g., TACI-Fc) can be used to treat scleroderma, myasthenia gravis, GVHD (including acute or chronic GVHD), transplant-related immune responses; antiphospholipid antibody syndrome; multiple sclerosis; Sjögren's syndrome; IgG4-related diseases; type 1 diabetes; rheumatoid arthritis (including glucocorticoid therapy (GC)RA) or acute lupus nephritis.

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

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

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

In some embodiments, a therapeutic amount of the pharmaceutical composition is administered. Typically, the precise amount of the composition of the invention to be administered can be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, degree of infection, and physical condition. The optimal dosage and treatment regimen for a particular patient can be readily determined by a person skilled in the medical field by monitoring the patient's symptoms and adjusting the treatment accordingly.

The subject composition can be administered by any convenient method, including via aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The composition described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (i.v.), or intraperitoneally. In one embodiment, the therapeutic composition is administered to the patient via intradermal or subcutaneous injection. In another embodiment, the therapeutic composition is administered via intravenous injection.

In some embodiments, the pharmaceutical composition is administered as a monotherapy (i.e., as a single agent) or as a combination therapy (i.e., in combination with one or more other immunosuppressants). In some embodiments, the other agents are glucocorticoids (e.g., prednisone, dexamethasone, and hydrocortisone), cell inhibitors (such as cell inhibitors affecting the proliferation of T cells and/or B cells (e.g., purine analogs, alkylating agents, or antimetabolites)), antibodies (e.g., anti-CD20, anti-CD25, or anti-CD3 monoclonal antibodies), cyclosporine, tacrolimus, sirolimus, everolimus, interferon, opioids, TNF-binding proteins, mycophenolate mofetil, small biologics (such as fingolimod or polysaccharin), cytokines (such as interferon β-1a), integrin agonists, or integrin antagonists.

VII. Products and Reagent Kits

This document also provides articles comprising the pharmaceutical compositions described herein in suitable packaging. Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed polyester film (Mylar) or plastic bags), etc. These articles may be further sterilized and/or sealed.

Kits are also provided that contain the pharmaceutical compositions (or articles) described herein, and the kits may also contain one or more instructions for use of the compositions (as described herein). The kits described herein may also include other materials required from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for performing any of the methods described herein.

VIII. Exemplary Implementation

The provided implementation plan includes:

1. An immunomodulatory protein comprising at least one TACI polypeptide, the TACI polypeptide being a truncated wild-type TACI extracellular domain or a variant thereof, wherein the truncated wild-type TACI extracellular domain contains a cysteine-rich domain 2 (CRD2) but lacks the complete cysteine-rich domain 1 (CRD1), wherein the variant TACI polypeptide comprises one or more amino acid substitutions in the truncated wild-type TACI extracellular domain.

2. An immunomodulatory protein comprising at least one TACI polypeptide, the TACI polypeptide being a truncated wild-type TACI extracellular domain or a variant thereof, wherein, with respect to the position shown in SEQ ID NO:122, the truncated wild-type TACI extracellular domain comprises a continuous sequence of amino acid residues 71-104 contained within amino acid residues 67-118, wherein the variant TACI polypeptide comprises one or more amino acid substitutions in the truncated wild-type TACI extracellular domain.

3. The immunomodulatory protein according to embodiment 1 or embodiment 2, wherein the length of the truncated wild-type TACI extracellular domain is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50 or 51 amino acids.

4. The immunomodulatory protein according to any one of embodiments 1-3, wherein the truncated wild-type TACI extracellular domain is composed of amino acid residues 68-110 shown in SEQ ID NO:122.

5. The immunomodulatory protein according to any one of embodiments 1-4, wherein the TACI polypeptide consists of the amino acid sequence shown in SEQ ID NO:13, or a variant thereof containing one or more amino acid substitutions in the sequence shown in SEQ ID NO:13.

6. An immunomodulatory protein comprising at least one TACI polypeptide, said TACI polypeptide being a truncated TACI polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13, or a variant thereof containing one or more amino acid substitutions in the sequence shown in SEQ ID NO:13.

7. An immunomodulatory protein according to any one of embodiments 1-5, wherein the truncated TACI polypeptide or a variant thereof binds to APRIL, BAFF, or BAFF/APRIL heterotrimer.

8. The immunomodulatory protein according to any one of embodiments 1-7, wherein the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence shown in SEQ ID NO:1.

9. The immunomodulatory protein according to any one of embodiments 1-7, wherein the TACI polypeptide is a truncated wild-type TACI extracellular domain consisting of the sequence shown in SEQ ID NO:13.

10. An immunomodulatory protein comprising a truncated TACI polypeptide consisting of the sequence shown in SEQ ID NO:13.

11. The immunomodulatory protein according to any one of embodiments 1-7, wherein the TACI polypeptide is a variant TACI polypeptide, wherein the variant TACI polypeptide has increased binding affinity to one or both of APRIL and BAFF compared to the truncated TACI polypeptide.

12. The immunomodulatory protein according to any one of embodiments 1-7 and 11, wherein the variant TACI polypeptide comprises 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 number shown in SEQ ID NO:122.

13. The immunomodulatory protein according to embodiment 12, wherein one or more amino acid substitutions are 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.

14. The immunomodulatory protein according to embodiment 12 or embodiment 13, wherein the one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V or C86Y.

15. The immunomodulatory protein according to any one of embodiments 12-13, wherein the one or more amino acid substitutions are D85E/K98T, I87L/K98T, L82P/I87L, G76S/P97S, K77E/R84L/F103Y, Y79F/Q99E, L83S/F103S, K77E/R84Q, K77E/A101D, K77E/F78Y/Y102D, Q75E/R84Q, Q75R/R84G/I92V, K77E/A101D/Y102D, R84Q/S88N/A101D, R84Q/F103V, K77E/Q95R/A101D, or I87M/A101D.

16. The immunomodulatory protein according to any one of embodiments 12-15, wherein one or more amino acid substitutions are K77E/F78Y/Y102D.

17. The immunomodulatory protein according to any one of embodiments 12-15, wherein one or more amino acid substitutions are Q75E/R84Q.

18. The immunomodulatory protein according to any one of embodiments 12-16, wherein the variant TACI polypeptide is shown in SEQ ID NO:26.

19. The immunomodulatory protein according to any one of embodiments 12-15 and 17, wherein the variant TACI polypeptide is shown in SEQ ID NO:27.

20. The immunomodulatory protein according to embodiment 1, wherein the TACI polypeptide is a variant TACI polypeptide comprising one or more amino acid substitutions in the extracellular domain (ECD) of the reference TACI polypeptide or its specific binding fragment thereof, the amino acid substitutions being located 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 numbers shown in SEQ ID NO: 122.

21. An immunomodulatory protein comprising at least one variant TACI polypeptide, wherein the at least one variant TACI polypeptide comprises one or more amino acid substitutions in an extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof, the amino acid substitutions being located 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 numbers shown in SEQ ID NO:122.

22. The immunomodulatory protein according to embodiment 20 or embodiment 21, wherein the reference TACI polypeptide is a truncated polypeptide, the truncated polypeptide consisting of the extracellular domain of TACI or its specific binding portion that binds to APRIL, BAFF, or BAFF/APRIL heterotrimer.

23. An immunomodulatory protein according to any one of embodiments 20-22, wherein 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 comprising one or both of the CRD1 and CRD2 domains, said portion being bound to APRIL, BAFF, or a BAFF/APRIL heterotrimer.

24. The immunomodulatory protein according to any one of embodiments 20-23, wherein the reference TACI polypeptide lacks an N-terminal methionine.

25. An immunomodulatory protein according to any one of embodiments 20-24, wherein the reference TACI polypeptide comprises the CRD1 domain and the CRD2 domain.

26. The immunomodulatory protein according to any one of embodiments 20-25, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1.

27. An immunomodulatory protein according to any one of embodiments 20-25, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1.

28. An immunomodulatory protein according to any one of embodiments 20-24, wherein the reference TACI polypeptide is substantially composed of the CRD2 domain.

29. An immunomodulatory protein according to any one of embodiments 20-24 and 28, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13.

30. An immunomodulatory protein according to any one of embodiments 20-24 and 28, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13.

31. The immunomodulatory protein according to any one of embodiments 20-30, wherein the one or more amino acid substitutions are selected from W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y or their conserved amino acid substitutions.

32. The immunomodulatory protein according to any one of embodiments 20-31, wherein the one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V or C86Y.

33. The immunomodulatory protein according to any one of embodiments 20-32, wherein the one or more amino acid substitutions include at least amino acid substitution for K77E.

34. The immunomodulatory protein according to any one of embodiments 20-32, wherein the one or more amino acid substitutions include at least amino acid substitution R84G.

35. The immunomodulatory protein according to any one of embodiments 20-32, wherein the one or more amino acid substitutions include at least amino acid substitution R84Q.

36. The immunomodulatory protein according to any one of embodiments 20-35, wherein one or more amino acid substitutions are D85E/K98T, I87L/K98T, R60G/Q75E/L82P, R60G/C86Y, W40R/L82P/F103Y, W40R/Q59R/T61P/K98T, L82P/I87L, G76S/P97S, K77E/R84L/F10 3Y, Y79F/Q99E, L83S/F103S, K77E/R84Q, K77E/A101D, K77E/F78Y/Y102D, Q75E/R84Q, Q75R/R8 4G/I92V, K77E/A101D/Y102D, R84Q/S88N/A101D, R84Q/F103V, K77E/Q95R/A101D or I87M/A101D.

37. An immunomodulatory protein according to any one of embodiments 20-32, 33 and 36, wherein one or more amino acid substitutions are K77E/F78Y/Y102D.

38. The immunomodulatory protein according to any one of embodiments 20-32, 35 and 36, wherein one or more amino acid substitutions are Q75E/R84Q.

39. An immunomodulatory protein according to any one of embodiments 20-38, wherein the variant TACI polypeptide has increased binding affinity to one or both of APRIL and BAFF compared to the reference TACI polypeptide.

40. The immunomodulatory protein according to embodiment 11 or embodiment 39, wherein the variant TACI polypeptide has increased binding affinity to APRIL.

41. The immunomodulatory protein according to embodiment 11 or embodiment 39, wherein the variant TACI polypeptide has increased binding affinity to BAFF.

42. The immunomodulatory protein according to embodiment 11 or embodiment 39, wherein the variant TACI polypeptide has increased binding affinity for APRIL and BAFF.

43. The immunomodulatory protein according to any one of embodiments 11 and 39-42, wherein the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times.

44. An immunomodulatory protein according to any one of embodiments 1-7 and 11-43, wherein:

The variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120; or

The variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO:14-20, 23-35, 92-100.

45. An immunomodulatory protein according to any one of embodiments 1-7 and 11-43, wherein:

The variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120; or

The variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO:14-20, 23-35, 92-100.

46. An immunomodulatory protein according to any one of embodiments 1-7, 11-43 and 45, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:26.

47. An immunomodulatory protein according to any one of embodiments 1-7, 11-43 and 45, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:27.

48. An immunomodulatory protein according to any one of embodiments 1-7, 11-43 and 45, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:107.

49. An immunomodulatory protein according to any one of embodiments 1-7, 11-43 and 45, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:20.

50. An immunomodulatory protein according to any one of embodiments 1-7 and 11-49, wherein the immunomodulatory protein comprises a heterologous portion linked to the at least one TACI polypeptide.

51. The immunomodulatory protein according to embodiment 50, wherein the heterologous portion is a half-life extended portion, a polymerized domain, a targeting portion that binds to molecules on the cell surface, or a detectable marker.

52. The immunomodulatory protein according to embodiment 51, wherein the extended half-life portion comprises a polymerizing domain, albumin, albumin-binding polypeptide, Pro/Ala/Ser (PAS), C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin-binding small molecule, or a combination thereof.

53. An immunomodulatory protein according to any one of embodiments 1-7 and 11-52, wherein the immunomodulatory protein is a TACI-Fc fusion protein, wherein at least one TACI polypeptide is linked to the Fc region of an immunoglobulin.

54. The immunomodulatory protein according to any one of embodiments 3-5, 7-9, 11-20 and 22-53, wherein the immunomodulatory protein is a dimer.

55. The immunomodulatory protein according to embodiment 53, wherein the immunoglobulin Fc region is a homodimeric Fc region.

56. The immunomodulatory protein according to embodiment 53, wherein the immunoglobulin Fc region is a heterodimeric Fc region.

57. The immunomodulatory protein according to embodiments 3-5, 7-9, 11-20, 22-53 and 54-55, wherein the immunomodulatory protein is a homodimer, and each polypeptide of the dimer is identical.

58. An immunomodulatory protein according to any one of embodiments 53, 54-55 and 57, wherein the immunoglobulin Fc is an IgG1 Fc domain, or a variant Fc exhibiting reduced binding affinity to the Fc receptor and/or reduced effector function, optionally as compared to the wild-type IgG1 Fc domain.

59. An immunomodulatory protein according to any one of embodiments 53, 54-55 and 57-58, wherein the immunoglobulin Fc is an IgG1 Fc domain and the Fc comprises the amino acid sequence shown in SEQ ID NO:81.

60. An immunomodulatory protein according to any one of embodiments 53, 54-55 and 57-58, wherein the immunoglobulin Fc is a variant IgG1 Fc domain comprising one or more amino acid substitutions selected according to EU numbers from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G and V302C.

61. The immunomodulatory protein according to embodiment 60, wherein the immunoglobulin Fc region contains amino acid substitutions of L234A, L235E and G237A according to EU numbers or amino acid substitutions of R292C, N297G and V302C according to EU numbers.

62. An immunomodulatory protein according to any one of embodiments 53, 54-55, 57-58 and 60-61, wherein the Fc is a variant Fc comprising the amino acid sequence shown in SEQ ID NO:73.

63. The immunomodulatory protein according to any one of embodiments 1-62, wherein:

The immunomodulatory protein blocks the binding of APRIL, BAFF, or APRIL/BAFF heterotrimer to BCMA or TACI; and/or

The immunomodulatory protein reduces the level of circulating APRIL, BAFF, or APRIL/BAFF in the blood after administration to the subject.

64. The immunomodulatory protein according to any one of embodiments 1-62, wherein the immunomodulatory protein reduces or inhibits B cell maturation, differentiation and/or proliferation.

65. One or more nucleic acid molecules, said nucleic acid molecules encoding an immunomodulatory protein according to any one of embodiments 1-64.

66. The nucleic acid molecule according to embodiment 65, wherein the nucleic acid molecule is a synthetic nucleic acid.

67. The nucleic acid molecule according to embodiment 65 or embodiment 66, wherein the nucleic acid molecule is cDNA.

68. A vector comprising a nucleic acid molecule according to any one of embodiments 65-67.

69. The carrier according to embodiment 68, wherein the carrier is an expression carrier.

70. The vector according to embodiment 68 or embodiment 69, wherein the vector is a mammalian expression vector or a viral vector.

71. A cell comprising a nucleic acid according to any one of embodiments 65-67 or a vector according to any one of embodiments 68-70.

72. The cell according to embodiment 71, wherein the cell is a mammalian cell.

73. The cell according to embodiment 71 or embodiment 72, wherein the cell is a human cell.

74. A method for producing an immunomodulatory protein, the method comprising introducing a nucleic acid molecule according to any one of embodiments 65-67 or a vector according to any one of embodiments 68-70 into a host cell under conditions in which the protein is expressed.

75. The method according to embodiment 74, the method further comprising isolating or purifying the immunomodulatory protein from the cells.

76. An immunomodulatory protein generated by the method according to embodiment 74 or embodiment 75.

77. A pharmaceutical composition comprising an immunomodulatory protein according to any one of embodiments 1-64 and 76.

78. 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 in an extracellular domain (ECD) of a reference TACI polypeptide or a specific binding fragment thereof, the amino acid substitutions being located 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 numbers shown in SEQ ID NO:122.

79. The variant TACI-Fc fusion protein according to embodiment 78, wherein the reference TACI polypeptide is a truncated polypeptide, the truncated polypeptide consisting of the extracellular domain of TACI or its specific binding portion that binds to APRIL, BAFF, or BAFF/APRIL heterotrimer.

80. The variant TACI-Fc fusion protein according to embodiment 78 or embodiment 79, wherein 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 comprising one or both of the CRD1 and CRD2 domains, said portion being bound to APRIL, BAFF, or a BAFF/APRIL heterotrimer.

81. A variant TACI-Fc fusion protein according to any one of embodiments 78-80, wherein the reference TACI polypeptide lacks an N-terminal methionine.

82. A variant TACI-Fc fusion protein according to any one of embodiments 78-81, wherein the reference TACI polypeptide comprises the CRD1 domain and the CRD2 domain.

83. A variant TACI-Fc fusion protein according to any one of embodiments 78-82, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1.

84. A variant TACI-Fc fusion protein according to any one of embodiments 78-82, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1.

85. A variant TACI-Fc fusion protein according to any one of embodiments 78-81, wherein the reference TACI polypeptide is substantially composed of the CRD2 domain.

86. A variant TACI-Fc fusion protein according to any one of embodiments 78-81 and 85, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13.

87. A variant TACI-Fc fusion protein according to any one of embodiments 78-81 and 85, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13.

88. The variant TACI-Fc fusion protein according to any one of embodiments 78-87, wherein the one or more amino acid substitutions are selected from W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y or their conserved amino acid substitutions.

89. The variant TACI-Fc fusion protein according to any one of embodiments 78-88, wherein the one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V or C86Y.

90. A variant TACI-Fc fusion protein according to any one of embodiments 78-89, wherein the one or more amino acid substitutions include at least amino acid substitution K77E.

91. A variant TACI-Fc fusion protein according to any one of embodiments 78-89, wherein the one or more amino acid substitutions include at least amino acid substitution R84G.

92. A variant TACI-Fc fusion protein according to any one of embodiments 78-92, wherein the one or more amino acid substitutions include at least amino acid substitution R84Q.

93. A variant TACI-Fc fusion protein according to any one of embodiments 78-92, wherein one or more amino acid substitutions are D85E/K98T, I87L/K98T, R60G/Q75E/L82P, R60G/C86Y, W40R/L82P/F103Y, W40R/Q59R/T61P/K98T, L82P/I87L, G76S/P97S, K77E/R84L /F103Y, Y79F/Q99E, L83S/F103S, K77E/R84Q, K77E/A101D, K77E/F78Y/Y102D, Q75E/R84Q, Q75R/R84G/I92V, K77E/A101D/Y102D, R84Q/S88N/A101D, R84Q/F103V, K77E/Q95R/A101D or I87M/A101D.

94. The variant TACI-Fc fusion protein according to any one of embodiments 78-90 and 93, wherein one or more amino acid substitutions are K77E/F78Y/Y102D.

95. The variant TACI-Fc fusion protein according to any one of embodiments 78-90, 92 and 93, wherein one or more amino acid substitutions are Q75E/R84Q.

96. A variant TACI-Fc fusion protein according to any one of embodiments 78-95, wherein the variant TACI polypeptide has increased binding affinity to one or both of APRIL and BAFF compared to the reference TACI polypeptide.

97. The variant TACI-Fc fusion protein according to any one of embodiments 78-96, wherein the variant TACI polypeptide has increased binding affinity to APRIL.

98. A variant TACI-Fc fusion protein according to any one of embodiments 78-96, wherein the variant TACI polypeptide has increased binding affinity to BAFF.

99. The variant TACI-Fc fusion protein according to any one of embodiments 78-96, wherein the variant TACI polypeptide has increased binding affinity for APRIL and BAFF.

100. The variant TACI-Fc fusion protein according to any one of embodiments 96-99, wherein the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times.

101. The variant TACI-Fc fusion protein according to any one of embodiments 96-100, wherein:

The variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120; or

The variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO:14-20, 23-35, 92-100.

102. The variant TACI-Fc fusion protein according to any one of embodiments 96-100, wherein:

The variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO: 2-12, 21, 22, 101-120; or

The variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO:14-20, 23-35, 92-100.

103. The variant TACI-Fc fusion protein according to any one of embodiments 96-100, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:26.

104. The variant TACI-Fc fusion protein according to any one of embodiments 96-100, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:27.

105. The variant TACI-Fc fusion protein according to any one of embodiments 96-100, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:107.

106. The variant TACI-Fc fusion protein according to any one of embodiments 96-100, wherein the variant TACI polypeptide consists of or is substantially composed of the sequence shown in SEQ ID NO:20.

107. The Fc fusion protein according to any one of embodiments 78-106, wherein the adapter comprises a peptide adapter and the peptide adapter is selected from GSGGS (SEQ ID NO:76), GGGGS (G4S; SEQ ID NO:77), GSGGGGS (SEQ ID NO:74), GGGGSGGGGS (2xGGGGS; SEQ ID NO:78), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO:79), GGGGSGGGGSGGGGSGGGGS (4xGGGGS; SEQ ID NO:84), GGGGSGGGGSGGGGSGGGGSGGGS (5xGGGGS; SEQ ID NO:91), GGGGSSA (SEQ ID NO:80), or a combination thereof.

108. The Fc fusion protein according to any one of embodiments 78-107, wherein the Fc fusion protein is a dimer.

109. The Fc fusion protein according to any one of embodiments 78-108, wherein the immunoglobulin Fc region is a homodimeric Fc region.

110. The Fc fusion protein according to any one of embodiments 78-109, wherein the immunoglobulin Fc is an IgG1 Fc domain, or a variant Fc exhibiting reduced binding affinity to the Fc receptor and/or reduced effector function, optionally as compared to the wild-type IgG1 Fc domain.

111. The Fc fusion protein according to any one of embodiments 78-110, wherein the immunoglobulin Fc is an IgG1 Fc domain and the Fc contains the amino acid sequence shown in SEQ ID NO:81.

112. The Fc fusion protein according to any one of embodiments 78-106 and 107-111, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:168.

113. The Fc fusion protein according to any one of embodiments 78-106 and 107-111, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:170.

114. The Fc fusion protein according to any one of embodiments 78-110, wherein the immunoglobulin Fc is a variant IgG1 Fc domain comprising one or more amino acid substitutions selected according to EU numbers from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C.

115. The Fc fusion protein according to embodiment 114, wherein the immunoglobulin Fc region contains amino acid substitutions of L234A, L235E and G237A according to EU numbers or amino acid substitutions of R292C, N297G and V302C according to EU numbers.

116. The Fc fusion protein according to any one of embodiments 78-115, wherein the immunoglobulin Fc is shown in SEQ ID NO:71.

117. The Fc fusion protein according to any one of embodiments 78-113 and 114-116, wherein the Fc is a variant Fc comprising the amino acid sequence shown in SEQ ID NO:73.

118. The Fc fusion protein according to any one of embodiments 78-106, 107-110, 114, 115 and 117, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:167.

119. The Fc fusion protein according to any one of embodiments 78-106, 107-110, 114, 115 and 117, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:169.

120. The Fc fusion protein according to any one of embodiments 78-119, wherein the Fc fusion protein is a dimer.

121. The Fc fusion protein according to any one of embodiments 78-120, wherein the Fc fusion protein is a homodimer.

122. The Fc fusion protein according to any one of embodiments 78-121, wherein the Fc fusion protein neutralizes APRIL and BAFF.

123. The Fc fusion protein according to embodiment 122, wherein:

The IC50 for neutralizing APRIL 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 foregoing; and/or

The IC50 for neutralizing BAFF 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 the aforementioned values.

124. The Fc fusion protein according to any one of embodiments 78-122, wherein:

The Fc fusion protein blocks the binding of APRIL, BAFF, or 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 subject.

125. The Fc fusion protein according to any one of embodiments 78-124, wherein the immunomodulatory protein reduces or inhibits B cell maturation, differentiation and/or proliferation.

126. One or more nucleic acid molecules, said nucleic acid molecules encoding an Fc fusion protein according to any one of embodiments 78-125.

127. The nucleic acid molecule according to embodiment 126, wherein the nucleic acid molecule is a synthetic nucleic acid.

128. The nucleic acid molecule according to embodiment 126 or embodiment 127, wherein the nucleic acid molecule is cDNA.

129. A vector comprising a nucleic acid molecule according to any one of embodiments 126-128.

130. The carrier according to embodiment 129, wherein the carrier is an expression carrier.

131. The vector according to embodiment 129 or embodiment 130, wherein the vector is a mammalian expression vector or a viral vector.

132. A cell comprising a nucleic acid according to any one of embodiments 126-128 or a vector according to any one of embodiments 129-131.

133. The cell according to embodiment 132, wherein the cell is a mammalian cell.

134. The cell according to embodiment 132 or embodiment 133, wherein the cell is a human cell.

135. A method for producing an Fc fusion protein, the method comprising introducing a nucleic acid molecule according to any one of embodiments 126-128 or a vector according to any one of embodiments 129-131 into a host cell under conditions in which the protein is expressed.

136. The method according to embodiment 135, the method further comprising isolating or purifying the Fc fusion protein from the cells.

137. An Fc fusion protein produced by the method according to embodiment 135 or embodiment 136.

138. A pharmaceutical composition comprising the Fc fusion protein according to any one of embodiments 78-125 and 137.

139. The pharmaceutical composition according to embodiment 77 or embodiment 138, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

140. The pharmaceutical composition according to any one of embodiments 77, 138 and 139, wherein the pharmaceutical composition is sterile.

141. An article comprising a pharmaceutical composition according to any one of embodiments 77 and 138-140 in a vial or container.

142. The article of manufacture according to embodiment 141, wherein the vial or container is sealed.

143. A kit comprising a pharmaceutical composition according to any one of embodiments 77 and 138-140 and instructions for use.

144. A kit comprising the article of manufacture and instructions for use as described in embodiment 141 or embodiment 142.

145. A method for reducing the immune response of a subject, the method comprising administering an immunomodulatory protein according to any one of embodiments 1-64 or 76 to a subject in need.

146. A method for reducing the immune response of a subject, the method comprising administering an Fc fusion protein according to any one of embodiments 78-126 and 137 to a subject in need.

147. A method for reducing the immune response of a subject, the method comprising administering a pharmaceutical composition according to any one of embodiments 77 and 137-140 to a subject in need.

148. The method according to any one of embodiments 145-147, wherein the B cell immune response is reduced in the subject, thereby reducing or inhibiting B cell maturation, differentiation and/or proliferation.

149. The method according to any one of embodiments 145-148, wherein the circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer are reduced in the subject.

150. A method for reducing circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer in a subject, the method comprising administering a pharmaceutical composition according to any one of embodiments 77 and 137-140 to the subject.

151. The method according to embodiment 57 or embodiment 147, wherein the T-cell immune response is reduced in the subject, thereby reducing or inhibiting T-cell co-stimulation.

152. The method according to any one of embodiments 145-151, wherein reducing the immune response treats the subject's disease or condition.

153. A method of treating a disease or condition of a subject, the method comprising administering an immunomodulatory protein according to any one of embodiments 1-64 or 76 to the subject in need.

154. A method of treating a disease or condition in a subject, the method comprising administering the Fc fusion protein according to any one of claims 78-115 and 121 to the subject in need.

155. A method of treating a disease or ailment of a subject, the method comprising administering a pharmaceutical composition according to any one of embodiments 77 and 137-140 to the subject in need.

156. The method according to any one of embodiments 150-153, wherein the disease or symptom is an autoimmune disease, B-cell carcinoma, antibody-mediated symptom, nephropathy, graft rejection, graft-versus-host disease, or viral infection.

157. The method according to embodiment 156, wherein the disease or condition is selected from the following autoimmune diseases: systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris.

158. The method according to embodiment 156, wherein the disease or condition is B-cell cancer and the cancer is myeloma.

IX. Implementation Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1. Identification of affinity-modified TACI

This embodiment describes a mutant DNA construct for generating the human TACI TNFR domain (TD) for translation and expression on the surface of yeast cells serving as a yeast display library, introducing the DNA library into the yeast, and selecting yeast cells expressing an affinity-modified variant (TACI vTD) containing at least one TD of the extracellular domain (ECD) of TACI. The selected TACI vTD is then formatted as an Fc fusion protein.

Generation of mutant DNA constructs of the A.TACI TNFR domain

Library containing random amino acid substitutions was constructed to identify variants of the extracellular domain (ECD) of TACI. Constructs were generated based on wild-type human TACI sequences containing ECD portions of TACI, the ECD portions comprising (1) two cysteine-rich protein domains (CRDs, CRD1/CRD2) as shown in SEQ ID NO:1 (corresponding to residues 29-110 as shown in UniProt accession number O14836), or (2) a single CRD (CRD2) as shown in SEQ ID NO:13 (corresponding to residues 68-110 as shown in UniProt accession number O14836).

TACI ECD(29-110)(SEQ ID NO:1):

VAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

TACI ECD(68-110)(SEQ ID NO:13):

SLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRS

DNA encoding the wild-type TACI ECD domain was cloned into the modified yeast expression vector PBYDS03 (Life Technologies, USA) between the BamHI and KpnI sites, which positions the TACI ECD at the N-terminus of the yeast surface anchoring domain Sag1 (the C-terminal domain of yeast α-lectin), which has an in-frame HA fusion tag at the N-terminus of the TACI ECD sequence and a c-Myc fusion tag at the C-terminus of the TACI ECD sequence. Expression in this vector was controlled by an inducible GAL1 promoter. After validating the correct DNA sequence, error-prone PCR was performed using the wild-type TACI ECD DNA construct as a template to introduce random mutations across the TACI ECD sequence at a frequency of 2–5 mutations per gene copy. The desired error rate was achieved using the Genemorph II kit (Agilent, USA) in combination with MnCl2 in incremental increments of 0.0–0.6 mM. Following error-prone PCR, the mutagenic DNA was purified by gel electrophoresis using a gel electrophoresis and PCR purification kit (Macherey-Nagel, Germany). The isolated DNA fragment was then amplified by PCR using primers containing a 48 bp overlap region homologous to pBYDS03 in a OneTaq 2xPCR master mix (New England Biolabs, USA) for preparation of large-scale yeast electroporation. The TACI ECD DNA insert was gel purified and resuspended in sterile deionized water at a nominal concentration of 500 ng/μL.

To prepare the vector for transformation, pBYDS03 was digested with BamHI-HF and KpnI-HF restriction enzymes (New England Biolabs, USA), and the large vector fragment (expected size: 7671 bp) was gel-purified and dissolved in sterile deionized water at a nominal concentration of 500 ng/μL. For yeast transformation, 12 μg of the library DNA insert was mixed with 4 μg of linearized vector for each electroporation well.

To introduce a random DNA library into yeast, *Saccharomyces cerevisiae* strain BJ5464 (ATCC.org; ATCC number 208288) was prepared immediately prior to electroporation, as detailed in Benatulil, L. et al., *Protein Eng Des Sel.* 2010 Apr; 23(4):155-159. Briefly, the overnight stationary phase culture of BJ5464 was passaged in 100 mL of YPD medium (10 g/L yeast nitrogen source, 20 g/L peptone, and 20 g/L D-(+)-glucose) to an OD ₆₀₀ of 0.3 and placed in a platform shaker at 30°C and 300 rpm until the inoculated culture reached an OD ₆₀₀ of 1.6. After approximately 5 hours, the cells were harvested by centrifugation and kept on ice for the remainder of the protocol, unless otherwise stated. After harvesting, the cells were washed twice with 50 mL of ice-cold water and once with electroporation buffer (1 M sorbitol, 1 mM CaCl2). The collected cells were conditioned as follows: resuspended in 20 mL of 0.1 M LiAc/10 mM DTT and shaken at 225 rpm in a culture flask at 30 °C for 30 min. The conditioned cells were immediately centrifuged, washed twice with electroporation buffer, and resuspended in approximately 100–200 μL of electroporation buffer to a volume of 1 mL. This conditioned cell suspension was sufficient for two electroporation reactions in a 400 μL cuvette.

For each electroporation, 12 μg of the library DNA insert and 4 μg of the linearized pBYDS03 vector (described above) were mixed with 400 μl of electrocompetent BJ5464 cells and transferred to a pre-chilled BioRadGenePulser cuvette with a 2 mm electrode gap. The mixture was kept on ice for 5 min, followed by electroporation at 2500 V using a BTX ECM399 exponentially decaying wave electroporation system. Immediately after electroporation, cells were added to 8 mL of a 1:1 mixture of 1 M sorbitol: 1XYPD and incubated at room temperature without shaking for 10 min, then placed on a platform shaker at 225 rpm and 30 °C for 1 h. Cells were collected by centrifugation and resuspended in 250 mL of SCD-Leu medium to accommodate the LEU2 selective label carried by the modified plasmid pBYDS03. Prepare 1 liter of SCD-Leu medium containing 14.7 g m sodium citrate, 4.29 g m citrate monohydrate, 20 g m dextrose, 6.7 g m yeast nitrogen source, and 1.6 g m leucine-free yeast synthesis-deficient medium supplement. Sterilize the medium by filtration using a 0.22 μm vacuum filter before use. Estimate the library size by spotting freshly recovered cells serially diluted on SCD-Leu agar plates in the range of ^10⁻⁵ to ^10⁻¹⁰ and extrapolating by colony counting after three days. Allow the remainder of the electroporation culture to saturate, and subculture the cells from this culture 1/100th in the same medium and grow to saturate again to minimize the fraction of untransformed cells and allow plasmid isolation from cells that may contain two or more library variants. To maintain library diversity, use an inoculum containing at least 10 times more cells than the calculated library size for this subculture step. Cells from the second saturated culture were resuspended in fresh medium containing 25% (w/v) glycerol to a density of 1 x ^10¹⁰ /mL and frozen and stored at -80°C (frozen library stock).

Thaw cells at least 10 times the estimated library size from the stock library, resuspend them in non-inducible SCD-Leu medium to 0.5 x ^10⁷ cells/mL, and grow overnight. The next day, centrifuge cells equal to 10 times the library size at 2000 RPM for 2 minutes and resuspend them in inducible SCDG-Leu medium to 0.5 x ^10⁷ cells/mL. Prepare one liter of SCDG-Leu induction medium containing 5.4 g _{m Na₂HPO₄} , 8.56 g m _NaH₂PO₄ · _H₂O , ₂₀ g m galactose, 2.0 g m dextrose, 6.7 g m yeast nitrogen source, and 1.6 g m leucine-free yeast synthesis-deficient supplement, dissolved in water and sterilized via a 0.22 μm membrane filter. Grow the culture overnight in the induction medium at 30°C to induce expression of the library proteins on the yeast cell surface.

Following overnight induction of the TACI ECD library, TACI ECD variants with the ability to bind their exogenous recombinant anti-structural proteins were enriched by magnetic separation using Dynabeads ^™ His-tagged magnetic beads preloaded with BAFF-9xHis to sort a number of cells equivalent to 10-fold of the estimated library diversity. The output from the magnetic separation was used in a subsequent FACS selection protocol involving four rounds of positive selection alternating between BAFF-9xHis and APRIL-FLAG, where the anti-structural concentration was simultaneously reduced by 10-fold in each round (e.g., FACS1: 50 nM APRIL-FLAG; FACS4: 0.05 nM BAFF-9xHis). The incubation volume was adjusted to maintain an anti-structural concentration at least 10-fold stoichiometric excess over the total number of TACI ECD variant molecules displayed by yeast (assuming 100,000 copies of protein per cell) to avoid ligand depletion artifacts that could reduce library distinguishability. The binding of BAFF-9xHis and APRIL-FLAG to the TACI ECD variants was detected using a PE-conjugated anti-6xHis tag antibody (Biolegend, USA) and a PE-conjugated anti-FLAG tag antibody, respectively. Variants isolated from FACS3 and FACS4 outputs were used for DNA sequencing and subsequent cloning for recombinant Fc fusion expression.

A second cycle of random mutagenesis was performed on the yeast cell outputs selected from the FACS4 BAFF-9xHis variants. Each sorting used the same positive selection protocol as the first cycle, alternating reverse structures, but with the reverse structure order reversed (e.g., FACS1: 50 nM BAFF-9xHis; FACS4: 0.05 nM APRIL-FLAG). Additional variants were selected from the FACS3 and FACS4 yeast cell outputs.

B. Reformat the selected output as an FC fusion.

The TACI ECD variant inserts from the FACS3 and FACS4 outputs of both cycle 1 and cycle 2 selections as described above were subcloned into an Fc fusion vector for sequence analysis of individual cloning. To generate a recombinant immunomodulatory protein (e.g., variant TACIECD-Fc) as an Fc fusion protein containing an ECD with at least one affinity-modified domain of TACI, encoding DNA was generated to encode the following protein: the variant TACI domain, followed by a 7-amino acid linker (GSGGGGS; SEQ ID NO: 74), followed by a human IgG1 effector Fc sequence containing mutants L234A, L235E, and G237A according to the Eu indexing system for immunoglobulin proteins. Because the construct does not include any antibody light chain that can form a covalent bond with cysteine, the human IgG1 Fc also contains a cysteine residue replaced by a serine residue at position 220 according to the EU index numbering system for immunoglobulin proteins (C220S) (corresponding to position 5 (C5S) of the wild-type or unmodified Fc shown in SEQ ID NO:71). The Fc region also lacks the C-terminal lysine at position 447, which is normally encoded in the wild-type human IgG1 constant region gene (named K447del) (corresponding to position 232 of the wild-type or unmodified Fc shown in SEQ ID NO:71). The effectorless (inert) IgG1 Fc in the fusion construct is shown in SEQ ID NO:73.

The selected TACI ECD FACS-sorted output cell library was grown to final density in SCD-Leu selective medium, and plasmid DNA was isolated using a yeast plasmid DNA isolation kit (Zymoresearch, USA). For Fc fusion generation, PCR amplification of affinity-matured TACI ECD variants was performed using primers containing a 40 bp homologous region at either end of an Fc fusion vector (encoding and frame-in-frame with the Fc region) digested with AfeI and BamHI, for in vitro recombination using GibsonAssembly master mix (New England Biolabs). GibsonAssembly reactants were added to *E. coli* strain NEB5α (New England Biolabs, USA) for heat shock transformation according to the manufacturer's instructions.

Diluted transformants were plated on LB agar containing 100 μg/mL carbenicillin (Teknova, USA) to isolate single colonies for selection. Typically, up to 96 colonies from each transformation were then allowed to saturate overnight at 37°C in LB broth (Teknova catalog number L8112) containing 100 μg/mL carbenicillin in 96-well plates, and aliquots from each well were submitted for DNA sequencing to identify one or more mutations in all clones.

After sequence analysis and identification of the target clone, plasmid DNA was prepared using the MidiPlus kit (Qiagen).

The recombinant Fc fusion protein variant was generated from suspension-adapted human embryonic kidney (HEK) 293 cells using the Expi293 expression system (Invitrogen, USA). The supernatant was harvested and the Fc protein was captured onto a Mab SelectSure column (GE Healthcare catalog number 17543801). The protein was eluted from the column using 50 mM acetate (pH 3.6). The MabSelectSure eluent was combined, and the pH was adjusted to above pH 5.0. This material was then purified on a preparative SEC column to produce a highly purified monomeric material. This material was buffer-exchanged to 10 mM acetate, 9% sucrose (pH 5.0). Protein purity was assessed using an analytical SEC column. The material was bottled and stored at -80°C.

The amino acid substitutions in the selected TACI vTDs identified and generated by the selection process are shown in Table 1. The binding and functional activity of the selected vTDs, formatted as Fc fusion proteins, were tested as described in Example 2.

Example 2. Evaluation of the activity of the Fc fusion protein.

This embodiment describes the characterization of the activity of molecules containing TACI domains (such as soluble wild-type (WT) or variant TACI vTD) formatted as Fc fusions using cell line-based in vitro bioassays.

Jurkat cells with a reporter for luciferase based on the nuclear factor κ light chain enhancer (NF-κB) of activated B cells were purchased (BPS Bioscience). Jurkat/NK-κB cells were transduced with lentivirus to produce stable cell surface expression of mouse TACI (Jurkat/NF-κB/TACI). Cells expressing mouse TACI responded to both human and mouse APRIL or BAFF. Upon binding of recombinant human or mouse APRIL or BAFF to TACI, the endogenous NK-κB transcription factor in Jurkat cells bound to the DNA response element controlling the transcription of the firefly luciferase gene. Luciferase production was quantified by adding a substrate containing luciferin, which emits light measurable using a microplate reader upon oxidation. A schematic diagram of the Jurkat/NF-κB/TACI assay is shown in Figure 1.

The recombinant human and mouse APRIL and BAFF ligands were purchased: human APRIL (Tonbo Biosciences); human BAFF (Biolegend); mouse APRIL (ProSci Incorporated); and mouse BAFF (R&D Systems).

To determine the bioactivity of molecules containing the TACI WT or vTD domains, 30 μL of recombinant human or mouse APRIL or BAFF at different concentrations (range 1–10 nM) were incubated together with 30 μL of fixed or incremental (range 40 nM–66 pM) molecules containing the TACI domain. The ligands and soluble receptors were incubated with shaking at room temperature (RT) for 20 min. 50 μL of the medium (RPMI 1640 + 5% fetal bovine serum [FBS]) was transferred to 50 μL of a 96-well white flat-bottomed plate containing ^1.5 x 10⁵ Jurkat/NF-κB/TACI cells/well. The wells were mixed and the plate was incubated at 37°C (C) in a humidified 5% _CO₂ chamber for 5 h. Remove the plate from the incubator and add 100 μL of cell lysate and luciferase substrate solution (Bio-Glo ^™ luciferase assay system, Promega) to each well. Incubate the plate on a track-shaker for 10 minutes. Determine the relative luminescence (RLU) of each test sample by measuring luminescence at an integration time of 1 second per well using a Cytation 3 (BioTek Instruments) imaging reader. A reduced RLU relative to the control protein in the presence of TACI WT or vTD indicates blockade and inhibition of ligand signaling via transduced TACI receptor in Jurkat/NF-κB/TACI cells.

As shown in Figure 2, the exemplary TACI-Fc vTD inhibits ligand signaling at a level equal to or higher than that of Fc fusion proteins containing WT TACI domains.

Example 3. Evaluation of the bioactivity of TACI-containing molecules against TACI-mediated stimuli and their blocking effect on TACI.

Functional characterization of TACI-containing WT or vTD proteins for blocking APRIL or BAFF-mediated ligand signaling via the TACI receptor in Jurkat/NF-κB/TACI cells was evaluated using cell line-based bioassays described in Example 2. APRIL or BAFF-mediated ligand signaling was quantified by monitoring luciferase production in cells. Binding to the TACI-Fc fusion containing the vTD shown in SEQ ID NO: 26 (26 TACI CRD2-Fc) was evaluated. For comparison, WT TACI-Fc containing only the CRD2 domain of TACI (13 TACI CRD2-Fc) was also evaluated.

As shown in Figure 3, the exemplary TACI vTD demonstrates increased inhibition of both human APRIL and BAFF. As shown in Figure 4, the exemplary TACI vTD-Fc molecule inhibits ligand signaling in mouse APRIL and BAFF. In summary, the results demonstrate the ability of TACI vTD molecules to block TACI-mediated ligand signaling in APRIL and BAFF.

In another similar study, the ability of the exemplary generated molecule as described in Example 1 to block APRIL or BAFF-mediated ligand signaling in Jurkat/NF-κB/TACI cells was evaluated. For comparison, a control molecule containing a wild-type TACI ECD fused with the Fc sequence shown in SEQ ID NO:73 was generated. In one control, the fusion protein contained WTTACI (TACI 30-110, SEQ ID NO:130; corresponding to the TACI ECD portion in teltascept, SEQ ID NO:132). In another control, the fusion protein contained WT TACI (TACI 13-118, SEQ ID NO:131, corresponding to the TACI ECD portion in teltascept). The activity was compared with the control molecule. The activity was also compared with the anti-BAFF monoclonal antibody belimumab.

Exemplary TACI molecules (WT or variant TACI vTD) were progressively increased (between 100,000 pM and 32 pM) and added to 2 nM recombinant human APRIL or BAFF, and assays were performed as described above for the Jurkat/NF-κB assay. As shown in Figure 5, the exemplary molecules containing TACI vTD exhibited enhanced APRIL and BAFF blockade greater than that of TACI 30-100-Fc, TACI 13-118-Fc, and belimumab. WT TACI-Fc (13TACI CRD2-Fc), containing only the CRD2 domain of TACI, also exhibited enhanced APRIL blockade greater than that of TACI 30-100-Fc and TACI 13-118-Fc.

These results are consistent with the finding that the minimal CRD2 domain (containing amino acid residues 68-110) exhibits improved APRIL blocking compared to TACI ECD molecules that also contain portions of the CRD1 domain, as present in asceticip and teltasceptip. Table E1 provides the half-maximal inhibitory concentration (IC50) values for the exemplary molecules described in Figure 5 that inhibit APRIL and BAFF-mediated TACI signaling. The relative blocking (Δasceticip) of each tested molecule compared to asceticip is also shown in parentheses.

Example 4. TACI in an in vivo mouse lupus model Evaluation of the activity of vTD-Fc.

This embodiment describes an assessment of the effect of the exemplary TACI vTD-Fc molecule on the immune response in an in vivo mouse (NZB/NZW)F1 spontaneous lupus model. (NZBxNZW)F1 mice spontaneously develop an autoimmune disease very similar to human SLE and are considered one of the best mouse models for this disease. (NZB/NZW)F1 mice begin to have high circulating concentrations of anti-dsDNA antibodies at approximately 20 weeks of age, and the initial clinical signs of the disease are detectable at approximately 23 weeks of age. The mice develop hemolytic anemia, proteinuria, and progressive glomerulonephritis mediated by immune complex deposition in the glomerular basement membrane.

(NZB/NZW)F1 mice were administered 14 mg/kg Fc control or a molar-matched dose of TACI vTD-Fc (26TACI CRD2-Fc) (17 mg/kg) via intraperitoneal (IP) injection twice weekly. Treatment began at group assignment (week 22) and continued until the end of the study. The study ended at 43 weeks of age, but some animals were euthanized earlier in the study when they were dying.

Urine and serum samples were collected at different time points between 20 and 40 weeks of age. Starting at 20 weeks of age, protein concentrations in the urine of all mice in the study were determined weekly using urine analysis test strips (Roche Chemstrip 2GP, catalog number 11895397160). The mean proteinuria score over time in each treatment group is presented in Figure 6A, and the mean percentage change in body weight in each group (weight loss is associated with progressive disease) is plotted in Figure 6B. The percentage of survival of mice in each treatment group is plotted in Figure 6C. Serum titers of anti-double-stranded (ds)DNA IgG were measured by Hooke Laboratories, Inc. (Lawrence, MA) using their in-house kit, and the results are presented in Figure 6D. Blood urea nitrogen (BUN) levels were increased in these mice with progressive disease. BUN levels in each treatment group at the end of the study (or at the time of premature death of mice) are shown in Figure 6E. Statistical analysis was performed using Student's t-test; **** indicates p < 0.0001 and *** indicates p = 0.0008.

At termination, kidneys were collected from each mouse and histologically analyzed in repeated periodic acid Schiff (PAS) stained sections using the criteria described in Alperovich G et al., 2007. Lupus 16:18-24. All kidney sections were blinded and analyzed by pathologists unaware of treatment and clinical scores. Glomerular lesions (mesangial dilatation, intracapillary proliferation, glomerular deposition, and extracapillary proliferation) and tubulointerstitial lesions (interstitial infiltration, tubular atrophy, and interstitial fibrosis) were semi-quantitatively analyzed and graded using a 0-3 scoring system, where 0 = no change, 1 = mild change, 2 = moderate change, and 3 = severe change. The total histological score for each mouse was calculated as the sum of individual scores (maximum total score 21). Kidney scores for total glomerular lesions, total tubular and interstitial lesions, and total nephrotic lesions are shown in Figure 6F; significantly improved renal histopathology was observed in animals treated with TACI vTD-Fc compared to mice treated with Fc (p<0.0001 compared to the Fc group).

For Figures 6G-6I, at the end of the study, the right kidney was collected from each mouse, weighed, dissected transversely, and frozen in a single optimal cutting temperature compound (OCT) block. The sections were then sectioned and stained with immunohistochemical staining for mouse IgG and mouse complement C3 to assess glomerular IgG and C3 deposition, respectively. Kidney sections were permeated with acetone and stained with either FITC-conjugated rat monoclonal anti-mouse complement component C3 (Cedarlane) diluted 1:25 in primary antibody diluent (Leica Biosystems) or AF594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) diluted 1:200 in primary antibody diluent. Glomerular deposition of IgG and C3 was analyzed using a semi-quantitative scoring system of 0 to 4, based on the method described by Kelkka et al. (2014) AntioxidRedox Signal. 21:2231-45, where 0 = no deposition, 1 = mild mesangial deposition, 2 = significant mesangial deposition, 3 = mesangial and mild capillary deposition, and 4 = severe mesangial and mesangial capillary deposition. Significantly reduced glomerular IgG and C3 were observed in animals treated with 26TACI CRD2-Fc compared to Fc control mice (p < 0.0001 for IgG compared to the Fc control; and p = 0.0005 for C3); statistically significant differences in the data were analyzed using Student's t-test.

The results confirmed that in the (NZB/NZW)F1 mouse SLE model, TACI vTD-Fc significantly suppressed proteinuria, maintained body weight, enhanced overall survival, reduced anti-dsDNA autoantibodies and BUN, reduced IgG and C3 renal deposition, and prevented or improved nephropathy. The exemplary molecule also effectively reduced subsets of B cells and T cells, including plasma cells, follicular helper T cells, germinal center cells, and memory T cells (data not shown), in the spleen and lymph nodes of these mice.

Example 5: Evaluation of the activity of TACI 13-118-Fc with the addition of the identified mutation.

The effects of the TACI mutations identified in Example 1 (see Table 1) were evaluated to determine their ability to regulate the activity of Fc fusion proteins containing longer TACIECD sequences (containing both CRD1 and CRD2 domains). In this example, exemplary mutations K77E, F78Y, and Y102D were introduced into reference TACI ECD 13-118, which are fused with the exemplary Fc sequence shown in SEQ ID NO:73. The activity was compared with TACIvTD-Fc fusion proteins containing only the CRD2 domain with the same mutation (shown in SEQ ID NO:26) or with WT TACI (30-110, SEQ ID NO:130; corresponding to the TACI ECD portion in asceticip, SEQ ID NO:132), each of which is also fused with the Fc sequence shown in SEQ ID NO:73. Blockage of APRIL or BAFF-mediated ligand signaling via the TACI receptor in Jurkat/NF-κB/TACI cells was evaluated using cell line-based bioassays described in Example 2. Quantification of APRIL or BAFF-mediated ligand signaling via the TACI receptor was achieved by monitoring luciferase production in cells.

As shown in Figure 7, the introduction of K77E, F78Y, and Y102D mutations into TACI 13-118ECD to generate variants (K77E/F78Y/T102D) of TACI 13-118, relative to the corresponding WT TACI 13-118ECD (diamond) or the alternative ECD control WT TACI 30-110 (upper triangle), improves APRIL and BAFF blocking (respectively). However, even when these mutations are incorporated into TACI 13-118ECD, shorter variants of TACI with the same mutations but containing only the CRD2 domain (vTD shown in SEQ ID NO:26) of TACI exhibit the greatest APRIL and BAFF blocking (lower triangle) in this assay. These results confirm that the smallest CRD2-containing domain confers improved activity in blocking APRIL and BAFF-mediated TACI signaling; however, the mutant K77E/F78Y/Y102D further enhances APRIL and BAFF blocking in the variant TACI ECD incorporating the mutant.

Table E2 provides the half-maximal inhibitory concentration (IC50) values for the exemplary molecules described in Figure 7 that inhibit APRIL and BAFF-mediated TACI signaling. A comparison of each molecule with the WT TACI-Fc control (Δ acecept) is also shown.

Example 6: Comparative evaluation of TACI vTD-Fc in an in vivo KLH immune model

This embodiment describes the assessment of the effect of a single-domain Fc fusion protein (described in Example 1) of an exemplary test on the immune response to keyhole cyanin (KLH) in mice. A mouse KLH immune model can be used to evaluate the effect of immunomodulatory molecules on antigen-specific responses to the T-cell-dependent antigen KLH following one or two injections of KLH. Two injections of KLH (each at least 7 days apart) provide a model that can evaluate both the primary immune response following the first KLH injection and the secondary immune response over a time period following the second injection. This embodiment describes a study evaluating the activity of various molecules containing a single TACI domain (such as soluble wild-type (WT) or variant TACI vTD formatted as an Fc fusion) in response to two KLH injections (on study days 0 and 12) without adjuvant. These test articles were compared with control proteins administered at molar-matched levels of Fc isotypes. The activity of the test articles observed in the mouse KLH model can generally predict the immunomodulatory effects of the test articles in humans.

To initiate the KLH study, 10-week-old female C57/BL6NJ mice (The Jackson Laboratories, Sacramento, California) were randomized to 12 groups of 5 mice each. Mice were administered 0.25 mg of KLH (EMD Millipore, catalog number 374825-25MG) via intraperitoneal (IP) injection on days 0 and 12; the original commercial KLH stock solution was diluted to the appropriate concentration with DuPont phosphate-buffered saline (DPBS) prior to injection. Mice were administered the test sample via IP injection as outlined in Table E3 (on days 4 and 11). Six mice remained untreated/uninjected as initial controls (group 13). Serum samples were collected on days 5 (24 h after dose 1), 12 (24 h after dose 2/24 h before KLH booster), and 20 to evaluate drug exposure, ADA, and/or anti-KLH antibody levels. One animal in group 10 received an incomplete dose of the test sample and was therefore removed from the study.

N/A = Not applicable

On day 20, all mice were anesthetized with isoflurane and blood was collected into serum separator tubes. Mice were euthanized, and their spleens were removed, weighed, and placed in DPBS on ice. Whole blood was centrifuged, serum was collected, and stored at -80°C until analysis for anti-KLH levels was performed by enzyme-linked immunosorbent assay (ELISA). Spleens were prepared into single-cell suspensions, and red blood cells (RBCs) were lysed using RBC lysis buffer (Biolegend, catalog number 420301) according to the manufacturer's instructions. Cells in each sample were counted using acridine orange/propidium iodide (AO/PI) staining (Nexcelom, catalog number CS2-0106-5mL) with dual fluorescence activity.

Each spleen sample was then stained for flow cytometry analysis of the immune cell subset using the following method: 1 x ^10⁶ live cells were placed into the wells of two 96-well plates (Corning, catalog number 3797; one plate for the B cell-specific group and one plate for the T cell-specific group), centrifuged at 1500 x g for 10 seconds, the supernatant was removed, and the cell pellet was washed twice with DPBS. The pellet was resuspended in 100 μL of live-dead staining reagent (LIVE/DEAD fixable light green dead cell staining kit, Life Technologies Corp., 1:1000 diluted in DPBS) and incubated in the dark at room temperature for 10 min. After washing twice with flow cytometry buffer (175 μL each time), the tumor pellet was resuspended in Mouse BD Fc Block (1:50 diluted with flow buffer) and incubated in the dark at RT for another 5 min. Without performing any further washing, add 50 μL of the following mixture of flow cytometry antibodies (diluted in flow cytometry buffer) to each well of the B cell or T cell group. For the B cell group, the following antibody combinations were used in the mixture: anti-mouse CD19 BUV395 (clone 1D3, Becton-Dickinson; 1:100), anti-mouse CD138 BV421 (clone 281-2, BioLegend Inc.; 1:100, final concentration), anti-mouse CD3ε BV510 (clone 17A2, BioLegend Inc.; 1:100, final concentration), anti-mouse IgD BV605 (clone 11-26c.2a, BioLegend Inc.; 1:100, final concentration), anti-mouse B220 BV785 (clone RA3-6B2, BioLegend Inc.; 1:100, final concentration), anti-mouse CD95 FITC (clone SA367H8, BioLegend Inc.; 1:100, final concentration), and anti-mouse CD23 PerCP. Cy5.5 (clone B3B4, BioLegend Inc.; 1:100, final concentration), anti-mouse GL7 PE (clone GL7, BioLegend Inc.; 1:100, final concentration), anti-mouse Gr1 PE Cy7 (clone RB6-8C5, BioLegend Inc.; 1:100, final concentration), anti-mouse CD21 APC (clone 7E9, BioLegend Inc.; 1:100, final concentration), and anti-mouse IgM APC Cy7 (clone RMM-1, BioLegend Inc.; 1:100, final concentration). For the T-cell group, the following antibody combinations were used in the mixture: anti-mouse PD-1 BV421 (clone 29F.1A12, BioLegend Inc.; 1:100, final concentration), anti-mouse CD11b BV510 (clone M1/70, BioLegend Inc.; 1:100, final concentration), anti-mouse CD3ε BV605 (clone 145-2C11, BioLegend Inc.; 1:100, final concentration), anti-mouse CD8 BV785 (clone 53-6.7, BioLegend Inc.; 1:100, final concentration), anti-mouse CD44 FITC (clone IM7, BioLegend Inc.; 1:100, final concentration), anti-mouse CD4 PerCP Cy5.5 (clone GK1.5, BioLegend Inc.; 1:100, final concentration), and anti-mouse CD62L PE (clone MEL-14, BioLegend Inc.). The cell mixtures included anti-mouse CXCR5 PE Dazzle (clone L138D7, BioLegend Inc.; 1:100, final concentration), anti-mouse CD25 PE Cy7 (clone PC61.5, BioLegend Inc.; 1:100, final concentration), and anti-mouse CD45 AF700 (clone 30-F11, BioLegend Inc.; 1:100, final concentration). The cells were incubated with one of the antibody mixtures in the dark on ice with gentle mixing for 45 min, followed by two washes with flow cytometry buffer (175 μL each). The cell pellet was resuspended in 200 μL of flow cytometry buffer and collected on an LSRII flow cytometer. Data were analyzed using FlowJo software version 10.2 (FlowJo LLC, USA) and visualized using GraphPad Prism software (version 8.1.2). Key cell subset analysis included: total B cells (B220 ⁺ cells), marginal zone (MZ) B cells (B220 ⁺ , CD19 ⁺ , ^CD23- , ^high CD21, ^high IgM cells), germinal center (GC) B cells (B220 ⁺ , CD19 ⁺ , GL7 ⁺ , CD95 ⁺ cells), T follicular helper (Tfh) cells (CD45 ⁺ , CD3 ⁺ , CD4 ⁺ , PD-1 ⁺ , CD185 ⁺ cells), CD4 ⁺ T effector memory ( _Tem ) cells (CD45 ⁺ , CD3 ⁺ , CD4 ⁺ , CD44 ⁺ , ^CD62L- cells), and CD8 ⁺ _Tem cells (CD45 ⁺ , CD3 ⁺ , CD8 ⁺ , CD44 ⁺ , ^CD62L- cells).

Statistically significant differences between groups were determined using one-way ANOVA and the uncorrected Fisher minimum significance (LSD) multiple comparison test with GraphPad Prism software (version 8.1.2) (p<0.05).

To determine the extent to which the test sample inhibited the KLH-mediated antibody immune response compared to the Fc isotype control (SEQ ID NO:73), the concentration of anti-KLH antibodies in serum samples was evaluated in two ELISA assays. The ELISA assays measured IgM-specific or IgG1-specific anti-KLH levels in serum. Mouse serum samples at various dilutions were incubated in KLH-coated plates, washed, and detected with either 1:2000 goat anti-mouse IgG1:HRP or 1:5000 goat anti-mouse IgM:HRP. Colorimetric development was achieved using a TMB substrate kit (SeraCare), and the ELISA plates were analyzed on an iD3 microplate reader (MolecularDevices LLC). Since no standard curve was available for these assays, optical density (OD) was used to compare anti-KLH antibody levels; a higher OD indicated a higher level of anti-KLH antibodies in the serum sample. For anti-KLH IgM OD levels, data are presented in Figures 10A (primary response) and 10B (secondary response), and statistical analyses performed by one-way ANOVA and uncorrected Fisher's LSD multiple comparison test are presented in Tables E4 and E5, respectively. Anti-KLH IgG1 OD levels are presented in Figures 10C (primary response) and 10D (secondary response), and statistical analyses performed by one-way ANOVA and uncorrected Fisher's LSD multiple comparison test are presented in Tables E6 and E7. The results confirmed that during the primary immune response, each assay significantly reduced serum anti-KLH IgM levels compared to the Fc control treatment. Among all assays, 29TACI-CRD2-Fc (SEQ ID NO:29) showed the largest reduction, while TACI 30-110-Fc and TACI 13-118-Fc treatments had the smallest effects (Figure 10A). For the re-response on day 20, measured 9 days after the second and last doses of the test samples, all test samples except TACI 13-118-Fc induced a significant reduction in anti-KLH IgM levels, with all test samples except TACI 30-110-Fc and TACI 13-118-Fc showing a reduction (Figure 10B). During the primary immunization response, each test sample also significantly reduced anti-KLH IgG1 levels compared to the Fc control, with all test samples except TACI 30-110-Fc and TACI 13-118-Fc still showing the largest reduction (Figure 10C). For the re-response against KLH, all test samples except TACI 30-110-Fc and TACI 13-118-Fc significantly reduced anti-KLH IgG1 levels (Figure 10D). These results indicate that, in this mouse immunization model, most molecules containing TACIvTD are effective in reducing T-cell-dependent antibody immune responses to KLH, with 26TACI CRD2-Fc, 27TACI CRD2-Fc, and 29TACI CRD2-Fc exhibiting the most significant effects.

As shown in Figures 11A and 11B, at the end of the study (day 20), mice treated with all test products except TACI 30-110-Fc or TACI 13-118-Fc had significantly smaller spleens compared to the Fc control mice, as assessed by weight and cell number, respectively (Table E8). Mice treated with each test product also had significantly fewer spleen cells compared to the Fc control group. The smaller spleen indicates a reduction in lymphocytes, which may have an immunomodulatory role in the pathogenesis of autoimmune and inflammatory diseases (particularly those driven by B cells and/or T cells) associated with enhanced immune responses. Statistical analyses of spleen weight and total cell number are shown in Tables E8 and E9, respectively.

Of particular importance to the pathogenesis of autoimmune and inflammatory diseases are the cell types that promote B cell survival and differentiation, antibody production, and T cell effector memory. These cell types include, but are not limited to, the following: total B cells, marginal zone (MZ) B cells, germinal center (GC) B cells, T follicular helper (Tfh) cells, and CD4 ⁺ and CD8 ⁺ T effector memory ( _Tem ) cells. Expected mechanisms of action include the efficacy of therapeutics that reduce these cell types in the treatment of a variety of autoantibody-mediated diseases. Compared with the other treatment groups, treatment with any TACI vTD-Fc assay significantly reduced the number of multiple splenic B cell subsets, including effects on ^the following: transition-2 (B220 ⁺ CD19 ⁺ CD23 ⁺ CD21 ^high IgM), follicles (B220 ⁺ CD19 ⁺ CD23 ⁺ CD21 ⁺ IgM ⁺ ), marginal zone (B220 ⁺ CD19 ⁺ CD23 ^negative CD21 ^{high IgM} ), germinal center (B220 ⁺ CD19 ⁺ GL7 ⁺ CD95 ⁺ ), and plasma cells (B220 ^low CD19 ⁺ CD138 ^high ) (Figures 12 and 13). These TACI vTD-molecules were as effective (not shown) or more effective than the two WT TACI-Fc molecules (TACI 13-188-Fc and TACI 30-110-Fc) in reducing the percentage (not shown) or number of these populations important for B cell survival, differentiation, and antibody production. Statistical analysis of flow cytometry data from day 20 spleen cells is shown in Tables E10–E28.

Compared to the Fc control group, the spleen CD3+, CD4+, or CD8+ T cell populations were largely unaffected by the six assays containing TACIvTD (Figs. 14A-14C), and Tcm and Tem memory T cells were unaffected compared to the Fc control group (Fig. 15). All assays reduced the number of follicular helper T cells (CD45 ⁺ , CD3 ⁺ , CD4 ⁺ , PD-1 ⁺ , CD185 ⁺ ) compared to the Fc control; these follicular helper T cells interact with B cells in the germinal centers and are significant contributors to T cell-dependent antibody responses (Fig. 14D).

In summary, these results indicate that single-domain Fc fusion molecules containing TACI vTD that inhibit B cell and/or T cell activity can reduce in vivo immune responses and changes in cell subsets mediated by the T cell-dependent antigen KLH (i.e., changes in serum anti-KLH levels and immune cell subsets). These results are consistent with the evaluation of single-domain TACI B cell inhibitory molecules as clinical therapeutics for the treatment of autoimmune and inflammatory diseases involving overactive lymphocytes.

Example 7. Evaluation of the bioactivity of TACI-containing molecules against TACI-mediated stimuli and their blocking effect on TACI.

Additional TACI vTDs are generated, containing one or more mutations present in the exemplary TACI vTDs shown in SEQ ID NO:26 (K77E, F78Y, Y102D), SEQ ID NO:27 (Q75E, R84Q), or SEQ ID NO:29 (K77E, A101D, Y102D). Combinations of single, double, and triple mutations from the mutations of Q75E, K77E, F78Y, R84Q, A101D, and Y102D are generated. The resulting TACI vTDs are further formatted into TACI vTD-Fc fusion proteins having an Fc domain. The exemplary generated Fc fusion protein is generated substantially as described in Example 1. In summary, to generate a recombinant immunomodulatory protein as an Fc fusion protein, encoding DNA was generated to encode the following protein: a variant TACI domain, followed by a 7-amino acid linker (GSGGGGS; SEQ ID NO:74), followed by a human IgG1 effector Fc sequence containing mutants L234A, L235E, and G237A according to the Eu indexing system for immunoglobulin proteins (SEQ ID NO:73). For comparison, the following molecules were also tested: (1) WT TACI(68-110)-Fc (TACI 68-110, SEQ ID NO:13, TACI-Fc SEQ ID NO:171); and (2) TACI-Fc having the exemplary mutants K77E, F78Y, and Y102D introduced in reference TACI ECD 13-118, which was fused with the exemplary Fc sequence shown in SEQ ID NO:73; see Example 5. Other controls include: (3) WT TACI(13-118)-Fc (TACI 13-118, SEQ ID NO:131; corresponding to the TACI ECD portion in teltascip); (4) WT TACI(30-110)-Fc (TACI 30-110, SEQ ID NO:130; corresponding to the TACIECD portion in asceticip, SEQ ID NO:132); (5) BAFF-R ECD and (6) belimumab.

The effects of the generated molecules on blocking APRIL or BAFF-mediated ligand signaling via the TACI receptor in Jurkat/NF-κB/TACI cells were evaluated, essentially as described in Example 2. Exemplary TACI vTD-Fc molecules were incremented from 100,000 to 6 pM and mixed with 30 nM human APRIL or 10 nM human BAFF, then added to Jurkat/NF-κB/TACI cells after 30 minutes. APRIL or BAFF-mediated ligand signaling was quantified by monitoring luciferase production in the cells.

The half-maximal inhibitory concentrations (IC50) of the molecules used as exemplary tests are summarized in Table E29. The percentage change in IC50 compared to the reference control WT TACI(68-110)-Fc (TACI 68-110, SEQ ID NO:13, TACI-Fc SEQ ID NO:171) is shown in parentheses (ΔWT). Similar to the results described above, wild-type minimal CRD2 WT TACI(68-110)-Fc exhibits superior APRIL and BAFF blocking compared to control molecules used in other tests, including those with sequences similar to teltascipes and acetaxel. As indicated, certain mutations and combinations of mutations were associated with a further significant increase in the ability to block APRIL or BAFF-mediated ligand signaling. In summary, the results demonstrate the ability of TACI vTD molecules to block APRIL and BAFF TACI-mediated ligand signaling.

Example 8. Evaluation in a Sjögren's syndrome model in non-obese diabetic mice

This embodiment describes the evaluation of an exemplary single-domain 26-TACI-vTD Fc fusion protein (TACI vTD SEQ ID NO:26; Fc fusion SEQ ID NO:167) in a short-term in vivo model of Sjögren's syndrome in NOD mice, including assessments of sialodenitis, serum levels of the test molecule, and pancreatitis.

The Sjögren's syndrome model was induced in female, diabetic NOD/ShiLtJ mice (approximately 6 weeks old) through repeated administration of anti-mPD-L1 antibody. Specifically, 0.1 mg of anti-mPD-L1 antibody was administered intraperitoneally on days 0, 2, 4, and 6. The test fusion protein was administered on days 0, 2, and 4 according to Table E30 below.

Abbreviations: IP = Intraperitoneal (IP); mg = Milligram; n/a = Not applicable

Blood samples (2-5 μL) were collected from the tail vein of mice on days 7, 8, 9, and 10. The samples were placed on ReliOnPrime blood glucose test strips, and blood glucose (mg/dL) was measured using the ReliOn Prime blood glucose testing system. On day 10, mice were sacrificed, and serum, submandibular gland (SMG), and pancreas were collected and analyzed.

The left stromal tumor (SMG) and pancreas were removed, dissected from adjacent lymph nodes, and placed in neutral buffered formalin (NBF) for approximately 72 hours, after which they were transferred to 70% ethanol. The fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) on slides.

The scoring system used to assess the severity of sialadenitis was based on Nandula et al. 2011 (Table 6 therein; reproduced as Table E31), and for pancreatitis it was based on Gutierrez et al. 2014 (Table 7 therein; reproduced as Table E32).

Student's t-test was used to determine statistically significant differences in histological scores between groups. Statistical analysis was performed using GraphPad software (version 8.1.2), and for all statistical tests, a p-value < 0.05 was considered statistically significant.

Treatment with the exemplary 26-TACI-CRD2 Fc fusion protein reduced the incidence of sialiditis (Figure 16A) and resulted in significantly lower mean histological scores compared to the Fc control (p<0.01) (Figure 16B). These results are consistent with the finding that in this Sjögren's syndrome model, treatment with the test molecule in NOD mice injected with anti-PD-L1 reduced both the incidence and severity of sialiditis.

The overall incidence of islet inflammation and the severity of islet inflammation after treatment with the tested molecules in these diabetic mice are shown in Figures 17A and 17B. The 26-TACI-CRD2 Fc fusion protein significantly reduced the severity of islet inflammation, as assessed by histological analysis (Figure 17B).

In summary, these results demonstrate that treatment with the tested exemplary TACI-Fc molecule reduces the incidence and severity of sialorrhea in this mouse model of Sjögren's syndrome. These findings suggest the potential of TACI molecules for therapeutic use in treating Sjögren's syndrome, and the potential of TACI-CTLA-4 multidomain stacked molecules as a therapeutic agent to influence the onset of type 1 diabetes in humans.

Example 9. Evaluation of exemplary monomers and tetramer constructs.

Additional TACI-Fc fusion proteins containing one (monomer) or four (tetrameric barbell and tetrameric tandem) TACI vTD domains were generated using WT TACI of the following different lengths: 68-110 (shown in SEQ ID NO:13), 29-110 (shown in SEQ ID NO:1), or 13-118 (shown in SEQ ID NO:131), and the TACI vTDs (K77E, F78Y, Y102D) shown in SEQ ID NO:26. The monomeric and tetrameric TACI WTs and TACI vTDs were formatted as TACI WT and TACI vTD-Fc fusion proteins with Fc domains. Exemplary generated Fc fusion proteins were generated substantially as described in Example 1 and are described in Tables E33A-E33C.

In short, to generate a recombinant monomeric immunomodulatory protein as a single-stranded Fc fusion protein, encoding DNA is generated to encode the following protein: a WT TACI or variant TACI domain, followed by a 12-amino acid linker (GSGGGGSGGGGS; SEQ ID NO:194), followed by the single-stranded Fc (scFc) shown in SEQ ID NO:218 (consisting of a human IgG1 effector Fc sequence containing mutants L234A, L235E, and G237A according to the Eu indexing system for immunoglobulin proteins (SEQ ID NO:73), followed by a (GGGGS) ₁₃ linker (SEQ ID NO:195), followed by a second human IgG1 effector Fc sequence (containing mutants L234A, L235E, and G237A according to the Eu indexing system for immunoglobulin proteins). A long linker (e.g., shown in SEQ ID NO:195) connects the C-terminus of the first Fc unit to the N-terminus of the second Fc unit, forming scFc. The generated molecules are summarized in Table E33A.

To generate a recombinant tetrameric immunomodulatory protein as an Fc fusion protein, the protein was generated in the following different formats:

In one format, coding DNA is generated to encode three different protein forms as follows: WT TACI (SEQ ID NO:198): WT TACI domain SEQ ID NO:13, followed by adapter (G4S)4 SEQ ID NO:84; followed by WT TACI domain SEQ ID NO:13; followed by adapter GSGGGGS SEQ ID NO:74; followed by human IgG1 effector Fc sequence containing mutants L234A, L235E, and G237A according to the Eu indexing system for immunoglobulin proteins (SEQ ID NO:73).

In one format, coding DNA is generated to encode three different protein forms as follows: WT TACI (SEQ ID NO:202): WT TACI domain SEQ ID NO:13, followed by adapter GSGGGGS SEQ ID NO:74; followed by human IgG1 effector Fc sequence containing mutants L234A, L235E and G237A according to the Eu indexing system for immunoglobulin proteins (SEQ ID NO:73), followed by adapter (G4S)4 SEQ ID NO:84, followed by WT TACI domain SEQ ID NO:13.

In one format, coding DNA is generated to encode three different protein forms: TACI vTD barbell (SEQ ID NO:201): TACI vTD shown in SEQ ID NO:26, followed by the adapter GSGGGGS SEQ ID NO:74; followed by the human IgG1 effector Fc sequence containing mutants L234A, L235E, and G237A according to the Eu indexing system for immunoglobulin proteins (SEQ ID NO:73), followed by the adapter (G4S)4 SEQ ID NO:84, followed by TACI vTD shown in SEQ ID NO:26.

A. Bioactivity of exemplary multi-domain molecules

In one experiment, Jurkat/NF-κB/TACI reporter cells were used, essentially as described in Example 1, to assess the blocking effect of the exemplary molecules shown in Tables E33A-E33C on APRIL or BAFF-mediated signaling. Activity was assessed for inhibition of soluble BAFF (trimer) or for inhibition of the oligomer of twenty BAFF trimers (BAFF 60-mer). Table E34 provides values for the half-maximal inhibitory concentration (IC50) for inhibiting APRIL and BAFF-mediated TACI signaling. In some cases, the proteins tested were not compared to their parental WT controls and are indicated as (-) in the table below. The results in Table E34 confirm that all generated formulations block BAFF and APRIL binding.

This invention is not intended to be limited to the specific embodiments disclosed herein, which are provided for example to illustrate various aspects of the invention. Various modifications to the compositions and methods will become apparent from the description and teaching herein. Such changes may be practiced without departing from the true scope and spirit of this disclosure, and such changes are intended to fall within the scope of this disclosure.

sequence list

<110> Takayama Immunoscience Co., Ltd.

<120> APRIL and BAFF inhibitory immunomodulatory proteins and their usage

<130> 761612003840

<140> Not yet allocated

<141> Submitted at the same time

<150> 63/080,643

<151> 2020-09-18

<150> 63/034,361

<151> 2020-06-03

<150> 63/022,373

<151> 2020-05-08

<160> 239

<170> PatentIn version 3.5

<210> 1

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI WT ECD

<400> 1

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 2

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L82P ECD

<400> 2

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Pro Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 3

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI D85E, K98T ECD

<400> 3

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Glu Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Thr Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 4

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI I87L, K98T ECD

<400> 4

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Leu Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Thr Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 5

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R60G, Q75E, L82P ECD

<400> 5

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Gly

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Glu Gly

35 40 45

Lys Phe Tyr Asp His Pro Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 6

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R60G, C86Y ECD

<400> 6

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Gly

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Tyr Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 7

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI A101D ECD

<400> 7

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Asp Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 8

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI C86Y ECD

<400> 8

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Tyr Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 9

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI W40R, L82P, F103Y ECD

<400> 9

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Arg Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Pro Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Tyr Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 10

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI W40R, Q59R, T61P, K98T ECD

<400> 10

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Arg Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Arg Arg

20 25 30

Pro Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Thr Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 11

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L82P, I87L ECD

<400> 11

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Pro Leu Arg Asp Cys Leu Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 12

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI G76S, P97S ECD

<400> 12

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Ser

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Ser Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 13

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI WT ECD

<400> 13

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 14

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI D85V ECD

<400> 14

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Val Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 15

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI E74V ECD

<400> 15

Ser Leu Ser Cys Arg Lys Val Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 16

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84L ECD

<400> 16

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Leu Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 17

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, R84L, F103Y ECD

<400> 17

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Leu Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Tyr Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 18

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Y79F, Q99E ECD

<400> 18

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Phe Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Glu

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 19

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Y79F ECD

<400> 19

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Phe Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 20

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84G ECD

<400> 20

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gly Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 21

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L83S, F103S ECD

<400> 21

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Ser Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Ser Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 22

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L82H ECD

<400> 22

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His His Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 23

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI A101D ECD

<400> 23

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 24

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, R84Q ECD

<400> 24

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 25

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, A101D ECD

<400> 25

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 26

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, F78Y, Y102D ECD

<400> 26

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 27

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Q75E, R84Q ECD

<400> 27

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 28

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Q75R, R84G, I92V ECD

<400> 28

Ser Leu Ser Cys Arg Lys Glu Arg Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gly Asp Cys Ile Ser Cys Ala Ser Val Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 29

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, A101D, Y102D ECD

<400> 29

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 30

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84Q ECD

<400> 30

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 31

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84Q, S88N, A101D ECD

<400> 31

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Asn Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 32

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E ECD

<400> 32

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 33

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84Q, F103V ECD

<400> 33

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Val Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 34

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, Q95R, A101D ECD

<400> 34

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Arg His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 35

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI I87M, A101D ECD

<400> 35

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Met Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 36

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI WT ECD

<400> 36

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 37

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI L82P ECD

<400> 37

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ctttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 38

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI D85E, K98T ECD

<400> 38

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga atgtataagc 180

tgtgcgtcta tttgtggaca acaccctaca caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 39

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI I87L, K98T ECD

<400> 39

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctgtttaagc 180

tgtgcgtcta tttgtggaca acaccctaca caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 40

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R60G, Q75E, L82P ECD

<400> 40

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caagggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagga aggcaaattt tacgaccatc ctttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 41

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R60G, C86Y ECD

<400> 41

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caagggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctacataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 42

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI A101D ECD

<400> 42

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgatt atttctgtga aaataagctt 240

cgatct 246

<210> 43

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI C86Y ECD

<400> 43

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctatataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 44

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI W40R, L82P, F103Y ECD

<400> 44

gtggcaatgc gctcatgccc agaggaacaa tatcggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ctttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggtca acaccctaaa caatgtgcttattactgtga aaataagctt 240

cgatct 246

<210> 45

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI W40R, Q59R, T61P, K98T ECD

<400> 45

gtggcaatgc gctcatgccc agaggaacaa tatcggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc cgaaggccat gcgcagcatt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaca caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 46

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI L82P, I87L ECD

<400> 46

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ctttgcgaga ctgtttaagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 47

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI G76S, P97S ECD

<400> 47

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aagcaaattt tacgaccatc ttttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggtca acactctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 48

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI WT ECD

<400> 48

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 49

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI D85V ECD

<400> 49

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agtctgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 50

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI E74V ECD

<400> 50

agtctctctt gcaggaaagt gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 51

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R84L ECD

<400> 51

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgct agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 52

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E, R84L, F103Y ECD

<400> 52

agtctctctt gcaggaaaga gcaaggcgaa ttttacgacc atcttttgct agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttattactg tgaaaataag 120

cttcgatct 129

<210> 53

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI Y79F, Q99E ECD

<400> 53

agtctctctt gcaggaaaga gcaaggcaaa tttttcgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaagaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 54

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI Y79F ECD

<400> 54

agtctctctt gcaggaaaga gcaaggcaaa tttttcgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 55

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R84G ECD

<400> 55

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttggg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 56

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI L83S, F103S ECD

<400> 56

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc tttcgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt attcctgtga aaataagctt 240

cgatct 246

<210> 57

<211> 246

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI L82H ECD

<400> 57

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc atttgcgaga ctgtataagc 180

tgtgcgtcta tctgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatct 246

<210> 58

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI A101D ECD

<400> 58

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg attatttctg tgaaaataag 120

cttcgatct 129

<210> 59

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E, R84Q ECD

<400> 59

agtctctctt gcaggaaaga gcaaggcgaa ttttacgacc atcttttgca agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 60

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E, A101D ECD

<400> 60

agtctctctt gcaggaaaga gcaaggcgaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg attatttctg tgaaaataag 120

cttcgatct 129

<210> 61

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E, F78Y, Y102D ECD

<400> 61

agtctctctt gcaggaaaga gcaaggcgaa tattacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg ctgatttctg tgaaaataag 120

cttcgatct 129

<210> 62

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI Q75E, R84Q ECD

<400> 62

agtctctctt gcaggaaaga ggaaggcaaa ttttacgacc atcttttgca agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 63

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI Q75R, R84G, I92V ECD

<400> 63

agtctctctt gcaggaaaga gcgaggcaaa ttttacgacc atcttttggg agactgtata 60

agctgtgcgt ctgtttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 64

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E, A101D, Y102D ECD

<400> 64

agtctctctt gcaggaaaga gcaaggcgaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg atgatttctg tgaaaataag 120

cttcgatct 129

<210> 65

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R84Q ECD

<400> 65

agtctctctt gcaggaaaga gcaaggcaaa ttttatgacc atcttttgca agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 66

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R84Q, S88N, A101D ECD

<400> 66

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgca agactgtata 60

aactgtgcgt ctatatgtgg acaacaccct aaacaatgtg attatttctg tgaaaataag 120

cttcgatct 129

<210> 67

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E ECD

<400> 67

agtctctctt gcaggaaaga gcaaggcgaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatct 129

<210> 68

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI R84Q, F103V ECD

<400> 68

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgca agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatgtctg tgaaaataag 120

cttcgatct 129

<210> 69

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI K77E, Q95R, A101D ECD

<400> 69

agtctctctt gcaggaaaga gcaaggcgaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acgacaccct aaacaatgtg attatttctg tgaaaataag 120

cttcgatct 129

<210> 70

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI I87M, A101D ECD

<400> 70

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtatg 60

agctgtgcgt caatttgtgg acaacaccct aaacaatgtg attatttctg tgaaaataag 120

cttcgatct 129

<210> 71

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG1 Fc

<400> 71

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 72

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> FLAG tags

<400> 72

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 73

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc C220S/L234A/L235E/G237A/K447del

<400> 73

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 74

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 74

Gly Ser Gly Gly Gly Gly Ser

1 5

<210> 75

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc C220S/L234A/L235E/G237A/E356D/M358L

<400> 75

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 76

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 76

Gly Ser Gly Gly Ser

1 5

<210> 77

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 77

Gly Gly Gly Gly Ser

1 5

<210> 78

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 78

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 79

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 79

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 80

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 80

Gly Gly Gly Gly Ser Ser Ala

1 5

<210> 81

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc (E356D and M358L)

<400> 81

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 82

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc (C5S (C220S), R77C, (R292C), N82G (N297G), V87C (V302C))

<400> 82

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys Glu Glu Gln

65 70 75 80

Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 83

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc with C220S/L234A/L235E/G237A

<400> 83

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 84

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 84

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 85

<211> 323

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc (G4S)2 TACI (516)

<400> 85

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu

245 250 255

Leu Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln

260 265 270

Arg Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln

275 280 285

Gly Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser

290 295 300

Ile Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys

305 310 315 320

Leu Arg Ser

<210> 86

<211> 294

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc (G4S)4 TACI (541)

<400> 86

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Leu Ser Cys Arg

245 250 255

Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu Arg Asp Cys Ile Ser

260 265 270

Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln Cys Ala Asp Phe Cys

275 280 285

Glu Asn Lys Leu Arg Ser

290

<210> 87

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 87

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

20 25 30

<210> 88

<211> 293

<212> PRT

<213> Artificial Sequence

<220>

<223> Full Length TACI

<400> 88

Met Ser Gly Leu Gly Arg Ser Arg Arg Gly Gly Arg Ser Arg Val Asp

1 5 10 15

Gln Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly Val Ala Met Arg

20 25 30

Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met

35 40 45

Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr Cys Ala Ala

50 55 60

Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp

65 70 75 80

His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His

85 90 95

Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Pro Val

100 105 110

Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser Gly Glu Val Glu Asn

115 120 125

Asn Ser Asp Asn Ser Gly Arg Tyr Gln Gly Leu Glu His Arg Gly Ser

130 135 140

Glu Ala Ser Pro Ala Leu Pro Gly Leu Lys Leu Ser Ala Asp Gln Val

145 150 155 160

Ala Leu Val Tyr Ser Thr Leu Gly Leu Cys Leu Cys Ala Val Leu Cys

165 170 175

Cys Phe Leu Val Ala Val Ala Cys Phe Leu Lys Lys Arg Gly Asp Pro

180 185 190

Cys Ser Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser Pro Ala Lys Ser

195 200 205

Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val Ser Thr Ser Pro

210 215 220

Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu Cys Arg Ala Pro

225 230 235 240

Thr Gln Glu Ser Ala Val Thr Pro Gly Thr Pro Asp Pro Thr Cys Ala

245 250 255

Gly Arg Trp Gly Cys His Thr Arg Thr Thr Val Leu Gln Pro Cys Pro

260 265 270

His Ile Pro Asp Ser Gly Leu Gly Ile Val Cys Val Pro Ala Gln Glu

275 280 285

Gly Gly Pro Gly Ala

290

<210> 89

<211> 230

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc K S139C, D141E, L143M, T151W

<400> 89

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro

225 230

<210> 90

<211> 230

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc H Y134C, D141E, L143M, T151S, L153A

<400> 90

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro

225 230

<210> 91

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 91

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 92

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L82P ECD

<400> 92

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Pro Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 93

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI D85E, K98T ECD

<400> 93

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Glu Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Thr Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 94

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI I87L, K98T ECD

<400> 94

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Leu Ser Cys Ala Ser Ile Cys Gly Gln His Pro Thr Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 95

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI A101D ECD

<400> 95

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 96

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI C86Y ECD

<400> 96

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Tyr Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 97

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L82P, I87L ECD

<400> 97

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Pro Leu

1 5 10 15

Arg Asp Cys Leu Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 98

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI G76S, P97S ECD

<400> 98

Ser Leu Ser Cys Arg Lys Glu Gln Ser Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Ser Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 99

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L83S, F103S ECD

<400> 99

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Ser

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Ser Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 100

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI L82H ECD

<400> 100

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His His Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 101

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI D85V ECD

<400> 101

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Val Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 102

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI E74V ECD

<400> 102

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Val Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 103

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84L ECD

<400> 103

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Leu Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 104

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, R84L, F103Y ECD

<400> 104

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Phe Tyr Asp His Leu Leu Leu Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Tyr Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 105

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Y79F, Q99E ECD

<400> 105

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Phe Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Glu Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 106

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Y79F ECD

<400> 106

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Phe Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 107

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84G ECD

<400> 107

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Gly Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 108

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI A101D ECD

<400> 108

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Asp Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 109

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, R84Q ECD

<400> 109

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Phe Tyr Asp His Leu Leu Gln Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 110

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, A101D ECD

<400> 110

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Asp Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 111

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, F78Y, Y102D ECD

<400> 111

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Tyr Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Asp Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 112

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Q75E, R84Q ECD

<400> 112

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Glu Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Gln Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 113

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Q75R, R84G, I92V ECD

<400> 113

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Arg Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Gly Asp Cys Ile Ser Cys Ala Ser Val

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 114

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, A101D, Y102D ECD

<400> 114

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Asp Asp Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 115

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84Q ECD

<400> 115

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Gln Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 116

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84Q, S88N, A101D ECD

<400> 116

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Gln Asp Cys Ile Asn Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Asp Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 117

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E ECD

<400> 117

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 118

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI R84Q, F103V ECD

<400> 118

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Gln Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Val Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 119

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI K77E, Q95R, A101D ECD

<400> 119

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Glu Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Arg His Pro Lys Gln Cys Asp Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 120

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI I87M, A101D ECD

<400> 120

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Met Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Asp Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser

<210> 121

<211> 660

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4 113 GSG4S Fc K/ BCMA 357 GSG4S Fc H

<400> 121

Lys Ala Met His Val Ala Gln Pro Ala Val Val Phe Ala Ser Ser His

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Trp Lys Ala Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Thr Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Gln Met Tyr Pro Pro Pro Tyr Leu Leu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys

130 135 140

Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu

145 150 155 160

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

165 170 175

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

180 185 190

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

195 200 205

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

210 215 220

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

225 230 235 240

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

245 250 255

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

260 265 270

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

275 280 285

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

290 295 300

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

305 310 315 320

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

325 330 335

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

340 345 350

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Leu Gln Met Ala

355 360 365

Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser Leu Leu Tyr Ala Cys

370 375 380

Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr Pro Pro Leu Thr Cys

385 390 395 400

Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser Val Lys Gly Thr Asn

405 410 415

Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Glu Pro

420 425 430

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

435 440 445

Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

450 455 460

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

465 470 475 480

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

485 490 495

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

500 505 510

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

515 520 525

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

530 535 540

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

545 550 555 560

Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

565 570 575

Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile

580 585 590

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

595 600 605

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys

610 615 620

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

625 630 635 640

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

645 650 655

Ser Leu Ser Pro

660

<210> 122

<211> 166

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI ECD (1-166)

<400> 122

Met Ser Gly Leu Gly Arg Ser Arg Arg Gly Gly Arg Ser Arg Val Asp

1 5 10 15

Gln Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly Val Ala Met Arg

20 25 30

Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met

35 40 45

Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr Cys Ala Ala

50 55 60

Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp

65 70 75 80

His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His

85 90 95

Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Pro Val

100 105 110

Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser Gly Glu Val Glu Asn

115 120 125

Asn Ser Asp Asn Ser Gly Arg Tyr Gln Gly Leu Glu His Arg Gly Ser

130 135 140

Glu Ala Ser Pro Ala Leu Pro Gly Leu Lys Leu Ser Ala Asp Gln Val

145 150 155 160

Ala Leu Val Tyr Ser Thr

165

<210> 123

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 1xEAAAK

<400> 123

Glu Ala Ala Ala Lys

1 5

<210> 124

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> 2xEAAAK

<400> 124

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10

<210> 125

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> 3xEAAAK

<400> 125

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

<210> 126

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> 4xEAAAK

<400> 126

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys

20

<210> 127

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> 5xEAAAK

<400> 127

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys

20 25

<210> 128

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Pound Fc

<400> 128

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 129

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> 臼Fc

<400> 129

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 130

<211> 81

<212> PRT

<213> Artificial Sequence

<220>

<223> Asesip (TACI section, 30-110)

<400> 130

Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly

1 5 10 15

Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr

20 25 30

Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys

35 40 45

Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys

50 55 60

Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg

65 70 75 80

Ser

<210> 131

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Tetracicept (TACI section, 13-118)

<400> 131

Ser Arg Val Asp Gln Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly

1 5 10 15

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

20 25 30

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

35 40 45

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

50 55 60

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

65 70 75 80

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

85 90 95

Arg Ser Pro Val Asn Leu Pro Pro Glu Leu

100 105

<210> 132

<211> 313

<212> PRT

<213> Artificial Sequence

<220>

<223> Asesip

<400> 132

Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly

1 5 10 15

Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr

20 25 30

Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys

35 40 45

Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys

50 55 60

Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg

65 70 75 80

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

85 90 95

Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

100 105 110

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

115 120 125

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

130 135 140

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

145 150 155 160

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

165 170 175

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

180 185 190

Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

195 200 205

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

210 215 220

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

225 230 235 240

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

245 250 255

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

260 265 270

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

275 280 285

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

290 295 300

Lys Ser Leu Ser Leu Ser Pro Gly Lys

305 310

<210> 133

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 133

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala

1 5 10

<210> 134

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc with C220S/E233P/L234V/L235A/G236del/S267K

<400> 134

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 135

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc (C5S (C220S), R77C, (R292C), N82G (N297G), V87C (V302C),

L232del (K447del))

<400> 135

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys Glu Glu Gln

65 70 75 80

Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 136

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc with C220S/L234A/L235E/G237A/K447del

<400> 136

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 137

<211> 230

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc with C220S/E233P/L234V/L235A/G236del/S267K/K447del

<400> 137

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly

225 230

<210> 138

<211> 235

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG2 Fc

<400> 138

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

1 5 10 15

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

20 25 30

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

35 40 45

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

50 55 60

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

65 70 75 80

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

85 90 95

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

100 105 110

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

115 120 125

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

130 135 140

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

145 150 155 160

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

165 170 175

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

180 185 190

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

195 200 205

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

210 215 220

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230 235

<210> 139

<211> 229

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4 Fc

<400> 139

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 140

<211> 229

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4 Fc S228P

<400> 140

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 141

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc K chain

<400> 141

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Lys Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 142

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc D chain

<400> 142

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Asp Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 143

<211> 237

<212> PRT

<213> Artificial Sequence

<220>

<223> Green fluorescent protein

<400> 143

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

1 5 10 15

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

20 25 30

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr

35 40 45

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Ser

50 55 60

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His

65 70 75 80

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

85 90 95

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

100 105 110

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

115 120 125

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr

130 135 140

Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile

145 150 155 160

Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln

165 170 175

Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val

180 185 190

Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys

195 200 205

Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr

210 215 220

Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 144

<211> 237

<212> PRT

<213> Artificial Sequence

<220>

<223> eGFP

<400> 144

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

1 5 10 15

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

20 25 30

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr

35 40 45

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr

50 55 60

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His

65 70 75 80

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

85 90 95

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

100 105 110

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

115 120 125

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr

130 135 140

Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile

145 150 155 160

Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln

165 170 175

Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val

180 185 190

Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys

195 200 205

Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr

210 215 220

Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 145

<211> 239

<212> PRT

<213> Artificial Sequence

<220>

<223> eGFP

<400> 145

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 146

<211> 45

<212> PRT

<213> Artificial Sequence

<220>

<223> Part: COMP

<400> 146

Asp Leu Gly Pro Gln Met Leu Arg Glu Leu Gln Glu Thr Asn Ala Ala

1 5 10 15

Leu Gln Asp Val Arg Glu Leu Leu Arg Gln Gln Val Arg Glu Ile Thr

20 25 30

Phe Leu Lys Asn Thr Val Met Glu Cys Asp Ala Cys Gly

35 40 45

<210> 147

<211> 33

<212> PRT

<213> Artificial Sequence

<220>

<223> Part: VASP

<400> 147

Asp Leu Gln Arg Val Lys Gln Glu Leu Leu Glu Glu Val Lys Lys Glu

1 5 10 15

Leu Gln Lys Val Lys Glu Glu Ile Ile Glu Ala Phe Val Gln Glu Leu

20 25 30

Arg

<210> 148

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> Section: ZZ12.6

<400> 148

Asp Val Gln Ala Ile Ser Ser Thr Ile Gln Asp Leu Gln Asp Gln Val

1 5 10 15

Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu

20 25 30

Leu Thr Arg Ser Gly Gly Gly Ser

35 40

<210> 149

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> HSA signal peptide

<400> 149

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser

<210> 150

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Igκ light chain

<400> 150

Met Asp Met Arg Ala Pro Ala Gly Ile Phe Gly Phe Leu Leu Val Leu

1 5 10 15

Phe Pro Gly Tyr Arg Ser

20

<210> 151

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Human azoxystrobin preprotein signal sequence

<400> 151

Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser

1 5 10 15

Ser Arg Ala

<210> 152

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 152

Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 153

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 153

Met Glu Leu Gly Leu Arg Trp Val Phe Leu Val Ala Ile Leu Glu Gly

1 5 10 15

Val Gln Cys

<210> 154

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 154

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser

<210> 155

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 155

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 156

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 156

Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly

1 5 10 15

Val Gln Ser

<210> 157

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 157

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 158

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 158

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly

1 5 10 15

Val Gln Cys

<210> 159

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG heavy chain signal peptide

<400> 159

Met Asp Leu Leu His Lys Asn Met Lys His Leu Trp Phe Phe Leu Leu

1 5 10 15

Leu Val Ala Ala Pro Arg Trp Val Leu Ser

20 25

<210> 160

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> IgGκ light chain signal sequence

<400> 160

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Ser Gly Ala Arg Cys

20

<210> 161

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> IgGκ light chain signal sequence

<400> 161

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala

20

<210> 162

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Gaussia luciferase

<400> 162

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala

<210> 163

<211> 18

<212> PRT

<213> Homo sapiens

<220>

<221> Unclassified features

<223> Human albumin

<400> 163

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser

<210> 164

<211> 18

<212> PRT

<213> Homo sapiens

<220>

<221> Unclassified features

<223> Human chymotrypsinogen

<400> 164

Met Ala Phe Leu Trp Leu Leu Ser Cys Trp Ala Leu Leu Gly Thr Thr

1 5 10 15

Phe Gly

<210> 165

<211> 14

<212> PRT

<213> Homo sapiens

<220>

<221> Unclassified features

<223> Human interleukin-2

<400> 165

Met Gln Leu Leu Ser Cys Ile Ala Leu Ile Leu Ala Leu Val

1 5 10

<210> 166

<211> 15

<212> PRT

<213> Homo sapiens

<220>

<221> Unclassified features

<223> Human trypsinogen-2

<400> 166

Met Asn Leu Leu Leu Ile Leu Thr Phe Val Ala Ala Ala Val Ala

1 5 10 15

<210> 167

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Fc

<400> 167

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 168

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Fc

<400> 168

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 169

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Fc

<400> 169

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 170

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Fc

<400> 170

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 171

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Fc

<400> 171

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 172

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI Fc

<400> 172

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 173

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc

<400> 173

Asp Lys Pro His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 174

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc

<400> 174

Asp Pro Lys Ser Cys Asp Lys Pro His Thr Cys Pro Leu Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 175

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc variant

<400> 175

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 176

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc variant

<400> 176

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 177

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 177

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 178

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 178

Ser Leu Ser Cys Arg Lys Glu Glu Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 179

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 179

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 180

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 180

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 181

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 181

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 182

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 182

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 183

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 183

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 184

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 184

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 185

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 185

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 186

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 186

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 187

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 187

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 188

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 188

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 189

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 189

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 190

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 190

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 191

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 191

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 192

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 192

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser

35 40

<210> 193

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Fc variant

<400> 193

Asp Lys Pro His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 194

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 194

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 195

<211> 65

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 195

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

50 55 60

Ser

65

<210> 196

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 196

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His

50 55 60

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

100 105 110

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

290 295 300

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

305 310 315 320

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

325 330 335

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp

340 345 350

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala

355 360 365

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

370 375 380

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

385 390 395 400

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

405 410 415

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

420 425 430

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

435 440 445

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

450 455 460

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

465 470 475 480

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

485 490 495

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

500 505 510

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

515 520 525

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

530 535 540

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

545 550 555 560

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

565 570 575

Gly

<210> 197

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 197

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 198

<211> 344

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 198

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Gly Gly Gly Ser

35 40 45

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

50 55 60

Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu Arg

65 70 75 80

Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln Cys

85 90 95

Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly Gly

100 105 110

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

115 120 125

Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

130 135 140

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

145 150 155 160

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

165 170 175

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

180 185 190

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

195 200 205

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

210 215 220

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

225 230 235 240

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

245 250 255

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

260 265 270

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

275 280 285

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

290 295 300

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

305 310 315 320

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

325 330 335

Lys Ser Leu Ser Leu Ser Pro Gly

340

<210> 199

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 199

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His

50 55 60

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

100 105 110

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

290 295 300

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

305 310 315 320

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

325 330 335

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp

340 345 350

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala

355 360 365

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

370 375 380

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

385 390 395 400

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

405 410 415

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

420 425 430

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

435 440 445

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

450 455 460

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

465 470 475 480

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

485 490 495

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

500 505 510

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

515 520 525

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

530 535 540

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

545 550 555 560

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

565 570 575

Gly

<210> 200

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 200

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 201

<211> 344

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 201

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Leu Ser

290 295 300

Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu Arg Asp Cys

305 310 315 320

Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln Cys Ala Asp

325 330 335

Phe Cys Glu Asn Lys Leu Arg Ser

340

<210> 202

<211> 344

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 202

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Leu Ser

290 295 300

Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu Arg Asp Cys

305 310 315 320

Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln Cys Ala Tyr

325 330 335

Phe Cys Glu Asn Lys Leu Arg Ser

340

<210> 203

<211> 616

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 203

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

1 5 10 15

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

20 25 30

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

35 40 45

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

50 55 60

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

65 70 75 80

Arg Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro

85 90 95

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

100 105 110

Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

340 345 350

Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

355 360 365

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

385 390 395 400

Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

405 410 415

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

420 425 430

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

435 440 445

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

450 455 460

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

465 470 475 480

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

485 490 495

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

500 505 510

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

515 520 525

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

530 535 540

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

545 550 555 560

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

565 570 575

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

580 585 590

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

595 600 605

Lys Ser Leu Ser Leu Ser Pro Gly

610 615

<210> 204

<211> 313

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 204

Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly

1 5 10 15

Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr

20 25 30

Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys

35 40 45

Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys

50 55 60

Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg

65 70 75 80

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

85 90 95

Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

100 105 110

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

115 120 125

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

130 135 140

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

145 150 155 160

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

165 170 175

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

180 185 190

Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

195 200 205

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

210 215 220

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

225 230 235 240

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

245 250 255

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

260 265 270

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

275 280 285

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

290 295 300

Lys Ser Leu Ser Leu Ser Pro Gly Lys

305 310

<210> 205

<211> 640

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 205

Ser Arg Val Asp Gln Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly

1 5 10 15

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

20 25 30

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

35 40 45

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

50 55 60

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

65 70 75 80

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

85 90 95

Arg Ser Pro Val Asn Leu Pro Pro Glu Leu Gly Ser Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly

340 345 350

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys

405 410 415

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro

420 425 430

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

435 440 445

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

450 455 460

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

465 470 475 480

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

485 490 495

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

500 505 510

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

515 520 525

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

530 535 540

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

545 550 555 560

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

565 570 575

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

580 585 590

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

595 600 605

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

610 615 620

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

625 630 635 640

<210> 206

<211> 333

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 206

Ser Arg Val Asp Gln Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly

1 5 10 15

Val Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu

20 25 30

Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg

35 40 45

Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly

50 55 60

Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile

65 70 75 80

Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu

85 90 95

Arg Ser Pro Val Asn Leu Pro Pro Glu Leu Asp Lys Thr His Thr Cys

100 105 110

Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu

115 120 125

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

130 135 140

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

145 150 155 160

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

165 170 175

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

180 185 190

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

195 200 205

Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

210 215 220

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

225 230 235 240

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

245 250 255

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

260 265 270

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

275 280 285

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

290 295 300

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

305 310 315 320

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 207

<211> 1731

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 207

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatctg gatccggtgg tggcgggtca gggggaggtg gaagcgagcc caagtcctca 180

gataagactc atacttgccc cccctgtccc gcccctgagg ccgaaggcgc accgtcagtg 240

tttttgttcc caccaaagcc taaggacaca ctgatgataa gcagaacacc tgaggtaacc 300

tgtgttgttg tggatgtaag ccacgaagat ccggaggtta agtttaactg gtacgtggac 360

ggtgtagaag tccataatgc taagacgaag ccgagggagg aacaatacaa ctccacgtat 420

agagtggtct ccgtcttgac cgtactccat caagactggc tcaacgggaa agagtacaag 480

tgtaaagtct ctaacaaagc tcttcctgca ccgattgaga aaaccatatc taaagccaag 540

ggacaaccga gagaaccaca ggtttacacg ctccccccca gtagagacga acttacaaaa 600

aaccaggtca gtcttacctg cctcgtcaaa ggcttctacc cctctgacat cgctgttgag 660

tgggaatcta acggccaacc tgagaacaat tacaaaacaa ccccgcctgt ccttgactca 720

gacggttccttttttcttta cagcaagctg accgtcgaca aatcacggtg gcaacaaggc 780

aatgtgtttt cctgttccgt gatgcacgag gcactgcaca accactacac tcaaaaatcc 840

ctttcccttt ccccaggggg aggtggaggg agcggtggag gtggtagcgg gggtggaggc 900

tcaggtggtg ggggttccgg cggtggcgga agtggaggcg gtggctctgg tggtggcgga 960

tctggcggag gaggcagcgg cggaggtggg tctgggggtg gaggctccgg aggcggggga 1020

agcggtggag gagggtcaga gcccaaaagc tccgacaaga ctcacacatg ccccccttgt 1080

ccagcgcctg aagctgaggg tgcgccctct gtcttccttt tcccccctaa gccgaaagat 1140

accctgatga tctcccgcac tcccgaagtc acatgtgttg ttgtcgacgt atctcatgaa 1200

gatcctgagg tgaaattcaa ctggtatgta gacggggtcg aagttcataa tgctaagact 1260

aagccacgag aagagcaata caactcaacg tatcgggtgg tgagcgttct gacggttctg 1320

caccaagatt ggcttaatgg aaaagagtat aagtgcaagg tgtccaacaa ggctcttccg 1380

gcacccatcg aaaagacgat ttccaaagcg aaaggccaac ctagggaacc gcaagtttac 1440

actttgcccc cgtcaagaga cgaacttacc aagaatcaag tttccctgac gtgccttgtg 1500

aagggcttct accctagcga tatagcagtt gagtgggaat ctaacggcca gcccgaaaat 1560

aattataaga ctactccgcc cgtgctggac agtgatggtt catttttcct gtattcaaaa 1620

ctcactgtgg acaaatctag atggcagcag ggtaatgtgt tctcttgttc agttatgcac 1680

gaggcattgc acaatcacta tacgcaaaaa agtttgtctc tctctccggg g 1731

<210> 208

<211> 843

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 208

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatctg gatccggtgg aggagggtca gagcccaaaa gctccgacaa gactcacaca 180

tgcccccctt gtccagcgcc tgaagctgag ggtgcgccct ctgtcttcct tttcccccct 240

aagccgaaag ataccctgat gatctcccgc actcccgaag tcacatgtgt tgttgtcgac 300

gtatctcatg aagatcctga ggtgaaattc aactggtatg tagacggggt cgaagttcat 360

aatgctaaga ctaagccacg agaagagcaa tacaactcaa cgtatcgggt ggtgagcgtt 420

ctgacggttc tgcaccaaga ttggcttaat ggaaaagagt ataagtgcaa ggtgtccaac 480

aaggctcttc cggcacccat cgaaaagacg atttccaaag cgaaaggcca acctagggaa 540

ccgcaagttt acactttgcc cccgtcaaga gacgaactta ccaagaatca agtttccctg 600

acgtgccttg tgaagggctt ctaccctagc gatatagcag ttgagtggga atctaacggc 660

cagcccgaaa ataattataa gactactccg cccgtgctgg acagtgatgg ttcatttttc 720

ctgtattcaa aactcactgt ggacaaatct agatggcagc agggtaatgt gttctcttgt 780

tcagttatgc acgaggcatt gcacaatcac tatacgcaaa aaagtttgtc tctctctccg 840

ggg 843

<210> 209

<211> 1032

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 209

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatctg ggggtggcgg atcaggaggt ggcggttcag gcggaggagg ttctgggggt 180

gggggttctt cattgtcctg cagaaaggaa caggggaaat tttacgatca cttgcttaga 240

gattgtataa gctgcgcgag catttgcggg caacacccta aacagtgtgc gtatttctgc 300

gagaataaac tccggtctgg atccggtgga ggagggtcag agcccaaaag ctccgacaag 360

actcacacat gccccccttg tccagcgcct gaagctgagg gtgcgccctc tgtcttcctt 420

ttccccccta agccgaaaga taccctgatg atctcccgca ctcccgaagt cacatgtgtt 480

gttgtcgacg tatctcatga agatcctgag gtgaaattca actggtatgt agacggggtc 540

gaagttcata atgctaagac taagccacga gaagagcaat acaactcaac gtatcgggtg 600

gtgagcgttc tgacggttct gcaccaagat tggcttaatg gaaaagagta taagtgcaag 660

gtgtccaaca aggctcttcc ggcacccatc gaaaagacga tttccaaagc gaaaggccaa 720

cctagggaac cgcaagttta cactttgccc ccgtcaagag acgaacttac caagaatcaa 780

gtttccctga cgtgccttgt gaagggcttc taccctagcg atatagcagt tgagtgggaa 840

tctaacggcc agcccgaaaa taattataag actactccgc ccgtgctgga cagtgatggt 900

tcatttttcc tgtattcaaa actcactgtg gacaaatcta gatggcagca gggtaatgtg 960

ttctcttgtt cagttatgca cgaggcattg cacaatcact atacgcaaaa aagtttgtct 1020

ctctctccgg gg 1032

<210> 210

<211> 1731

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 210

agtctctctt gcaggaaaga gcaaggcgaa tattacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg ctgatttctg tgaaaataag 120

cttcgatctg gatccggtgg tggcgggtca gggggaggtg gaagcgagcc caagtcctca 180

gataagactc atacttgccc cccctgtccc gcccctgagg ccgaaggcgc accgtcagtg 240

tttttgttcc caccaaagcc taaggacaca ctgatgataa gcagaacacc tgaggtaacc 300

tgtgttgttg tggatgtaag ccacgaagat ccggaggtta agtttaactg gtacgtggac 360

ggtgtagaag tccataatgc taagacgaag ccgagggagg aacaatacaa ctccacgtat 420

agagtggtct ccgtcttgac cgtactccat caagactggc tcaacgggaa agagtacaag 480

tgtaaagtct ctaacaaagc tcttcctgca ccgattgaga aaaccatatc taaagccaag 540

ggacaaccga gagaaccaca ggtttacacg ctccccccca gtagagacga acttacaaaa 600

aaccaggtca gtcttacctg cctcgtcaaa ggcttctacc cctctgacat cgctgttgag 660

tgggaatcta acggccaacc tgagaacaat tacaaaacaa ccccgcctgt ccttgactca 720

gacggttccttttttcttta cagcaagctg accgtcgaca aatcacggtg gcaacaaggc 780

aatgtgtttt cctgttccgt gatgcacgag gcactgcaca accactacac tcaaaaatcc 840

ctttcccttt ccccaggggg aggtggaggg agcggtggag gtggtagcgg gggtggaggc 900

tcaggtggtg ggggttccgg cggtggcgga agtggaggcg gtggctctgg tggtggcgga 960

tctggcggag gaggcagcgg cggaggtggg tctgggggtg gaggctccgg aggcggggga 1020

agcggtggag gagggtcaga gcccaaaagc tccgacaaga ctcacacatg ccccccttgt 1080

ccagcgcctg aagctgaggg tgcgccctct gtcttccttt tcccccctaa gccgaaagat 1140

accctgatga tctcccgcac tcccgaagtc acatgtgttg ttgtcgacgt atctcatgaa 1200

gatcctgagg tgaaattcaa ctggtatgta gacggggtcg aagttcataa tgctaagact 1260

aagccacgag aagagcaata caactcaacg tatcgggtgg tgagcgttct gacggttctg 1320

caccaagatt ggcttaatgg aaaagagtat aagtgcaagg tgtccaacaa ggctcttccg 1380

gcacccatcg aaaagacgat ttccaaagcg aaaggccaac ctagggaacc gcaagtttac 1440

actttgcccc cgtcaagaga cgaacttacc aagaatcaag tttccctgac gtgccttgtg 1500

aagggcttct accctagcga tatagcagtt gagtgggaat ctaacggcca gcccgaaaat 1560

aattataaga ctactccgcc cgtgctggac agtgatggtt catttttcct gtattcaaaa 1620

ctcactgtgg acaaatctag atggcagcag ggtaatgtgt tctcttgttc agttatgcac 1680

gaggcattgc acaatcacta tacgcaaaaa agtttgtctc tctctccggg g 1731

<210> 211

<211> 843

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 211

agtctctctt gcaggaaaga gcaaggcgaa tattacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg ctgatttctg tgaaaataag 120

cttcgatctg gatccggtgg aggagggtca gagcccaaaa gctccgacaa gactcacaca 180

tgcccccctt gtccagcgcc tgaagctgag ggtgcgccct ctgtcttcct tttcccccct 240

aagccgaaag ataccctgat gatctcccgc actcccgaag tcacatgtgt tgttgtcgac 300

gtatctcatg aagatcctga ggtgaaattc aactggtatg tagacggggt cgaagttcat 360

aatgctaaga ctaagccacg agaagagcaa tacaactcaa cgtatcgggt ggtgagcgtt 420

ctgacggttc tgcaccaaga ttggcttaat ggaaaagagt ataagtgcaa ggtgtccaac 480

aaggctcttc cggcacccat cgaaaagacg atttccaaag cgaaaggcca acctagggaa 540

ccgcaagttt acactttgcc cccgtcaaga gacgaactta ccaagaatca agtttccctg 600

acgtgccttg tgaagggctt ctaccctagc gatatagcag ttgagtggga atctaacggc 660

cagcccgaaa ataattataa gactactccg cccgtgctgg acagtgatgg ttcatttttc 720

ctgtattcaa aactcactgt ggacaaatct agatggcagc agggtaatgt gttctcttgt 780

tcagttatgc acgaggcatt gcacaatcac tatacgcaaa aaagtttgtc tctctctccg 840

ggg 843

<210> 212

<211> 1032

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 212

agtctctctt gcaggaaaga gcaaggcgaa tattacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg ctgatttctg tgaaaataag 120

cttcgatctg gatccggtgg aggagggtca gagcccaaaa gctccgacaa gactcacaca 180

tgcccccctt gtccagcgcc tgaagctgag ggtgcgccct ctgtcttcct tttcccccct 240

aagccgaaag ataccctgat gatctcccgc actcccgaag tcacatgtgt tgttgtcgac 300

gtatctcatg aagatcctga ggtgaaattc aactggtatg tagacggggt cgaagttcat 360

aatgctaaga ctaagccacg agaagagcaa tacaactcaa cgtatcgggt ggtgagcgtt 420

ctgacggttc tgcaccaaga ttggcttaat ggaaaagagt ataagtgcaa ggtgtccaac 480

aaggctcttc cggcacccat cgaaaagacg atttccaaag cgaaaggcca acctagggaa 540

ccgcaagttt acactttgcc cccgtcaaga gacgaactta ccaagaatca agtttccctg 600

acgtgccttg tgaagggctt ctaccctagc gatatagcag ttgagtggga atctaacggc 660

cagcccgaaa ataattataa gactactccg cccgtgctgg acagtgatgg ttcatttttc 720

ctgtattcaa aactcactgt ggacaaatct agatggcagc agggtaatgt gttctcttgt 780

tcagttatgc acgaggcatt gcacaatcac tatacgcaaa aaagtttgtc tctctctccg 840

gggggggtg gcggatcagg aggtggcggt tcaggcggag gaggttctgg gggtgggggt 900

tcttcattgt cctgcagaaa ggaacagggg gagtattacg atcacttgct tagagattgt 960

ataagctgcg cgagcatttg cgggcaacac cctaaacagt gtgcggattt ctgcgagaat 1020

aaactccggt ct 1032

<210> 213

<211> 1032

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 213

agtctctctt gcaggaaaga gcaaggcaaa ttttacgacc atcttttgcg agactgtata 60

agctgtgcgt ctatttgtgg acaacaccct aaacaatgtg cttatttctg tgaaaataag 120

cttcgatctg gatccggtgg aggagggtca gagcccaaaa gctccgacaa gactcacaca 180

tgcccccctt gtccagcgcc tgaagctgag ggtgcgccct ctgtcttcct tttcccccct 240

aagccgaaag ataccctgat gatctcccgc actcccgaag tcacatgtgt tgttgtcgac 300

gtatctcatg aagatcctga ggtgaaattc aactggtatg tagacggggt cgaagttcat 360

aatgctaaga ctaagccacg agaagagcaa tacaactcaa cgtatcgggt ggtgagcgtt 420

ctgacggttc tgcaccaaga ttggcttaat ggaaaagagt ataagtgcaa ggtgtccaac 480

aaggctcttc cggcacccat cgaaaagacg atttccaaag cgaaaggcca acctagggaa 540

ccgcaagttt acactttgcc cccgtcaaga gacgaactta ccaagaatca agtttccctg 600

acgtgccttg tgaagggctt ctaccctagc gatatagcag ttgagtggga atctaacggc 660

cagcccgaaa ataattataa gactactccg cccgtgctgg acagtgatgg ttcatttttc 720

ctgtattcaa aactcactgt ggacaaatct agatggcagc agggtaatgt gttctcttgt 780

tcagttatgc acgaggcatt gcacaatcac tatacgcaaa aaagtttgtc tctctctccg 840

gggggggtg gcggatcagg aggtggcggt tcaggcggag gaggttctgg gggtgggggt 900

tcttcattgt cctgcagaaa ggaacagggg aaattttacg atcacttgct tagagattgt 960

ataagctgcg cgagcatttg cgggcaacac cctaaacagt gtgcgtattt ctgcgagaat 1020

aaactccggt ct 1032

<210> 214

<211> 1848

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 214

gtggcaatgc gctcatgccc agaggaacaa tattggggatc cgcttcttgg gacgtgtatg 60

agctgcaaga ccatctgtaa tcatcaatcc caaaggacat gcgcagcttt ctgcaggagt 120

ctctcttgca ggaaagagca aggcaaattt tacgaccatc ttttgcgaga ctgtataagc 180

tgtgcgtcta tttgtggaca acaccctaaa caatgtgctt atttctgtga aaataagctt 240

cgatctggat ccggtggtgg cgggtcaggg ggaggtggaa gcgagcccaa gtcctcagat 300

aagactcata cttgcccccc ctgtcccgcc cctgaggccg aaggcgcacc gtcagtgttt 360

ttgttcccac caaagcctaa ggacacactg atgataagca gaacacctga ggtaacctgt 420

gttgttgtgg atgtaagcca cgaagatccg gaggttaagt ttaactggta cgtggacggt 480

gtagaagtcc ataatgctaa gacgaagccg agggaggaac aatacaactc cacgtataga 540

gtggtctccg tcttgaccgt actccatcaa gactggctca acgggaaaga gtacaagtgt 600

aaagtctcta acaaagctct tcctgcaccg attgagaaaa ccatatctaa agccaaggga 660

caaccgagag aaccacaggt ttacacgctc ccccccagta gagacgaact tacaaaaaac 720

caggtcagtc ttacctgcct cgtcaaaggc ttctacccct ctgacatcgc tgttgagtgg 780

gaatctaacg gccaacctga gaacaattac aaaacaaccc cgcctgtcct tgactcagac 840

ggttcctttt ttctttacag caagctgacc gtcgacaaat cacggtggca acaaggcaat 900

gtgttttcct gttccgtgat gcacgaggca ctgcacaacc actacactca aaaatccctt 960

tccctttccc cagggggagg tggagggagc ggtggaggtg gtagcggggg tggaggctca 1020

ggtggtgggg gttccggcgg tggcggaagt ggaggcggtg gctctggtgg tggcggatct 1080

ggcggaggag gcagcggcgg aggtgggtct gggggtggag gctccggagg cgggggaagc 1140

ggtggaggag ggtcagagcc caaaagctcc gacaagactc acacatgccc cccttgtcca 1200

gcgcctgaag ctgagggtgc gccctctgtc ttccttttcc cccctaagcc gaaagatacc 1260

ctgatgatct cccgcactcc cgaagtcaca tgtgttgttg tcgacgtatc tcatgaagat 1320

cctgaggtga aattcaactg gtatgtagac ggggtcgaag ttcataatgc taagactaag 1380

ccacgagaag agcaatacaa ctcaacgtat cgggtggtga gcgttctgac ggttctgcac 1440

caagattggc ttaatggaaa agagtataag tgcaaggtgt ccaacaaggc tcttccggca 1500

cccatcgaaa agacgatttc caaagcgaaa ggccaaccta gggaaccgca agtttacact 1560

ttgcccccgt caagagacga acttaccaag aatcaagttt ccctgacgtg ccttgtgaag 1620

ggcttctacc ctagcgatat agcagttgag tgggaatcta acggccagcc cgaaaataat 1680

tataagacta ctccgcccgt gctggacagt gatggttcat ttttcctgta ttcaaaactc 1740

actgtggaca aatctagatg gcagcagggt aatgtgttct cttgttcagt tatgcacgag 1800

gcattgcaca atcactatac gcaaaaaagt ttgtctctct ctccgggg 1848

<210> 215

<211> 939

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 215

gcaatgcgct catgcccaga ggaacaatat tgggatccgc ttcttgggac gtgtatgagc 60

tgcaagacca tctgtaatca tcaatcccaa aggacatgcg cagctttctg caggagtctc 120

tcttgcagga aagagcaagg caaattttac gaccatcttt tgcgagactg tataagctgt 180

gcgtctattt gtggacaaca ccctaaacaa tgtgcttatt tctgtgaaaa taagcttcga 240

tctgagccca aaagctccga caagactcac acatgccccc cttgtccagc gcctgaagct 300

gagggtgcgc cctctgtctt ccttttcccc cctaagccga aagataccct gatgatctcc 360

cgcactcccg aagtcacatg tgttgttgtc gacgtatctc atgaagatcc tgaggtgaaa 420

ttcaactggt atgtagacgg ggtcgaagtt cataatgcta agactaagcc acgagaagag 480

caatacaact caacgtatcg ggtggtgagc gttctgacgg ttctgcacca agattggctt 540

aatggaaaag agtataagtg caaggtgtcc aacaaggctc ttccgagctc catcgaaaag 600

acgatttcca aagcgaaagg ccaacctagg gaaccgcaag tttacacttt gcccccgtca 660

agagacgaac ttaccaagaa tcaagtttcc ctgacgtgcc ttgtgaaggg cttctaccct 720

agcgatatag cagttgagtg ggaatctaac ggccagcccg aaaataatta taagactact 780

ccgcccgtgc tggacagtga tggttcattt ttcctgtatt caaaactcac tgtggacaaa 840

tctagatggc agcagggtaa tgtgttctct tgttcagtta tgcacgaggc attgcacaat 900

cactatacgc aaaaaagttt gtctctctct ccggggaag 939

<210> 216

<211> 1920

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 216

agccgtgtgg accagggagga gcgctttcca cagggcctgt ggacgggggt ggcaatgcgc 60

tcatgcccag aggaacaata ttggggatccg cttcttggga cgtgtatgag ctgcaagacc 120

atctgtaatc atcaatccca aaggacatgc gcagctttct gcaggagtct ctcttgcagg 180

aaagagcaag gcaaatttta cgaccatctt ttgcgagact gtataagctg tgcgtctatt 240

tgtggacaac accctaaaca atgtgcttat ttctgtgaaa ataagcttcg atctcccgtg 300

aacctgccac ccgagctggg atccggtggt ggcgggtcag ggggaggtgg aagcgagccc 360

aagtcctcag ataagactca tacttgcccc ccctgtcccg cccctgaggc cgaaggcgca 420

ccgtcagtgt ttttgttccc accaaagcct aaggacacac tgatgataag cagaacacct 480

gaggtaacct gtgttgttgt ggatgtaagc cacgaagatc cggaggttaa gtttaactgg 540

tacgtggacg gtgtagaagt ccataatgct aagacgaagc cgagggagga acaatacaac 600

tccacgtata gagtggtctc cgtcttgacc gtactccatc aagactggct caacgggaaa 660

gagtacaagt gtaaagtctc taacaaagct cttcctgcac cgattgagaa aaccatatct 720

aaagccaagg gacaaccgag agaaccacag gtttacacgc tcccccccag tagagacgaa 780

cttacaaaaa accaggtcag tcttacctgc ctcgtcaaag gcttctaccc ctctgacatc 840

gctgttgagt gggaatctaa cggccaacct gagaacaatt acaaaacaac cccgcctgtc 900

cttgactcag acggttcctt ttttctttac agcaagctga ccgtcgacaa atcacggtgg 960

caacaaggca atgtgttttc ctgttccgtg atgcacgagg cactgcacaa ccactacact 1020

caaaaatccc tttccctttc cccaggggga ggtggaggga gcggtggagg tggtagcggg 1080

ggtggaggct caggtggtgg gggttccggc ggtggcggaa gtggaggcgg tggctctggt 1140

ggtggcggat ctggcggagg aggcagcggc ggaggtgggt ctgggggtgg aggctccgga 1200

ggcgggggaa gcggtggagg agggtcagag cccaaaagct ccgacaagac tcacacatgc 1260

cccccttgtc cagcgcctga agctgagggt gcgccctctg tcttcctttt cccccctaag 1320

ccgaaagata ccctgatgat ctcccgcact cccgaagtca catgtgttgt tgtcgacgta 1380

tctcatgaag atcctgaggt gaaattcaac tggtatgtag acggggtcga agttcataat 1440

gctaagacta agccacgaga agagcaatac aactcaacgt atcgggtggt gagcgttctg 1500

acggttctgc accaagattg gcttaatgga aaagagtata agtgcaaggt gtccaacaag 1560

gctcttccgg cacccatcga aaagacgatt tccaaagcga aaggccaacc tagggaaccg 1620

caagtttaca ctttgccccc gtcaagagac gaacttacca agaatcaagt ttccctgacg 1680

tgccttgtga agggcttcta ccctagcgat atagcagttg agtgggaatc taacggccag 1740

cccgaaaata attataagac tactccgccc gtgctggaca gtgatggttc atttttcctg 1800

tattcaaaac tcactgtgga caaatctaga tggcagcagg gtaatgtgtt ctcttgttca 1860

gttatgcacg aggcattgca caatcactat acgcaaaaaa gtttgtctct ctctccgggg 1920

<210> 217

<211> 999

<212> DNA

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 217

agccgtgtgg accagggagga gcgctttcca cagggcctgt ggacgggggt ggcaatgcgc 60

tcatgcccag aggaacaata ttggggatccg cttcttggga cgtgtatgag ctgcaagacc 120

atctgtaatc atcaatccca aaggacatgc gcagctttct gcaggagtct ctcttgcagg 180

aaagagcaag gcaaatttta cgaccatctt ttgcgagact gtataagctg tgcgtctatt 240

tgtggacaac accctaaaca atgtgcttat ttctgtgaaa ataagcttcg atctcccgtg 300

aacctgccac ccgagctgga caagactcac acatgccccc cttgtccagc gcctgaagct 360

gagggtgcgc cctctgtctt ccttttcccc cctaagccga aagataccct gatgatctcc 420

cgcactcccg aagtcacatg tgttgttgtc gacgtatctc atgaagatcc tgaggtgaaa 480

ttcaactggt atgtagacgg ggtcgaagtt cataatgcta agactaagcc acgagaagag 540

caatacaact caacgtatcg ggtggtgagc gttctgacgg ttctgcacca agattggctt 600

aatggaaaag agtataagtg caaggtgtcc aacaaggctc ttccgagctc catcgaaaag 660

acgatttcca aagcgaaagg ccaacctagg gaaccgcaag tttacacttt gcccccgtca 720

agagacgaac ttaccaagaa tcaagtttcc ctgacgtgcc ttgtgaaggg cttctaccct 780

agcgatatag cagttgagtg ggaatctaac ggccagcccg aaaataatta taagactact 840

ccgcccgtgc tggacagtga tggttcattt ttcctgtatt caaaactcac tgtggacaaa 900

tctagatggc agcagggtaa tgtgttctct tgttcagtta tgcacgaggc attgcacaat 960

cactatacgc aaaaaagttt gtctctctct ccggggaag 999

<210> 218

<211> 522

<212> PRT

<213> Artificial Sequence

<220>

<223> FC variant

<400> 218

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

290 295 300

Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro

305 310 315 320

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

325 330 335

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

340 345 350

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

355 360 365

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

370 375 380

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

385 390 395 400

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

405 410 415

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

420 425 430

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

435 440 445

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

450 455 460

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

465 470 475 480

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

485 490 495

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

500 505 510

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

515 520

<210> 219

<211> 222

<212> PRT

<213> Artificial Sequence

<220>

<223> FC variant

<400> 219

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

100 105 110

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

180 185 190

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 220

<211> 228

<212> PRT

<213> Artificial Sequence

<220>

<223> FC variant

<400> 220

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly

225

<210> 221

<211> 226

<212> PRT

<213> Artificial Sequence

<220>

<223> FC variant

<400> 221

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 222

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 222

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 223

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI variant

<400> 223

Ser Leu Ser Cys Arg Lys Glu Glu Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 224

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 224

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 225

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 225

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 226

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 226

Ser Leu Ser Cys Arg Lys Glu Glu Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 227

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 227

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 228

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 228

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 229

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 229

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 230

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 230

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 231

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 231

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 232

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 232

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 233

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 233

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Gln Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 234

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 234

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Asp Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 235

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 235

Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 236

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 236

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Tyr Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 237

<211> 281

<212> PRT

<213> Artificial Sequence

<220>

<223> TACI FC

<400> 237

Ser Leu Ser Cys Arg Lys Glu Gln Gly Glu Phe Tyr Asp His Leu Leu

1 5 10 15

Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His Pro Lys Gln

20 25 30

Cys Ala Asp Phe Cys Glu Asn Lys Leu Arg Ser Gly Ser Gly Gly Gly

35 40 45

Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

50 55 60

Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

65 70 75 80

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

85 90 95

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

100 105 110

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

115 120 125

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

130 135 140

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

145 150 155 160

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

165 170 175

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

180 185 190

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

195 200 205

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

210 215 220

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

225 230 235 240

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

245 250 255

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

260 265 270

Gln Lys Ser Leu Ser Leu Ser Pro Gly

275 280

<210> 238

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Hinges

<400> 238

Glu Pro Lys Ser Ser

1 5

<210> 239

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Hinges

<400> 239

Glu Pro Lys Ser Cys

1 5

Claims (119)

1. An immunomodulatory protein comprising at least one variant TACI polypeptide, wherein the at least one variant TACI polypeptide comprises one or more amino acid substitutions in the extracellular domain (ECD) of a reference TACI polypeptide, the amino acid substitutions being located 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 numbers shown in SEQ ID NO:122. 2. An immunomodulatory protein comprising a variant TACI-Fc fusion protein, the 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 in an extracellular domain (ECD) of a reference TACI polypeptide, the amino acid substitutions being located 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 numbers shown in SEQ ID NO:122. 3. The immunomodulatory protein according to claim 1 or claim 2, wherein the reference TACI polypeptide is a truncated polypeptide, the truncated polypeptide consisting of the extracellular domain of TACI or its specific binding portion that binds to APRIL, BAFF, or BAFF/APRIL heterotrimer. 4. The immunomodulatory protein according to any one of claims 1-3, wherein the reference TACI polypeptide comprises the amino acid sequence shown in SEQ ID NO:122, or a portion thereof comprising one or both of the CRD1 and CRD2 domains, said portion binding to APRIL, BAFF, or a BAFF/APRIL heterotrimer. 5. The immunomodulatory protein according to any one of claims 1-4, wherein the reference TACI polypeptide lacks an N-terminal methionine. 6. The immunomodulatory protein according to any one of claims 1-5, wherein the reference TACI polypeptide comprises the CRD1 domain and the CRD2 domain. 7. The immunomodulatory protein according to any one of claims 1-6, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1. 8. The immunomodulatory protein according to any one of claims 1-6, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:1. 9. The immunomodulatory protein according to any one of claims 1-5, wherein the reference TACI polypeptide is a truncated wild-type TACI extracellular domain containing a cysteine-rich domain 2 (CRD2) but lacking the complete cysteine-rich domain 1 (CRD1), wherein the variant TACI polypeptide comprises one or more amino acid substitutions in the truncated wild-type TACI extracellular domain. 10. The immunomodulatory protein according to any one of claims 1-5 and 9, wherein the reference TACI polypeptide is a truncated wild-type TACI extracellular domain, and at the position shown in SEQ ID NO:122, the truncated wild-type TACI extracellular domain consists of a continuous sequence including amino acid residues 71-104 contained within amino acid residues 67-118, wherein the variant TACI polypeptide comprises one or more amino acid substitutions in the truncated wild-type TACI extracellular domain. 11. The immunomodulatory protein according to claim 9 or claim 10, wherein the reference TACI polypeptide is a truncated wild-type TACI extracellular domain, the length of which is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50 or 51 amino acids. 12. The immunomodulatory protein according to any one of claims 1-5 and 9-11, wherein the reference TACI polypeptide is substantially composed of the CRD2 domain. 13. The immunomodulatory protein according to any one of claims 1-5 and 9-12, wherein the reference TACI polypeptide is a truncated wild-type TACI extracellular domain, the truncated wild-type TACI extracellular domain consisting of amino acid residues 68-110 shown in SEQ ID NO:122. 14. The immunomodulatory protein according to any one of claims 1-5 and 9-13, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13. 15. The immunomodulatory protein according to any one of claims 1-5 and 9-13, wherein the reference TACI polypeptide comprises the sequence shown in SEQ ID NO:13. 16. The immunomodulatory protein according to any one of claims 1-15, wherein the one or more amino acid substitutions are selected from W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions. 17. The immunomodulatory protein according to any one of claims 1-16, wherein the one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. 18. The immunomodulatory protein according to any one of claims 1-16, wherein the one or more amino acid substitutions comprise amino acid substitutions selected from the group consisting of Q75E, K77E, F78Y, R84G, R84Q, A101D and Y102D or any combination thereof. 19. The immunomodulatory protein according to any one of claims 1-16 and 18, wherein the one or more amino acid substitutions include at least amino acid substitution Q75E. 20. The immunomodulatory protein according to any one of claims 1-19, wherein the one or more amino acid substitutions include at least amino acid substitution for K77E. 21. The immunomodulatory protein according to any one of claims 1-16 and 18-20, wherein the one or more amino acid substitutions include at least the amino acid substitution F78Y. 22. The immunomodulatory protein according to any one of claims 1-17 and 19-21, wherein the one or more amino acid substitutions include at least amino acid substitution R84G. 23. The immunomodulatory protein according to any one of claims 1-21, wherein the one or more amino acid substitutions include at least amino acid substitution R84Q. 24. The immunomodulatory protein according to any one of claims 1-16 and 18-23, wherein the one or more amino acid substitutions include at least amino acid substitution A101D. 25. The immunomodulatory protein according to any one of claims 1-23, wherein the one or more amino acid substitutions include Q75E/R84Q, Q75E/K77E, Q75E/F78Y, Q75E/A101D, Q75E/Y102D, F77E/F78Y, K77E/R84Q, K77E/A101D, K77E/Y102D, F78Y/R84Q, F78Y/A101D, F78Y/Y102D, R84Q/A101D, R84Q/Y102D, or A101D/Y102D. 26. The immunomodulatory protein according to any one of claims 1-25, wherein the one or more amino acid substitutions are D85E/K98T, I87L/K98T, R60G/Q75E/L82P, R60G/C86Y, W40R/L82P/F103Y, W40R/Q59R/T61P/K98T, L82P/I87L, G76S/P97S, K77E/R84L/F10 3Y, Y79F/Q99E, L83S/F103S, K77E/R84Q, K77E/A101D, K77E/F78Y/Y102D, Q75E/R84Q, Q75R/R8 4G/I92V, K77E/A101D/Y102D, R84Q/S88N/A101D, R84Q/F103V, K77E/Q95R/A101D or I87M/A101D. 27. The immunomodulatory protein according to any one of claims 1-25, wherein the one or more amino acid substitutions are R84G, A101D, K77E/R84Q, K77E/A101D, K77E/F78Y, K77E/F78Y/Y102D, Q75E/R84Q, K77E/A101D/Y102D, R84Q, K77E, A101D, Q75E, K77E/F78Y/R84Q, F78Y, F78Y/R84Q, F78Y/A101D, F78Y/Y102D or K77E/Y102D. 28. The immunomodulatory protein according to any one of claims 1-18, 20, 21 and 25-27, wherein one or more amino acid substitutions are K77E/F78Y/Y102D. 29. The immunomodulatory protein according to any one of claims 1-19, 23 and 25-27, wherein one or more amino acid substitutions are Q75E/R84Q. 30. The immunomodulatory protein according to any one of claims 1-18, 20 and 24-27, wherein one or more amino acid substitutions are K77E/A101D/Y102D. 31. The immunomodulatory protein according to any one of claims 1-30, wherein the variant TACI polypeptide has increased binding affinity to one or both of APRIL and BAFF compared to the reference TACI polypeptide. 32. The immunomodulatory protein according to any one of claims 1-31, wherein the variant TACI polypeptide has increased binding affinity to APRIL. 33. The immunomodulatory protein according to any one of claims 1-32, wherein the variant TACI polypeptide has increased binding affinity to BAFF. 34. The immunomodulatory protein according to any one of claims 1-33, wherein the variant TACI polypeptide has increased binding affinity for APRIL and BAFF. 35. The immunomodulatory protein according to any one of claims 30-34, wherein the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times. 36. The immunomodulatory protein according to any one of claims 30-35, wherein the variant TACI polypeptide has up to 10 amino acid modifications compared to the reference TACI polypeptide. 37. The immunomodulatory protein according to any one of claims 30-35, wherein the variant TACI polypeptide has up to 5 amino acid modifications compared to the reference TACI polypeptide. 38. The immunomodulatory protein according to any one of claims 1-37, wherein the variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:122 or a specific binding fragment thereof comprising the CRD1 domain and/or the CRD2 domain. 39. The immunomodulatory protein according to any one of claims 1-37, wherein the variant TACI polypeptide has at least 95% sequence identity with SEQ ID NO:122 or a specific binding fragment thereof comprising the CRD1 domain and/or the CRD2 domain. 40. The immunomodulatory protein according to claim 38 or claim 39, wherein the specific binding fragment is shown in SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:130 or SEQ ID NO:131. 41. The immunomodulatory protein according to any one of claims 1-40, wherein the variant TACI polypeptide has at least 90% sequence identity with SEQ ID NO:13. 42. The immunomodulatory protein according to any one of claims 1-40, wherein the variant TACI polypeptide has at least 95% sequence identity with SEQ ID NO:13. 43. The immunomodulatory protein according to any one of claims 1-42, wherein: The variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO: 2-12, 21, 22 and 101-120; or The variant TACI polypeptide comprises the sequence shown in any one of SEQ ID NO:14-20, 23-35, 92-100, and 177-192. 44. The immunomodulatory protein according to any one of claims 1-43, wherein: The variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO: 2-12, 21, 22 and 101-120; or The variant TACI polypeptide consists of or is substantially composed of the sequences shown in any one of SEQ ID NO: 14-20, 23-35, 92-100 and 177-192. 45. The immunomodulatory protein according to any one of claims 1-44, wherein the variant TACI polypeptide is shown in SEQ ID NO:26. 46. The immunomodulatory protein according to any one of claims 1-44, wherein the variant TACI polypeptide is shown in SEQ ID NO:27. 47. An immunomodulatory protein comprising at least one TACI polypeptide, the TACI polypeptide being a truncated wild-type TACI extracellular domain, wherein the truncated wild-type TACI extracellular domain contains a cysteine-rich domain 2 (CRD2) but lacks the complete cysteine-rich domain 1 (CRD1). 48. An immunomodulatory protein comprising at least one TACI polypeptide, the TACI polypeptide being a truncated wild-type TACI extracellular domain, wherein, with respect to the position shown in SEQ ID NO:122, the truncated wild-type TACI extracellular domain comprises a continuous sequence of amino acid residues 71-104 contained within amino acid residues 67-118. 49. An immunomodulatory agent comprising a TACI-Fc fusion protein, the TACI-Fc fusion protein comprising a truncated wild-type TACI extracellular domain, an Fc region, and a linker between the TACI polypeptide and the Fc region, wherein the truncated wild-type TACI extracellular domain contains a cysteine-rich domain 2 (CRD2) but lacks the complete cysteine-rich domain 1 (CRD1). 50. An immunomodulatory agent comprising a TACI-Fc fusion protein, the TACI-Fc fusion protein comprising a truncated wild-type TACI extracellular domain, an Fc region, and a linker between the TACI polypeptide and the Fc region, wherein, with respect to the position shown in SEQ ID NO:122, the truncated wild-type TACI extracellular domain comprises a continuous sequence of amino acid residues 71-104 within amino acid residues 67-118. 51. The immunomodulatory protein according to any one of claims 47-50, wherein the length of the truncated wild-type TACI extracellular domain is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50 or 51 amino acids. 52. The immunomodulatory protein according to any one of claims 47-51, wherein the truncated wild-type TACI extracellular domain is composed of amino acid residues 68-110 shown in SEQ ID NO:122. 53. The immunomodulatory protein according to any one of claims 47-51, wherein the truncated wild-type TACI extracellular domain consists of the amino acid sequence shown in SEQ ID NO:13. 54. An immunomodulatory protein comprising at least one TACI polypeptide, the TACI polypeptide being a truncated TACI polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13. 55. The immunomodulatory protein according to any one of claims 47-54, wherein the truncated TACI polypeptide is bound to APRIL, BAFF, or a BAFF/APRIL heterotrimer. 56. The immunomodulatory protein according to any one of claims 1, 3-48 and 51-55, wherein the immunomodulatory protein comprises a heterologous portion linked to the at least one TACI polypeptide, optionally linked via a linker. 57. The immunomodulatory protein of claim 56, wherein the heterologous portion is a half-life extended portion, a polymerized domain, a targeting portion that binds to molecules on the cell surface, or a detectable marker. 58. The immunomodulatory protein of claim 57, wherein the extended half-life portion comprises a polymerizing domain, albumin, albumin-binding polypeptide, Pro/Ala/Ser (PAS), C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin-binding small molecule, or a combination thereof. 59. The immunomodulatory protein according to any one of claims 1, 3-48 and 51-58, wherein the immunomodulatory protein is a TACI-Fc fusion protein, wherein the at least one TACI polypeptide is linked to the Fc region of an immunoglobulin, optionally via a linker. 60. The immunomodulatory protein according to any one of claims 2, 49, 50, and 56-59, 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), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO:79), GGGGSGGGGSGGGGSGGGS (4xGGGGS; SEQ ID NO:84), GGGGSGGGGSGGGGSGGGSGGGS (5xGGGGS; SEQ ID NO:91), GGGGSSA (SEQ ID NO:80), or GSGGGGSGGGGS (SEQ ID NO:194) or combinations thereof. 61. The immunomodulatory protein according to any one of claims 2, 49, 50, 59 and 60, wherein the immunoglobulin Fc is an IgG1 Fc domain, or a variant Fc exhibiting reduced binding affinity to the Fc receptor and/or reduced effector function, optionally as compared to the wild-type IgG1 Fc domain. 62. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-61, wherein the immunoglobulin Fc is an IgG1 Fc domain and the Fc comprises the amino acid sequence shown in SEQ ID NO:81. 63. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-61, wherein the immunoglobulin Fc is a variant IgG1 Fc domain, the variant IgG1 Fc domain comprising one or more amino acid substitutions selected according to EU numbers from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G and V302C. 64. The immunomodulatory protein of claim 63, wherein the immunoglobulin Fc region comprises amino acid substitutions L234A, L235E and G237A according to EU numbers, optionally wherein the Fc region is shown in any one of SEQ ID NO: 73, 75, 83, 136 or 221. 65. The immunomodulatory protein according to claim 63 or claim 64, wherein the immunoglobulin Fc region further comprises amino acid substitutions A330S and P331S, optionally wherein the Fc region is shown in SEQ ID NO:175 or SEQ ID NO:176. 66. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-65, wherein the TACI-Fc fusion protein comprises the following structure: TACI polypeptide (TACI)-connector-Fc region. 67. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-62 and 66, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:168. 68. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-62 and 66, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:170. 69. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-61 and 63-66, wherein the Fc is a variant Fc comprising the amino acid sequence shown in SEQ ID NO:73. 70. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-61, 63-66 and 69, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:167. 71. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-61, 63-66 and 69, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:169. 72. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-71, wherein the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein. 73. An immunomodulatory protein, said immunomodulatory protein being a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO: 167, linked by covalent disulfide bonds. 74. An immunomodulatory protein, said immunomodulatory protein being a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:168, linked by covalent disulfide bonds. 75. An immunomodulatory protein, said immunomodulatory protein being a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO:169, linked by covalent disulfide bonds. 76. An immunomodulatory protein, said immunomodulatory protein being a homodimer comprising two identical copies of the TACI-Fc fusion protein shown in SEQ ID NO: 170, linked by covalent disulfide bonds. 77. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-65, wherein the TACI-Fc fusion protein comprises the following structure: (TACI)-connector-Fc region-connector-(TACI). 78. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-65 and 77, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:201. 79. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-65 and 77, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:202. 80. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-65, wherein the TACI-Fc fusion protein comprises the following structure: (TACI)-connector-(TACI)-connector-Fc region. 81. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-65 and 80, wherein the TACI-Fc fusion protein is shown in SEQ ID NO:198. 82. The immunomodulatory protein according to any one of claims 2, 49, 50, 59-65 and 77-81, wherein the immunomodulatory protein is a homodimer comprising two identical copies of the TACI-Fc fusion protein. 83. The immunomodulatory protein according to any one of claims 2, 49, 50 and 59-82, wherein the Fc fusion protein neutralizes APRIL and BAFF. 84. The immunomodulatory protein according to any one of claims 2, 49, 50, and 59-83, wherein: The IC50 for neutralizing APRIL 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 foregoing; and/or The IC50 for neutralizing BAFF 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 the aforementioned values. 85. The immunomodulatory protein according to any one of claims 1-84, wherein: The Fc fusion protein blocks the binding of APRIL, BAFF, or 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 subject. 86. The immunomodulatory protein according to any one of claims 1-85, wherein the immunomodulatory protein reduces or inhibits B cell maturation, differentiation and/or proliferation. 87. One or more nucleic acid molecules, said nucleic acid molecules encoding an immunomodulatory protein according to any one of claims 1-86. 88. A vector comprising the nucleic acid molecule according to claim 87. 89. The carrier according to claim 88, wherein the carrier is an expression carrier. 90. The vector of claim 88 or claim 89, wherein the vector is a mammalian expression vector or a viral vector. 91. A cell comprising the nucleic acid according to claim 87 or the vector according to any one of claims 88-90. 92. The cell of claim 91, wherein the cell is a mammalian cell. 93. The cell of claim 91 or claim 92, wherein the cell is a human cell. 94. A method for producing an immunomodulatory protein, the method comprising introducing a nucleic acid molecule according to claim 87 or a vector according to any one of claims 88-90 into a host cell under conditions in which the protein is expressed. 95. The method of claim 94, further comprising isolating or purifying the immunomodulatory protein from the cells. 96. An immunomodulatory protein produced by the method according to claim 94 or claim 95. 97. A pharmaceutical composition comprising an immunomodulatory protein according to any one of claims 1-86 or 96. 98. The pharmaceutical composition of claim 97, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient. 99. An article comprising a pharmaceutical composition according to claim 97 or claim 98 in a vial or container. 100. A kit comprising a pharmaceutical composition according to claim 97 or claim 98, or an article of manufacture according to claim 99, and instructions for use. 101. A method for reducing the immune response of a subject, the method comprising administering an immunomodulatory protein according to any one of claims 1-86 or a pharmaceutical composition according to claim 97 or 98 to a subject in need. 102. The method of claim 101, wherein the B-cell immune response is reduced in the subject, thereby reducing or inhibiting B-cell maturation, differentiation, and/or proliferation. 103. The method of claim 101 or claim 102, wherein the circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer are reduced in the subject. 104. The method according to any one of claims 101-103, wherein reducing the immune response treats the subject's disease, disorder, or condition. 105. A method for reducing circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer in a subject, the method comprising administering an immunomodulatory protein according to any one of claims 1-86 or a pharmaceutical composition according to claim 97 or 98 to the subject. 106. A method of treating a disease, disorder, or ailment of a subject, the method comprising administering an immunomodulatory protein according to any one of claims 1-86 or a pharmaceutical composition according to claim 97 or 98 to the subject in need. 107. The method of claim 104 or claim 106, wherein the disease, disorder or symptom is an autoimmune disease, an inflammatory condition, B-cell carcinoma, antibody-mediated symptom, kidney disease, graft rejection, graft-versus-host disease, or viral infection. 108. The method according to claim 106 or claim 107, wherein the disease, disorder, or condition is selected from: systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris. 109. The method of claim 106 or claim 107, wherein the disease, disorder, or symptom is B-cell cancer and the cancer is myeloma. 110. The pharmaceutical composition according to claim 97 or claim 98, wherein the pharmaceutical composition is used to reduce the immune response of a subject. 111. Use of the immunomodulatory protein according to any one of claims 1-86 or the pharmaceutical composition according to claim 97 or 98 in the manufacture of a medicament for reducing the immune response of a subject. 112. The pharmaceutical composition for the use according to claim 110 or the use according to claim 111, wherein the immune response is a B cell immune response, wherein reducing the immune response reduces or inhibits B cell maturation, differentiation and/or proliferation. 113. The pharmaceutical composition for the use according to any one of claims 110-112 or the use according to any one of claims 110-112, wherein reducing the immune response reduces the circulating levels of APRIL, BAFF, or APRIL/BAFF heterotrimer in the subject. 114. The pharmaceutical composition for the said use according to any one of claims 110-113 or the use according to any one of claims 110-113, wherein reducing the immune response treats the subject's disease, disorder, or condition. 115. The pharmaceutical composition according to claim 97 or claim 98, wherein the pharmaceutical composition is used to treat a disease, disorder or ailment of a subject. 116. Use of the immunomodulatory protein according to any one of claims 1-86 or the pharmaceutical composition according to claim 97 or claim 98 in the manufacture of a medicament for treating a disease, disorder or ailment of a subject. 117. The pharmaceutical composition for the said use according to claim 115 or the use according to claim 116, wherein the disease, disorder or condition is an autoimmune disease, an inflammatory condition, B-cell carcinoma, antibody-mediated disease, kidney disease, graft rejection, graft-versus-host disease, or viral infection. 118. The pharmaceutical composition for the said use according to any one of claims 115-117 or the use according to any one of claims 115-117, wherein the disease, disorder or condition is selected from: systemic lupus erythematosus (SLE); Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid antibody syndrome (APS), autoimmune polyendocrine syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris. 119. The pharmaceutical composition for the said use according to any one of claims 115-117 or the use according to any one of claims 115-117, wherein the disease, disorder or symptom is B-cell cancer and the cancer is myeloma.