DcR3 variants

Abstract

The present invention addresses the problem of providing: a DcR3 variant that has the binding activity (preferably neutralizing activity) of DcR3 to ligands and that exhibits improved in vivo kinetics and / or reduces the amount of generated aggregates compared to wild-type DcR3 when produced using mammal-derived cells as hosts; DNA for encoding the DcR3 variant; a vector including the DNA; a transformant obtained by introducing the vector; a variant production method using the transformant; and an agent that includes the variant as an active ingredient and that is for preventing or treating an autoimmune disease, an inflammatory disease, or an allergic disease. In order to solve this problem, the present invention provides a DcR3 variant including part of DcR3 and part of a TNF superfamily molecule.

Description

Technical Field

[0001] The present invention relates to a DcR3 variant, which is a variant of wild-type DcR3. More specifically, the present invention relates to a DcR3 variant that has binding activity (preferably neutralizing activity) against a ligand of DcR3 and shows a reduced amount of aggregate formation and / or improved pharmacokinetics when produced using mammalian-derived cells as a host, as compared to wild-type DcR3.

Background Art

[0002] Tumor necrosis factor (TNF) superfamily (TNFSF) and TNF receptor superfamily (TNFRSF) form 18 ligands and 29 receptor families that are structurally similar, respectively. Antibodies and Fc fusion proteins against many molecules included in this family have been developed and marketed, and have shown therapeutic effects in the treatment of various autoimmune diseases (Non-Patent Document 1).

[0003] Many of TNFRSF are expressed on the cell membrane and transmit signals downstream upon ligand binding, but some molecules are decoy receptors (DcRs) that do not participate in signal transduction. So far, four decoy receptors, DcR1, DcR2, DcR3, and OPG (Osteoprotegerin), have been identified.

[0004] OPG is a soluble decoy receptor for RANKL and TRAIL, and inhibits signal transduction by competing with the ligand binding of the RANKL receptor and the TRAIL receptor. On the other hand, DcR1 and DcR2 are decoy receptors that neutralize the ligand by competitively inhibiting the binding of the receptor that transmits signals to TRAIL, and DcR3 is a decoy receptor that neutralizes the ligand by competitively inhibiting the binding of the receptor that transmits signals to three molecules, LIGHT, TL1A, and FasL (Non-Patent Document 2).

[0005] DcR3 is a soluble molecule consisting of 300 amino acid residues. It has a signal peptide on the N-terminal side, followed by four cysteine-rich domains (CRD) characteristic of TNFRSF (CRD1, CRD2, CRD3, and CRD4), and a heparin sulfate-binding region (HBD) rich in basic amino acids containing a heparin sulfate-binding motif on the C-terminal side. LIGHT, TL1A, and FasL all bind via CRD2 and CRD3 of DcR3 (Non-Patent Documents 3, 4, 5).

[0006] In addition to its function as a decoy receptor by ligand neutralization, DcR3 has a function as an immunomodulatory molecule based on the activity of HBD. For example, it directly binds via HBD to glycosaminoglycans (GAG) such as heparin sulfate on the cell membranes of monocytes, macrophages, or dendritic cells, and induces Th2 induction by dendritic cell differentiation, induction of M2 macrophages, enhanced adhesion of monocytes, osteoclast differentiation, or decreased expression of MHC class II molecules, etc., and various immunosuppressive and immunostimulatory effects have been reported. (Non-Patent Documents 6, 12).

[0007] The DcR3 ligand has been reported to be involved in autoimmune diseases, inflammatory diseases, allergies, cancers, infectious diseases, or other various inflammatory reactions. For example, LIGHT, TL1A, and FasL are all included in susceptibility loci in inflammatory bowel disease (IBD), and in particular, for TL1A, the existence of multiple gene polymorphisms related to the disease state has been found. In addition, there are reports of increased expression of the DcR3 ligand in the blood or tissues of IBD patients, and improvement of the disease state by inhibition of the DcR3 ligand in a mouse enteritis model (Non-Patent Documents 6-9).

[0008] The expression of DcR3 in normal human tissues is at an extremely low level, but its expression is induced by infection or tissue damage. Furthermore, it has been found that the blood levels of DcR3 are elevated in various autoimmune or inflammatory diseases such as inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), atopic dermatitis (AD), or rheumatoid arthritis (RA). Although a DcR3 homolog has not been identified in mice, in mouse disease models such as type I diabetes models, multiple sclerosis models, or nephritis models, the improvement of the disease state in transgenic mice of human DcR3 and the drug efficacy by plasmid or recombinant DcR3 administration have been confirmed respectively (Non-Patent Documents 6, 10).

[0009] Genentech cloned the human DcR3 gene and showed that a fusion of DcR3 and the Fc region of human IgG1 binds to soluble human FasL and inhibits human FasL-dependent apoptosis in vitro (Patent Document 1).

[0010] Eli Lilly obtained FLINT, a protease-resistant DcR3 mutant, by a single amino acid mutation (R218Q) of wild-type DcR3, and reported that its pharmacokinetics were improved in mice and monkeys compared to wild-type DcR3. However, the blood half-lives when wild-type DcR3 and FLINT were administered to cynomolgus monkeys by intravenous injection at 0.5 mg / kg were extremely short, 9 hours and 12.3 hours respectively (Patent Documents 2, 3, Non-Patent Document 11).

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0012] [Non-Patent Document 1] Nature Reviews Drug Discovery, 2013, 12: p. 147 - 168 [Non-Patent Document 2] Nature Reviews Cancer, 2002.2: p. 420 - 430 [Non-Patent Document 3] Structure, 2011, 19: p. 162 - 171 [Non-Patent Document 4] Structure, 2014, 22: p. 1252 - 1262 [Non-Patent Document 5] Structure, 2016, 24: p. 2016 - 2023 [Non-Patent Document 6] Biochemical Pharmacology, 2011, 81: p. 838 - 847 [Non-Patent Document 7] Immunology, 2009, 128: p. 451 - 458 [Non-Patent Document 8] P.N.A.S., 2006, 103: p. 8441 - 8446 [Non-Patent Document 9] Am. J. Physiol. Gastrointest. Liver Physiol., 2003, 285: p. G754 - G760 [Non-Patent Document 10] Journal of Biomedical Science, 2017, 24: 39 [Non-Patent Document 11] Drug Metabolism and Disposition, 2003, 31: p. 502 - 507 [Non-Patent Document 12] J. Immunol., 2006, 176: p. 173 - 180 [Summary of the Invention] [Problems to be Solved by the Invention]

[0013] FLINT, an amino acid variant of wild-type DcR3, has extremely poor pharmacokinetics, and as a recombinant preparation whose mechanism of action is ligand neutralization, frequent administration is required, which is not desirable as a pharmaceutical. Therefore, DcR3 variants that can ensure an administration interval for a certain period by improving pharmacokinetics are expected to be useful as pharmaceuticals.

[0014] Moreover, as far as is currently known, no functional DcR3 variant has been found that shows a reduced amount of aggregate formation when expressed, isolated, and purified using mammalian-derived cells as a host, and that has neutralizing activity against DcR3 ligands.

[0015] The present invention aims to provide a DcR3 variant having binding activity (preferably neutralizing activity) against a DcR3 ligand, showing a reduced amount of aggregate formation compared to wild-type DcR3 when producing a DcR3 protein using mammalian-derived cells as a host, and / or showing improved pharmacokinetics, a DNA encoding the DcR3 variant, a vector containing the DNA, a transformant obtained by introducing the vector, a method for producing the variant using the transformant, and a pharmaceutical composition containing the variant as an active ingredient and a preventive or therapeutic agent for autoimmune diseases, inflammatory diseases, or allergies.

Means for Solving the Problems

[0016] To solve the above problems, the present invention provides the following inventions. [1] A DcR3 variant, which is a variant of wild-type Decoy Receptor 3 (hereinafter abbreviated as DcR3), and shows improved pharmacokinetics compared to the wild-type DcR3. [2] The DcR3 variant according to [1], which has one or more N-glycoside-linked complex sugar chains. [3] The DcR3 variant according to [1] or [2], which has neutralizing activity against at least one of LIGHT, TL1A, and FasL. [4] The DcR3 variant according to any one of [1] to [3], which has neutralizing activity against all of LIGHT, TL1A, and FasL. [5]The DcR3 variant according to any one of [1] to [3], which has no neutralizing activity against FasL and has neutralizing activity against at least one of LIGHT and TL1A. [6]The DcR3 variant according to any one of [1] to [3] and [5], which has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A. [7]In the cysteine-rich region of wild-type DcR3, a first chimeric cysteine-rich region consisting of an amino acid sequence in which at least a part of the cysteine-rich domain (hereinafter abbreviated as CRD) of wild-type DcR3 is replaced by at least a part of the cysteine-rich domain of a TNF receptor superfamily molecule other than DcR3, or a second chimeric cysteine-rich region consisting of an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence of the first chimeric cysteine-rich region, and the DcR3 variant containing the same. [8]The DcR3 variant according to [7], which has one or more N-glycoside bond complex-type sugar chains. [9]The DcR3 variant according to [7] or [8], which has neutralizing activity against at least one of LIGHT, TL1A and FasL.

[10] The DcR3 variant according to any one of [7] to [9], which has neutralizing activity against all of LIGHT, TL1A and FasL.

[11] The DcR3 variant according to any one of [7] to [9], which has no neutralizing activity against FasL and has neutralizing activity against at least one of LIGHT and TL1A.

[12] The DcR3 variant according to any one of [7] to [9] and

[11] , which has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A.

[13] The DcR3 variant according to any one of [7] to

[12] , wherein the TNF receptor superfamily molecule is OPG.

[14] At least a part of the cysteine-rich domain of the wild-type DcR3 is selected from all or part of CRD1, all or part of CRD2, all or part of CRD3, and all or part of CRD4. The DcR3 variant according to any one of [7] to

[13] , wherein at least a part of the cysteine-rich domain of the TNF receptor superfamily molecule is selected from all or part of CRD1, all or part of CRD2, all or part of CRD3, and all or part of CRD4.

[15] The first chimeric cysteine-rich region is Substitution at a part of CRD1 of the wild-type DcR3 with a part of CRD1 of the TNF receptor superfamily molecule corresponding to the part of CRD1 of the wild-type DcR3. Substitution of all of CRD1 of the wild-type DcR3 with all of CRD1 of the TNF receptor superfamily molecule. Substitution at a part of CRD2 of the wild-type DcR3 with a part of CRD2 of the TNF receptor superfamily molecule corresponding to the part of CRD2 of the wild-type DcR3. Substitution of all of CRD2 of the wild-type DcR3 with all of CRD2 of the TNF receptor superfamily molecule. Substitution at a part of CRD3 of the wild-type DcR3 with a part of CRD3 of the TNF receptor superfamily molecule corresponding to the part of CRD3 of the wild-type DcR3. Substitution of all of CRD3 of the wild-type DcR3 with all of CRD3 of the TNF receptor superfamily molecule. Substitution at a part of CRD4 of the wild-type DcR3 with a part of CRD4 of the TNF receptor superfamily molecule corresponding to the part of CRD4 of the wild-type DcR3, and Substitution of all of CRD4 of the wild-type DcR3 with all of CRD4 of the TNF receptor superfamily molecule. The DcR3 variant according to

[14] , having one or more substitutions selected from the above.

[16] The DcR3 variant according to

[15] , wherein in the first chimeric cysteine-rich region, part or all of the CRD2 of the wild-type DcR3 is retained.

[17] The DcR3 variant according to

[15] or

[16] , wherein in the first chimeric cysteine-rich region, part or all of the CRD3 of the wild-type DcR3 is retained.

[18] The DcR3 variant according to any one of

[14] to

[17] , wherein the first chimeric cysteine-rich region contains the amino acid sequence of any of the following (a), (b), (c), or (d), and the second chimeric cysteine-rich region contains the amino acid sequence of the following (e). (a) In the amino acid sequence of the cysteine-rich region of the wild-type DcR3, the amino acid sequence in which CRD1 of the wild-type DcR3 is replaced with CRD1 of OPG (b) In the amino acid sequence of the cysteine-rich region of the wild-type DcR3, the amino acid sequence in which CRD4 of the wild-type DcR3 is replaced with CRD4 of OPG (c) In the amino acid sequence of the cysteine-rich region of the wild-type DcR3, the amino acid sequence in which CRD1 of the wild-type DcR3 is replaced with CRD1 of OPG and CRD4 of the wild-type DcR3 is replaced with CRD4 of OPG (d) In the amino acid sequence of (a), (b), or (c), the portion from the 103rd to the 123rd amino acid from the N-terminus is replaced with the corresponding portion of the amino acid sequence of the cysteine-rich domain of OPG (e) In the amino acid sequence of (a), (b), (c), or (d), the amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added

[19] The amino acid sequence of (a) is the amino acid sequence consisting of the first 1 to 164 amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 26 or 50, The amino acid sequence of (b) is the amino acid sequence consisting of the first 1 to 164 amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 28 or 52, The amino acid sequence of (c) is an amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 30 or 54, The DcR3 variant according to

[18] , wherein the amino acid sequence of (d) is an amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 32 or 56.

[20] The DcR3 variant according to

[18] or

[19] , wherein the amino acid sequence of (e) has a substitution of one or more amino acids selected from the group consisting of the 57th Glu, 58th Arg, and 60th Arg from the N-terminus of the amino acid sequence of (a), (b), (c), or (d) with other amino acids.

[21] The DcR3 variant according to any one of

[18] to

[20] , wherein the amino acid sequence of (e) has substitutions of the 57th Glu and 58th Arg from the N-terminus of the amino acid sequence of (a), (b), (c), or (d) with other amino acids.

[22] The DcR3 variant according to any one of

[18] to

[21] , wherein the amino acid sequence of (e) has one or more substitutions selected from the group consisting of a substitution of the 57th Glu of the amino acid sequence of (a), (b), (c), or (d) from the N-terminus with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met, a substitution of the 58th Arg with Asp, Glu, or Thr, and a substitution of the 60th Arg with Lys.

[23] The DcR3 variant according to any one of

[18] to

[22] , wherein the amino acid sequence of (e) has a substitution of the 57th Glu of the amino acid sequence of (a), (b), (c), or (d) from the N-terminus with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met, and a substitution of the 58th Arg with Asp, Glu, or Thr.

[24] The DcR3 variant according to any one of

[18] to

[23] , wherein the amino acid sequence of (e) has a substitution selected from the following (f) to (i). (f) Substitution of the 131st and 144th Asn from the N-terminus of the amino acid sequence of (b), (c), or (d) with other amino acids (g) Substitutions of Asn at positions 131, 144, and 157 from the N-terminus of the amino acid sequence of (b), (c), or (d) with other amino acids (h) Substitutions of Thr at position 133 and Ser at position 146 from the N-terminus of the amino acid sequence of (b), (c), or (d) with other amino acids (i) Substitutions of Thr at position 133, Ser at position 146, and Thr at position 159 from the N-terminus of the amino acid sequence of (b), (c), or (d) with other amino acids

[25] The DcR3 variant according to any one of

[18] to

[24] , wherein the amino acid sequence of (e) has substitutions selected from the following (f’) to (i’). (f’) Substitutions of Asn at positions 131 and 144 from the N-terminus of the amino acid sequence of (b), (c), or (d) with Ser (g’) Substitutions of Asn at positions 131, 144, and 157 from the N-terminus of the amino acid sequence of (b), (c), or (d) with Ser (h’) Substitutions of Thr at position 133 and Ser at position 146 from the N-terminus of the amino acid sequence of (b), (c), or (d) with Ala (i’) Substitutions of Thr at position 133, Ser at position 146, and Thr at position 159 from the N-terminus of the amino acid sequence of (b), (c), or (d) with Ala

[26] The DcR3 variant according to any one of

[18] to

[25] , wherein the amino acid sequence of (e) is an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of the amino acid sequences described in SEQ ID NO: 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284, or 286.

[27] The DcR3 variant is a DcR3 variant comprising the first or second chimeric cysteine-rich region and part or all of the heparin sulfate-binding region of the wild-type DcR3 bound to the C-terminal side of the first or second chimeric cysteine-rich region, or a DcR3 variant comprising the first or second chimeric cysteine-rich region and not comprising the heparin sulfate-binding region of the wild-type DcR3, the DcR3 variant according to any one of [7] to

[26] .

[28] The DcR3 variant is (I) the amino acid sequence set forth in SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence, and (II) the amino acid sequence set forth in SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284 or 286, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence The DcR3 variant according to

[27] , comprising any one amino acid sequence selected from the group consisting of

[29] The DcR3 variant according to [7] to

[28] , comprising an Fc region derived from a human IgG1, IgG2 or IgG4 antibody, or a mutant Fc region consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of the Fc region.

[30] The DcR3 variant according to

[29] , wherein the Fc region or the mutant Fc region is bound to the C-terminal side of the first or second chimeric cysteine-rich region via another region or a linker.

[31] The DcR3 variant according to

[29] or

[30] , wherein the mutant Fc region has a substitution of Cys at position 220 indicated by the EU index with Ser in the amino acid sequence of the heavy chain of human IgG1.

[32] The variant Fc region has substitutions of Leu at position 234 with Ala, Leu at position 235 with Ala, and Gly at position 237 with Ala in the amino acid sequence of the heavy chain of human IgG1, and is the DcR3 variant described in

[31] .

[33] The variant Fc region has a substitution of Asn at position 434 with Ala in the amino acid sequence of the heavy chain of human IgG1, and is the DcR3 variant described in

[31] or

[32] .

[34] The variant Fc region has substitutions of Met at position 252 with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu in the amino acid sequence of the heavy chain of human IgG1, and is the DcR3 variant described in

[31] .

[35] The variant Fc region has substitutions of Ser at position 228 with Pro, Leu at position 235 with Glu, and Arg at position 409 with Lys in the amino acid sequence of the heavy chain of human IgG4, and is the DcR3 variant described in

[29] or

[30] .

[36] The DcR3 variant contains a variant Fc region consisting of the amino acid sequence described in SEQ ID NO: 72, 74, 156, 158, 160, 162, 164, 166, 311, 312 or 313, and is the DcR3 variant described in

[29] or

[30] .

[37] The DcR3 variant is the amino acid sequence described in SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 150, 168, 170, 172, 174, 176, 178, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 288, 290, 292, 294, 296, 298, 300, 302, 304, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 or 337, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence, and is the DcR3 variant described in any one of

[29] to

[36] .

[38] A DcR3 variant composition comprising the DcR3 variant described in any one of [1] to

[37] .

[39] The composition according to

[38] , comprising a DcR3 variant having one or more N-glycoside-linked complex sugar chains and a DcR3 variant having no N-glycoside-linked complex sugar chains.

[40] DNA encoding the DcR3 variant described in any one of [1] to

[37] .

[41] A recombinant vector containing the DNA according to

[40] .

[42] A transformant obtained by introducing the recombinant vector according to

[41] into a host cell.

[43] The transformant according to

[42] , wherein the host cell is a cell derived from a mammal.

[44] The transformant according to

[43] , wherein the cell derived from a mammal is a CHO cell. Culturing the transformant according to any one of

[45] to

[44] in a medium to produce and accumulate the DcR3 variant according to any one of [1] to

[37] , and purifying the DcR3 variant from the obtained culture solution. A method for producing a DcR3 variant or a DcR3 variant composition, characterized by the above. A DcR3 variant or a DcR3 variant composition produced by using the production method according to

[46] . A pharmaceutical composition containing, as an active ingredient, the DcR3 variant or the DcR3 variant composition according to any one of

[47] , [1] to

[39] , and

[46] . The pharmaceutical composition according to

[48] , which is a prophylactic or therapeutic agent for autoimmune diseases, inflammatory diseases, or allergic diseases. A method for preventing or treating an autoimmune disease, an inflammatory disease, or an allergic disease, which comprises administering the pharmaceutical composition according to

[47] or

[48] to a patient in need of prevention or treatment of an autoimmune disease, an inflammatory disease, or an allergic disease.

Advantages of the Invention

[0017] According to the present invention, there are provided a DcR3 variant having a binding activity (preferably a neutralizing activity) against a ligand of DcR3, and having a reduced amount of aggregate formation compared to wild-type DcR3 when producing the DcR3 protein using mammalian-derived cells as a host, and / or showing improved pharmacokinetics, a DNA encoding the DcR3 variant, a vector containing the DNA, a transformant obtained by introducing the vector, a method for producing the variant using the transformant, and a pharmaceutical composition containing the variant as an active ingredient and a prophylactic or therapeutic agent for autoimmune diseases, inflammatory diseases, or allergies.

Brief Description of the Drawings

[0018]

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Mode for Carrying Out the Invention

[0019] Hereinafter, preferred embodiments for carrying out the present invention will be described. Note that the embodiments described below show an example of a typical embodiment of the present invention, and the scope of the present invention is not construed narrowly thereby.

[0020] Among the embodiments described below, two or more embodiments can be combined, and such combinations are also included in the present invention.

[0021] The present invention relates to a DcR3 variant that is a variant of wild-type DcR3. Specifically, the present invention relates to a DcR3 variant having DcR3 ligand-binding activity (or neutralizing activity) and showing improved pharmacokinetics compared to wild-type DcR3 and / or reduced aggregability compared to wild-type DcR3 during production in mammalian cells, and a DcR3 variant containing a cysteine-rich domain in which mutations are introduced into one or more amino acids in the cysteine-rich domain of wild-type DcR3.

[0022] 1. Wild-type DcR3 DcR3 is generally also called Decoy Receptor 3, DCR3, TNFRSF6B (Tumor necrosis factor receptor superfamily member 6B), TR6 or M68. DcR3 is a soluble decoy receptor belonging to the TNF receptor superfamily and lacking a transmembrane domain. By binding to three ligands, LIGHT, TL1A, or FasL, it competitively inhibits the binding of each ligand to its receptor and neutralizes the ligand. Also, as a function different from ligand neutralization, it has been reported to directly bind to glycosaminoglycans (GAGs) such as heparan sulfate on the cell membranes of monocytes, macrophages, or dendritic cells via a heparan sulfate binding domain (HBD), inducing various immunosuppressive and immunostimulatory effects [Biochemical. Pharmacology, 2011, 81: p. 838 - 847, J. Immunol., 2006, 176: p. 173 - 180].

[0023] Naturally occurring DcR3 has CRD1, CRD2, CRD3, CRD4 and HBD in order from the N-terminal side. Naturally occurring DcR3 further has a region existing between CRD1 and CRD2, a region existing between CRD2 and CRD3, a region existing between CRD3 and CRD4, and a region existing between CRD4 and HBD. The cysteine-rich region of naturally occurring DcR3 is the region from the N-terminal of CRD1 to the C-terminal of CRD4, including CRD1, CRD2, CRD3 and CRD4, and also including the region existing between CRD1 and CRD2, the region existing between CRD2 and CRD3, and the region existing between CRD3 and CRD4. "Wild-type DcR3" means a molecule containing a cysteine-rich region and HBD, where the cysteine-rich region and HBD are wild-type (i.e., the cysteine-rich region and HBD are the same as those of naturally occurring DcR3). Therefore, in addition to naturally occurring DcR3, variants of naturally occurring DcR3 such as gene polymorphisms and isoforms are also included in "wild-type DcR3" as long as they contain the cysteine-rich region and HBD of naturally occurring DcR3. Also, immature DcR3 and mature DcR3 are included in "wild-type DcR3". "Immature DcR3" means DcR3 having a signal peptide, and "mature DcR3" means DcR3 with the signal peptide cleaved. The signal peptide can be any of a sequence derived from naturally occurring DcR3, an artificial sequence, a sequence derived from an expression vector, or a sequence derived from another protein suitable for the host cell expressing naturally occurring DcR3. Also, when the cleavage site differs depending on the signal peptide used, sequences with different amino acids at the N-terminus of the mature form are also included in "wild-type DcR3". The CRD of naturally occurring DcR3 and the CRD of wild-type DcR3 are also referred to as "wild-type CRD", and the cysteine-rich region of naturally occurring DcR3 and the cysteine-rich region of wild-type DcR3 are also referred to as "wild-type cysteine-rich region".

[0024] The wild-type DcR3 in the present invention is not limited in its origin, and examples include DcR3 derived from various eukaryotes. For example, DcR3 derived from amphibians such as frogs, birds such as chickens, or mammals such as primates including humans, or even-toed ungulates such as pigs and cows can be mentioned. When using the DcR3 variant of the present invention in humans, it is preferable to use DcR3 derived from humans as the wild-type DcR3.

[0025] The cDNA sequence of human-derived DcR3 is represented by SEQ ID NO: 1, and the corresponding mRNA sequence is registered in GenBank (NCBI, USA) under the accession number: NM_003823.3. The amino acid sequence of human-derived DcR3 is represented by SEQ ID NO: 2, and is registered in GenBank (NCBI, USA) under the accession number: NP_003814.1. Immature human DcR3 has a signal peptide at the N-terminus, followed by four CRDs (CRD1, CRD2, CRD3, CRD4) characteristic of the TNF receptor superfamily, and an HBD rich in basic amino acids at the C-terminus. Mature human DcR3 is the one in which the signal peptide of the immature form is cleaved. The amino acid sequence of mature human DcR3 is represented by, for example, SEQ ID NO: 4, and the nucleotide sequence of DNA encoding the amino acid sequence of mature human DcR3 is represented by, for example, SEQ ID NO: 3. In the present invention, among the amino acid sequence of human DcR3 (SEQ ID NO: 2), the region from the 30th to 70th positions from the N-terminus is defined as CRD1 (SEQ ID NO: 6), the region from the 73rd to 113th positions is defined as CRD2 (SEQ ID NO: 8), the region from the 115th to 150th positions is defined as CRD3 (SEQ ID NO: 10), the region from the 153rd to 193rd positions is defined as CRD4 (SEQ ID NO: 12), and the region from the 196th to 300th positions is defined as HBD (SEQ ID NO: 48) (Figure 2). The nucleotide sequences of DNA encoding the amino acid sequences of CRD1, CRD2, CRD3, CRD4, and HBD of human DcR3 are represented by, for example, SEQ ID NOs: 5, 7, 9, 11, and 47, respectively. In addition, there are multiple definitions of the amino acid sequence of CRD other than those described above, but any definition of the amino acid sequence of CRD can be used for the DcR3 variant of the present invention using known information [UniProt O95407, GenBank NP_003814.1, Structure, 2011, 19: p. 162-171].

[0026] As described above, ligands of DcR3 include LIGHT, TL1A, and FasL, all of which belong to TNFSF.

[0027] LIGHT (lymphotoxin - like, exhibits inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for HVEM, a receptor expressed by T lymphocytes) is generally also called TNFSF14 (Tumor necrosis factor superfamily member 14), LTg, HVEM - L or CD258. The mRNA sequence of human LIGHT and its corresponding cDNA sequence are registered in GenBank (NCBI, USA) under accession number: NM_003807.4, and the amino acid sequence is registered under accession number: NP_003798.2. Soluble LIGHT is generated after being expressed on the cell membrane as membrane - bound LIGHT and then the extracellular region is shed by proteases. The cleavage site in membrane - bound LIGHT is between the 82nd and 83rd amino acids of NP_003798.2. Both the soluble and membrane - bound forms are functional.

[0028] TL1A (Tumor necrosis factor (TNF) - like cytokine 1A) is generally also called TNFSF15 (TNF superfamily member 15), TL1, VEGI or VEGI - 251. The mRNA sequence of human TL1A and its corresponding cDNA sequence are registered in GenBank (NCBI, USA) under accession number: NM_005118.3, and the amino acid sequence is registered under accession number: NP_005109.2. Soluble TL1A is generated after being expressed on the cell membrane as membrane - bound TL1A and then the extracellular region is shed by proteases. The cleavage site in membrane - bound TL1A is between the 71st and 72nd amino acids of NP_005109.2, and both the soluble and membrane - bound forms are functional.

[0029] FasL (Fas ligand) is generally also referred to as FASLG, TNFSF6 (Tumor necrosis factor superfamily member 6), CD178, or APT1LG1. The mRNA sequence of human FasL and the corresponding cDNA sequence are registered in GenBank (NCBI, USA) under the accession number: NM_000639.2, and the amino acid sequence is registered under the accession number: NP_000630.1. Soluble FasL is generated by the shedding of the extracellular region by protease after being expressed on the cell membrane as membrane-bound FasL. The cleavage site in membrane-bound FasL is between the 81st and 82nd, or between the 129th and 130th amino acids of NP_000630.1. In vivo, membrane-bound FasL is mainly reported as the functional ligand.

[0030] Note that polymorphisms or isoforms are often observed in genes encoding eukaryotic proteins. Genes in which such polymorphisms cause mutations in the nucleotide sequence or amino acid sequence are also included in the genes encoding LIGHT, TL1A, or FasL in the present invention.

[0031] 2. DcR3 variants The DcR3 variant of the present invention contains a chimeric cysteine-rich region.

[0032] 2-1. Chimeric cysteine-rich region The chimeric cysteine-rich region of the present invention consists of an amino acid sequence in which one or more amino acids have been mutated in the amino acid sequence of the cysteine-rich region of wild-type DcR3. The phrase "a mutation has been introduced into a certain amino acid sequence / nucleotide sequence" means that one or more amino acids / nucleotides have been substituted, deleted, inserted, or added in that sequence. "Substitution, deletion, insertion, or addition" also includes combinations of two or more mutations selected from substitution, deletion, insertion, and addition. The mutation is introduced into at least one or more CRDs selected from CRD1, CRD2, CRD3, and CRD4 in the cysteine-rich region of wild-type DcR3. In the cysteine-rich region of wild-type DcR3, a mutation may or may not be introduced into the region existing between CRD1 and CRD2, the region existing between CRD2 and CRD3, and the region existing between CRD3 and CRD4. When a mutation is introduced into one or more regions selected from the region existing between CRD1 and CRD2, the region existing between CRD2 and CRD3, and the region existing between CRD3 and CRD4, the number of amino acids constituting each region after the introduction of the mutation is usually 1 to 10, preferably 1 to 7, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid sequence of each region after the introduction of the mutation is not particularly limited. The mutations introduced into the cysteine-rich region of wild-type DcR3 include both naturally occurring mutations and artificially induced mutations. Examples of the chimeric cysteine-rich region in the present invention include a chimeric cysteine-rich region consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence of the cysteine-rich region of wild-type DcR3. Examples of such a chimeric cysteine-rich region include the first and second chimeric cysteine-rich regions described below.

[0033] 2-1-1. The First Chimeric Cysteine-Rich Region The first chimeric cysteine-rich region consists of an amino acid sequence in which at least a part of the CRD of wild-type DcR3 is replaced with another peptide or protein in the amino acid sequence of the cysteine-rich region of wild-type DcR3. That is, the first chimeric cysteine-rich region includes an amino acid sequence derived from the cysteine-rich region of wild-type DcR3 and an amino acid sequence derived from another peptide or protein.

[0034] Among the cysteine-rich regions of wild-type DcR3, the portion replaced with another peptide or protein is preferably at least a part of at least one CRD selected from CRD1, CRD2, CRD3, and CRD4. Therefore, among the cysteine-rich regions of wild-type DcR3, the portion replaced with another peptide or protein can be selected from all or part of CRD1, all or part of CRD2, all or part of CRD3, and all or part of CRD4.

[0035] The amino acid sequence of the first chimeric cysteine-rich region also includes an amino acid sequence in which, in the amino acid sequence of the cysteine-rich region of wild-type DcR3, in addition to at least a part of the CRD of wild-type DcR3, a portion other than the wild-type CRD is replaced with another peptide or protein. The portion other than the CRD replaced with another peptide or protein can be selected from the regions existing between CRD1 and CRD2, the regions existing between CRD2 and CRD3, and the regions existing between CRD3 and CRD4. The portion other than the CRD replaced with another peptide or protein may be one portion or two or more portions. For example, an amino acid sequence in which the amino acid sequence of the region existing between CRD1 and CRD2, the region existing between CRD2 and CRD3, or the region existing between CRD3 and CRD4 is replaced with another peptide or protein is also included in the amino acid sequence of the first chimeric cysteine-rich region.

[0036] Among the cysteine-rich regions of wild-type DcR3, the portion to be replaced with other peptides or proteins may be one portion or two or more portions. When at least a part of the CRD in the cysteine-rich region of wild-type DcR3 is replaced, the replaced portion may be all or part of one CRD or all or part of a plurality of CRDs. However, all or part of CRD2 of wild-type DcR3 involved in the binding to LIGHT, TL1A, and FasL, and / or all or part of the region of CRD3 are retained, and it is more preferable that all or part of the other CRDs are replaced with other peptides or proteins.

[0037] Other peptides or proteins to be substituted may be either natural or artificial peptides or proteins, and examples include at least a part of the CRD derived from a protein other than DcR3. In a preferred embodiment, the other peptide or protein to be substituted is at least a part of the CRD of a TNF receptor superfamily (TNFRSF) molecule other than DcR3. That is, in a preferred embodiment, the first chimeric cysteine-rich region consists of an amino acid sequence in which at least a part of the cysteine-rich domain of wild-type DcR3 is substituted with at least a part of the cysteine-rich domain of a TNFRSF molecule other than DcR3 in the amino acid sequence of the cysteine-rich region of wild-type DcR3. At least a part of the CRD of the TNFRSF molecule can be selected from all or part of CRD1, all or part of CRD2, all or part of CRD3, and all or part of CRD4. Accordingly, the first chimeric cysteine-rich region may have one or more substitutions selected from substitution of a part of CRD1 of wild-type DcR3 with the corresponding part of CRD1 of the TNFRSF molecule, substitution of all of CRD1 of wild-type DcR3 with all of CRD1 of the TNFRSF molecule, substitution of a part of CRD2 of wild-type DcR3 with the corresponding part of CRD2 of the TNFRSF molecule, substitution of all of CRD2 of wild-type DcR3 with all of CRD2 of the TNFRSF molecule, substitution of a part of CRD3 of wild-type DcR3 with the corresponding part of CRD3 of the TNFRSF molecule, substitution of all of CRD3 of wild-type DcR3 with all of CRD3 of the TNFRSF molecule, substitution of a part of CRD4 of wild-type DcR3 with the corresponding part of CRD4 of the TNFRSF molecule, and substitution of all of CRD4 of wild-type DcR3 with all of CRD4 of the TNFRSF molecule.

[0038] Examples of the first chimeric cysteine-rich region include a chimeric cysteine-rich region having a substitution of the CRD1 of wild-type DcR3 with the CRD1 of a TNFRSF molecule (in this chimeric cysteine-rich region, preferably, the other CRDs of wild-type DcR3 are retained), a chimeric cysteine-rich region having a substitution of the CRD4 of wild-type DcR3 with the CRD4 of a TNFRSF molecule (in this chimeric cysteine-rich region, preferably, the other CRDs of wild-type DcR3 are retained), or a chimeric cysteine-rich region having a substitution of the CRD1 of wild-type DcR3 with the CRD1 of a TNFRSF molecule and a substitution of the CRD4 of wild-type DcR3 with the CRD4 of a TNFRSF molecule (in this chimeric cysteine-rich region, preferably, the other CRDs of wild-type DcR3 are retained). In addition to one or more of these substitutions, a chimeric cysteine-rich region further having a substitution of a part of the CRD2 of wild-type DcR3 with a part of the CRD2 of the corresponding TNFRSF molecule and / or a substitution of a part of the CRD3 of wild-type DcR3 with a part of the CRD3 of the corresponding TNFRSF molecule is also included in the first chimeric cysteine-rich region.

[0039] Twenty-nine receptors belong to human TNFRSF. Each receptor has a CRD in its N-terminal extracellular domain. Typically, each CRD has six Cys residues forming three disulfide bonds, and each receptor has one to four CRDs [Trends Biochem Sci, 2002. 27: p-19-26.].

[0040] As human TNFRSF, for example, DcR1 (TNFRSF10C, TRAIL-R3, LIT, TRID, CD263), DcR2 (TNFRSF10D, TRAIL-R4, TRUNDD, CD264), TNFR type I (TNFRSF1A, TNF-R, CD120a, TNFAR, TNF-R55, TNFR60), TNFR type II (TNFRSF1B, TNFBR, CD120b, TNFR80, p75, TNF-R75), LTBR (Lymphotoxin beta receptor, TNFRSF3, TNFR III, TNFCR, TNFR-RP, TNFR2-RP), OX-40 (TNFRSF4, ACT35, TXGP1L, CD134), CD40 (TNFRSF5, Bp50, p50), Fas (Fas cell surface death receptor, TNFRSF6, CD95, APO-1, APT1, FAS1), CD27 (TNFRSF7, S152, Tp55), CD30 (TNFRSF8, Ki-1), 4-1BB (TNFRSF9, CD137, ILA), DR4 (TNFRSF10A, Apo2, TRAILR-1, CD261), DR5 (TNFRSF10B, TRAIL-R2, KILLER, TRICK2A, TRICKB, CD262), RANK (TNFRSF11A, CD265, FEO), FN14 (TNFRSF12A, TweakR, CD266), TACI (TNFRSF13B, CD267, IGAD2), BAFFR (TNFRSF13C, CD268), HVEM (TNFRSF14, ATAR, TR2, LIGHTR, HVEA, CD270), NGFR (nerve growth factor receptor, TNFRSF16, p75NTR, CD271), BCMA (TNFRSF17, BCM, CD269, TNFRSF13A), GITR (TNFRSF18, AITR, CD357), TROY (TNFRSF19, TAJ-alpha, TAJ, TRADE), RELT (TNFRSF19L), DR6 (Death Receptor 6, TNFRSF21, CD358), DR3 (Death Receptor 3, TNFRSF25, TRAMP, WSL-1, LARD, WSL-LR, DDR3, TR3, APO-3), EDAR (ectodysplasin Areceptor, ED3, DL, ED5, EDA3, Edar, ED1R, EDA1R), EDAR2R (ectodysplasin A2 receptor, XEDAR, EDAA2R, EDA - A2R, TNFRSF27), or Osteoprotegerin (OPG, TNFRSF11B, TR1, OCIF).

[0041] In the first chimeric cysteine - rich region, other peptides or proteins that replace at least a part of wild - type DcR3 include, but are not limited to, among TNFRSF, OPG is particularly preferred.

[0042] OPG includes, but is not limited to its origin, OPGs derived from various eukaryotes. For example, OPGs derived from amphibians such as frogs, birds such as chickens, or mammals, for example, primates including humans, even - toed ungulates such as pigs and cows, or rodents including mice, etc.

[0043] The cDNA sequence of human - derived OPG is represented by SEQ ID NO: 13, and the corresponding mRNA sequence is registered in GenBank (NCBI, USA) with accession number: NM_002546.3. The amino - acid sequence of human - derived OPG is represented by SEQ ID NO: 14, and is registered in GenBank (NCBI, USA) with accession number: NP_002537.3.

[0044] Immature human OPG (SEQ ID NO: 14) has a signal peptide at the N-terminus. Mature human OPG is the one in which the signal peptide of the immature form has been cleaved. The amino acid sequence of mature human OPG is represented by, for example, SEQ ID NO: 16, and the nucleotide sequence of the DNA encoding the amino acid sequence of mature human OPG is represented by, for example, SEQ ID NO: 15. In the present invention, among the amino acid sequence (SEQ ID NO: 14) of immature human OPG, the region from the 22nd to the 62nd positions from the N-terminus is defined as CRD1 (SEQ ID NO: 18), the region from the 65th to the 105th positions is defined as CRD2 (SEQ ID NO: 20), the region from the 107th to the 142nd positions is defined as CRD3 (SEQ ID NO: 22), and the region from the 145th to the 185th positions is defined as CRD4 (SEQ ID NO: 24). The nucleotide sequences of the DNAs encoding the amino acid sequences of CRD1, CRD2, CRD3, and CRD4 of human OPG are represented by, for example, SEQ ID NO: 17, 19, 21, and 23, respectively. In addition, there are multiple definitions for the amino acid sequences of CRDs other than those described above, but any definition of the CRD amino acid sequence can be used for the DcR3 variants of the present invention using known information [UniProt O00300, GenBank NP_002537.3].

[0045] Note that polymorphisms and isoforms are often observed in genes encoding eukaryotic proteins. Genes in which the nucleotide sequence or amino acid sequence has been mutated due to such polymorphisms in the genes used in the present invention are also included in the genes encoding OPG of the present invention.

[0046] OPG binds to RANKL and suppresses bone resorption by osteoclasts by neutralizing its activity [J. Immunol., 2012, 189: p. 245 - 252]. In addition, OPG also binds to TRAIL and inhibits apoptosis mediated by TRAIL by neutralizing its activity [Am. J. Cancer Res., 2012, 2: p. 45 - 64]. Since neutralization of either ligand may cause unwanted activities and the like, it is desirable that the DcR3 variants of the present invention do not have neutralizing activity against either RANKL or TRAIL.

[0047] In a preferred embodiment, the first chimeric cysteine-rich region comprises, or consists of, the amino acid sequence of any one of the following (a), (b), (c), or (d). (a) An amino acid sequence in which CRD1 of wild-type DcR3 is replaced with CRD1 of OPG in the amino acid sequence of the cysteine-rich region of wild-type DcR3 (in this amino acid sequence, preferably, the other CRDs of wild-type DcR3 are retained). (b) An amino acid sequence in which CRD4 of wild-type DcR3 is replaced with CRD4 of OPG in the amino acid sequence of the cysteine-rich region of wild-type DcR3 (in this amino acid sequence, preferably, the other CRDs of wild-type DcR3 are retained). (c) An amino acid sequence in which CRD1 of wild-type DcR3 is replaced with CRD1 of OPG and CRD4 of wild-type DcR3 is replaced with CRD4 of OPG in the amino acid sequence of the cysteine-rich region of wild-type DcR3 (in this amino acid sequence, preferably, the other CRDs of wild-type DcR3 are retained). (d) An amino acid sequence in which a part of CRD2 of wild-type DcR3 is replaced with the corresponding part of CRD2 of OPG and / or a part of CRD3 of wild-type DcR3 is replaced with the corresponding part of CRD3 of OPG in the amino acid sequence of any one of (a), (b), or (c) above (in this amino acid sequence, preferably, the other CRDs of wild-type DcR3 are retained).

[0048] Examples of the amino acid sequence of (d) above include, for example, an amino acid sequence in which the portion from the 103rd to the 123rd positions from the N-terminus in the amino acid sequence of (a), (b), or (c) is replaced with the corresponding portion in the amino acid sequence of the CRD of OPG. This amino acid sequence is such that the portion from the 18th to the 36th positions of CRD3 of wild-type DcR3 and the 2 amino acid residues following the C-terminal side of the amino acid sequence of CRD3 are replaced with a part of OPG corresponding to this amino acid sequence.

[0049] Specific examples of the amino acid sequence in (a) above include the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 26 or 50. Specific examples of the amino acid sequence in (b) above include the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 28 or 52. Specific examples of the amino acid sequence in (c) above include the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30 or 54. Specific examples of the amino acid sequence in (d) above include the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 32 or 56.

[0050] Note that SEQ ID NO: 26 represents the amino acid sequence of chimeric B-HBD (a DcR3 variant in which CRD1 in wild-type DcR3 (SEQ ID NO: 4) is replaced with CRD1 of OPG), SEQ ID NO: 28 represents the amino acid sequence of chimeric C-HBD (a DcR3 variant in which CRD4 in wild-type DcR3 (SEQ ID NO: 4) is replaced with CRD4 of OPG), SEQ ID NO: 30 represents the amino acid sequence of chimeric A-HBD (a DcR3 variant in which CDR1 and CDR4 in wild-type DcR3 (SEQ ID NO: 4) are replaced with CDR1 and CDR4 of OPG, respectively), SEQ ID NO: 32 represents the amino acid sequence of 103-123OPG-HBD (a DcR3 variant in which the amino acid sequence containing the 18th to 36th portions and the 2 amino acids on the C-terminal side of CRD3 in chimeric A-HBD is replaced with human OPG), SEQ ID NO: 50 represents the amino acid sequence of chimeric B (a DcR3 variant in which CRD1 in wild-type DcR3 (SEQ ID NO: 4) is replaced with CRD1 of OPG and the heparan sulfate binding region is deleted), SEQ ID NO: 52 represents the amino acid sequence of chimeric C (a DcR3 variant in which CRD4 in wild-type DcR3 (SEQ ID NO: 4) is replaced with CRD4 of OPG and the heparan sulfate binding region is deleted), SEQ ID NO: 54 represents the amino acid sequence of chimeric A (a DcR3 variant in which CDR1 and CDR4 in wild-type DcR3 (SEQ ID NO: 4) are replaced with CDR1 and CDR4 of OPG, respectively, and the heparan sulfate binding region is deleted), and SEQ ID NO: 56 represents the amino acid sequence of 103-123OPG (a DcR3 variant in which the amino acid sequence containing the 18th to 36th portions and the 2 amino acids on the C-terminal side of CRD3 in chimeric A is replaced with human OPG).

[0051] In a preferred embodiment, the DcR3 variant containing the first chimeric cysteine-rich region includes a DcR3 variant having binding activity to at least one or more of LIGHT, TL1A, and FasL, a DcR3 variant having binding activity to all of LIGHT, TL1A, and FasL, a DcR3 variant having no binding activity to FasL and having binding activity to any one of LIGHT and TL1A, or a DcR3 variant having no binding activity to FasL and having binding activity to both LIGHT and TL1A.

[0052] In the present invention, "having binding activity to a ligand" means that the binding activity of a DcR3 variant containing a first chimeric cysteine-rich region to the ligand is equivalent to that of wild-type DcR3 to the ligand and is not significantly decreased, and that it is significantly enhanced compared to the binding activity of wild-type DcR3 to the ligand. For example, when measured by the surface plasmon resonance method (SPR method), if the dissociation constant (K D ) value of the DcR3 variant is less than 3 times that of wild-type DcR3, it can be determined that the DcR3 variant has binding activity to the ligand.

[0053] In the present invention, the expression that a DcR3 variant "has no binding activity to a ligand" means that the binding activity of a DcR3 variant containing a first chimeric cysteine-rich region to the ligand is not detected, and that it is significantly decreased compared to the binding activity of wild-type DcR3 to the ligand. For example, when measured by the SPR method, if the KD value of the DcR3 variant is more than 3 times that of wild-type DcR3, or if the Rmax of the DcR3 variant is less than 5, it is determined that the binding activity to the ligand is significantly decreased, and the DcR3 variant can be defined as having no binding activity to the ligand.

[0054] In a particularly preferred embodiment, examples of the DcR3 variant containing a first chimeric cysteine-rich region include DcR3 variants with reduced binding to FasL. The "FasL-binding reduced variant" means a DcR variant containing a chimeric cysteine-rich region that has no binding activity to FasL and has binding activity to one or more of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region that has no binding activity to FasL and has binding activity to LIGHT and TL1A.

[0055] In a preferred embodiment, the DcR3 variant of the present invention is a DcR3 variant having neutralizing activity against at least one of LIGHT, TL1A, and FasL, a DcR3 variant having neutralizing activity against all of LIGHT, TL1A, and FasL, a DcR3 variant having no neutralizing activity against FasL and having neutralizing activity against any one of LIGHT and TL1A, or a DcR3 variant having no neutralizing activity against FasL and having neutralizing activity against LIGHT and TL1A.

[0056] In the present invention, the expression "neutralizing activity against a certain ligand" means that when the ligand binds to the DcR3 variant, it inhibits the binding of the ligand to the receptor on the cell membrane surface, and by inhibiting the binding of the ligand to the receptor on the cell membrane surface, it inhibits the cell functions induced by the binding of the ligand to the receptor on the cell membrane surface, that is, the biological activity of the ligand (for example, biological activities such as cytokine production, enhanced proliferation, and apoptosis induction in cells).

[0057] In the present invention, the expression that the DcR3 variant "has neutralizing activity" is used in the sense that the neutralizing activity of the DcR3 variant against the ligand has no significant difference compared with the neutralizing activity of the wild-type DcR3 against the ligand, and is significantly enhanced compared with the neutralizing activity of the wild-type DcR3 against the ligand.

[0058] In the present invention, the expression that the DcR3 variant "has no neutralizing activity" is used in the sense that the neutralizing activity of the DcR3 variant against the ligand is significantly decreased compared with the neutralizing activity of the wild-type DcR3 against the ligand.

[0059] In a particularly preferred embodiment, the DcR3 variant in the present invention is a DcR3 variant with reduced binding affinity for FasL. The "FasL-binding affinity-reduced variant" means a DcR3 variant that has no neutralizing activity against FasL and has neutralizing activity against one or more of LIGHT and TL1A, or a DcR3 variant that has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A.

[0060] 2-1-2. The Second Chimeric Cysteine-Rich Region The second chimeric cysteine-rich region is one in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence of the first chimeric cysteine-rich region.

[0061] As a method for obtaining a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of the first chimeric cysteine-rich region, site-directed mutagenesis methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA., 79, 6409, (1982), Gene, 34, 315 (1985), Proc. Natl. Acad. Sci. USA., 82, 488 (1985)] can be mentioned.

[0062] The mutations (modifications) added to the first chimeric cysteine-rich region include any of natural mutations and artificial amino acid substitutions, deletions, insertions or additions. As the amino acid sequence of the second chimeric cysteine-rich region, in the amino acid sequence of the first chimeric cysteine-rich region, one or more, preferably 1 to 30, more preferably 1 to 10, still more preferably 1 to 5, still more preferably 1 to 3 amino acid substitutions, deletions, insertions or additions are made, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, for example 93% or more, 95% or more, 97% or more, 98% or more or 99% or more identity with the amino acid sequence of the first chimeric cysteine-rich region. As a method for describing an amino acid substituent, for example, when the 131st Asn from the N-terminus of the amino acid sequence to be substituted is substituted with Ser, it can be represented as N131S.

[0063] In a preferred embodiment, the second chimeric cysteine-rich region contains, or consists of, the amino acid sequence of (e) below. (e) An amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence of (a), (b), (c) or (d) above.

[0064] Examples of the mutations (modifications) added to the first chimeric cysteine-rich region include addition or deletion of a sugar chain attachment site. By adding or deleting a sugar chain attachment site to the first chimeric cysteine-rich region, the biological activity or its properties of the DcR3 variant of the present invention, the pharmacokinetics such as the blood half-life, or the physical or chemical properties such as the protein stability can be controlled.

[0065] Glycosylation generally refers to the N-glycosidic linkage of sugar chains to asparagine residues of peptides or proteins, and / or the O-glycosidic linkage of sugar chains to serine or threonine residues. Examples of O-type sugar chains added to the DcR3 variant include core 1, core 2, etc., and examples of N-type sugar chains include high-mannose type, hybrid type, or complex type sugar chains, with the complex type sugar chain being preferred.

[0066] Examples of the mutation (modification) added to the first chimeric cysteine-rich region include substituting at least one or more amino acids in the amino acid sequence of the first chimeric cysteine-rich region with amino acids to which sugar chains can be added by N-glycosidic linkage or O-glycosidic linkage, thereby adding sugar chains, and particularly preferably adding N-glycosidic linkage type sugar chains.

[0067] In addition, for example, substituting at least one or more, preferably two or more, amino acids involved in N-glycosidic linkage in the amino acid sequence of the chimeric cysteine-rich region with other amino acids to remove sugar chains is also included in the present invention. Generally, when expressing a peptide or protein using yeast, insect cells, or mammalian cells, N-glycosidic linkage of sugar chains occurs by recognizing the sequence Asn-X-Thr / Ser (where X is any amino acid residue other than Pro). For example, N-glycosidic linkage type sugar chains can be removed by substituting Asn, Ser, or Thr in the Asn-X-Thr / Ser sequence present in the DcR3 variant with other amino acids.

[0068] As the second chimeric cysteine-rich region, in order to reduce the aggregates of the DcR3 variant of the present invention, a chimeric cysteine-rich region that retains the sugar chain N-glycosidically linked to Asn at the 157th position from the N-terminus of the amino acid sequence of the first chimeric cysteine-rich region (for example, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in SEQ ID NO: 30) is preferred.

[0069] In a preferred embodiment, the amino acid sequence of the second chimeric cysteine-rich region from which the glycosylation site has been removed (one form of the amino acid sequence of (e) above) is (f) substitution of the 131st and 144th Asn from the N-terminus of the amino acid sequence of (b), (c) or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54 or 56) with other amino acids, (g) substitution of the 131st, 144th and 157th Asn from the N-terminus of the amino acid sequence of (b), (c) or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54 or 56) with other amino acids, (h) substitution of the 133rd Thr and 146th Ser from the N-terminus of the amino acid sequence of (b), (c) or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54 or 56) with other amino acids, and (i) substitution of the 133rd Thr, 146th Ser and 159th Thr from the N-terminus of the amino acid sequence of (b), (c) or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54 or 56) with other amino acids having a substitution selected from the above.

[0070] In a more preferred embodiment, the amino acid sequence from which the glycosylation site has been removed (one form of the amino acid sequence of (e) above) is (f’) substitution of the 131st and 144th Asn from the N-terminus of the amino acid sequence of (b), (c) or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54 or 56) with Ser, (g’) Substitution of Asn at the 131st, 144th, and 157th positions from the N-terminus of the amino acid sequence of (b), (c), or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54, or 56) with Ser, (h’) Substitution of Thr at the 133rd position and Ser at the 146th position from the N-terminus of the amino acid sequence of (b), (c), or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54, or 56) with Ala, and (i’) Substitution of Thr at the 133rd position, Ser at the 146th position, and Thr at the 159th position from the N-terminus of the amino acid sequence of (b), (c), or (d) above (for example, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences shown in SEQ ID NO: 28, 30, 32, 52, 54, or 56) with Ala having a substitution selected from the above.

[0071] Examples of the amino acid sequence having the substitution of (f’) above include, for example, in the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30 or SEQ ID NO: 54, the amino acid sequence in which Asn at the 131st and 144th positions from the N-terminus is substituted with Ser (N131S / N144S) (among the amino acid sequences described in SEQ ID NO: 34 or SEQ ID NO: 58, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus), and the like.

[0072] Note that SEQ ID NO: 34 represents the amino acid sequence of N131S / N144S-HBD (a DcR3 variant in which Asn at the 131st and 144th positions from the N-terminus is substituted with Ser in chimeric A-HBD), and SEQ ID NO: 58 represents the amino acid sequence of N131S / N144S (a DcR3 variant in which Asn at the 131st and 144th positions from the N-terminus is substituted with Ser in chimeric A).

[0073] Examples of the amino acid sequence having the substitution of the above (h') include, for example, among the amino acid sequences described in SEQ ID NO: 30 or SEQ ID NO: 54, in the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus, the amino acid sequence in which the 133rd Thr and 146th Ser from the N-terminus are substituted with Ala (T133A / S146A) (among the amino acid sequences described in SEQ ID NO: 36 or SEQ ID NO: 60, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus), and the like.

[0074] Note that SEQ ID NO: 36 represents the amino acid sequence of T133A / S146A-HBD (a DcR3 variant in which the 133rd Thr and 146th Ser from the N-terminus are substituted with Ala in chimeric A-HBD), and SEQ ID NO: 60 represents the amino acid sequence of T133A / S146A (a DcR3 variant in which the 133rd Thr and 146th Ser from the N-terminus are substituted with Ala in chimeric A).

[0075] Examples of the amino acid sequence having the substitution of the above (g') include, for example, among the amino acid sequences described in SEQ ID NO: 30 or SEQ ID NO: 54, in the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus, the amino acid sequence in which the 131st, 144th, and 157th Asn from the N-terminus are substituted with Ser (N131S / N144S / N157S) (among the amino acid sequences described in SEQ ID NO: 38 or SEQ ID NO: 62, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus), and the like.

[0076] Note that SEQ ID NO: 38 represents the amino acid sequence of N131S / N144S / N157S-HBD (a DcR3 variant in which the 131st, 144th, and 157th Asn from the N-terminus are substituted with Ser in chimeric A-HBD), and SEQ ID NO: 62 represents the amino acid sequence of N131S / N144S / N157S (a DcR3 variant in which the 131st, 144th, and 157th Asn from the N-terminus are substituted with Ser in chimeric A).

[0077] Examples of the amino acid sequence having the substitution of (i') above include, for example, among the amino acid sequences described in SEQ ID NO: 30 or SEQ ID NO: 54, in the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus, the amino acid sequence in which Thr at the 133rd position, Ser at the 146th position, and Thr at the 159th position from the N-terminus are substituted with Ala (T133A / S146A / T159A) (among the amino acid sequences described in SEQ ID NO: 40 or SEQ ID NO: 64, the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus), and the like.

[0078] Note that SEQ ID NO: 40 represents the amino acid sequence of T133A / S146A / T159A-HBD (a DcR3 variant in which Thr at the 133rd position, Ser at the 146th position, and Thr at the 159th position from the N-terminus in chimeric A-HBD are substituted with Ala), and SEQ ID NO: 64 represents the amino acid sequence of T133A / S146A / T159A (a DcR3 variant in which Thr at the 133rd position, Ser at the 146th position, and Thr at the 159th position from the N-terminus in chimeric A are substituted with Ala).

[0079] In the mutation (modification) added to the first chimeric cysteine-rich region, for example, no mutation is introduced into CRD2 and CRD3 of the first chimeric cysteine-rich region involved in the binding to LIGHT, TL1A, and FasL, and a DcR3 variant containing a chimeric cysteine-rich region in which the binding activity between LIGHT, TL1A, and FasL and the chimeric cysteine-rich region is not reduced can be obtained by introducing a mutation into CRD1 and / or CRD4 of the first chimeric cysteine-rich region. On the other hand, a DcR3 variant containing a chimeric cysteine-rich region in which the binding activity between LIGHT, TL1A, or FasL and the chimeric cysteine-rich region is changed can be obtained by introducing a mutation into CRD2 or / and CRD3 of the first chimeric cysteine-rich region involved in the binding to LIGHT, TL1A, or FasL. That is, by introducing the above mutations, a DcR3 variant containing a chimeric cysteine-rich region having desirable binding characteristics to LIGHT, TL1A, or FasL can be obtained.

[0080] In a preferred embodiment, examples of the DcR3 variant containing the second chimeric cysteine-rich region include a DcR3 variant containing a chimeric cysteine-rich region having binding activity to at least one of LIGHT, TL1A, and FasL, a DcR3 variant containing a chimeric cysteine-rich region having binding activity to all of LIGHT, TL1A, and FasL, a DcR3 variant containing a chimeric cysteine-rich region having no binding activity to FasL and having binding activity to any one of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region having no binding activity to FasL and having binding activity to LIGHT and TL1A. The meanings of the expressions "having binding activity to a ligand" and "having no binding activity to a ligand" for the DcR3 variant of the present invention are as described above.

[0081] In a particularly preferred embodiment, examples of the DcR3 variant containing the second chimeric cysteine-rich region include a DcR3 variant with reduced binding to FasL. The "FasL binding-reduced variant" means a DcR3 variant containing a chimeric cysteine-rich region having no binding activity to FasL and having binding activity to at least one of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region having no binding activity to FasL and having binding activity to LIGHT and TL1A.

[0082] In a preferred embodiment, examples of the DcR3 variant containing the second chimeric cysteine-rich region include a DcR3 variant containing a chimeric cysteine-rich region having neutralizing activity against at least one or more of LIGHT, TL1A, and FasL, a DcR3 variant containing a chimeric cysteine-rich region having neutralizing activity against all of LIGHT, TL1A, and FasL, a DcR3 variant containing a chimeric cysteine-rich region having no neutralizing activity against FasL and having neutralizing activity against any one of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region having no neutralizing activity against FasL and having neutralizing activity against LIGHT and TL1A. The meanings of the expressions "having neutralizing activity against a ligand" and "having no neutralizing activity against a ligand" for the DcR3 variant of the present invention are as described above.

[0083] In a particularly preferred embodiment, examples of the DcR3 variant containing the second chimeric cysteine-rich region include a DcR3 variant with reduced neutralizing activity against FasL. The "variant with reduced FasL binding ability" means a DcR3 variant containing a chimeric cysteine-rich region having no neutralizing activity against FasL and having neutralizing activity against at least one or more of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region having no neutralizing activity against FasL and having neutralizing activity against LIGHT and TL1A.

[0084] For example, a DcR3 variant containing a chimeric cysteine-rich region, which is a FasL-binding affinity-reduced variant, can be obtained by preparing a modified form in which the binding site of DcR3 or the DcR3 variant with each ligand, as inferred from crystal structure analysis or the like, is substituted with Ala or another amino acid, and measuring the binding activity and neutralizing activity against LIGHT, TL1A, or FasL ligand. Alternatively, a gene library in which the periphery of the ligand-binding site of DcR3 or the DcR3 variant is randomly converted to other amino acids can be prepared, displayed on phage, yeast, mammalian cells, etc., and screened using the binding activity and neutralizing activity against LIGHT, TL1A, or FasL ligand as indicators.

[0085] Examples of the amino acid sequence of the chimeric cysteine-rich region included in the FasL-binding affinity-reduced variant (one form of the amino acid sequence in (e) above) include amino acid sequences having substitutions of one or more amino acids selected from the group consisting of Glu at the 57th position, Arg at the 58th position, and Arg at the 60th position from the N-terminus of the amino acid sequence in (a), (b), (c), or (d) above with other amino acids.

[0086] The other amino acid substituted for Glu at the 57th position is not particularly limited and can be appropriately selected from 19 types of amino acids excluding Glu among 20 types of amino acids (Glu, Ala, Asp, Lys, Leu, Cys, Phe, Gly, His, Ile, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr). However, it is preferably selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, more preferably selected from Lys, Leu, Arg, and Val, even more preferably selected from Lys, Arg, and Val, and even more preferably selected from Lys and Arg.

[0087] The other amino acid to be substituted for the 58th Arg is not particularly limited and can be appropriately selected from 19 amino acids excluding Arg among the 20 types of amino acids, but it is preferably selected from Asp, Glu, and Thr, and more preferably selected from Asp and Glu.

[0088] The other amino acid to be substituted for the 60th Arg is not particularly limited and can be appropriately selected from 19 amino acids excluding Arg among the 20 types of amino acids, but it is preferably Lys.

[0089] In a preferred embodiment, one or more amino acids selected from the group consisting of the 57th Glu, 58th Arg, and 60th Arg is one amino acid consisting of the 57th Glu.

[0090] In yet another preferred embodiment, one or more amino acids selected from the group consisting of the 57th Glu, 58th Arg, and 60th Arg are two amino acids consisting of the 57th Glu and 58th Arg. In this embodiment, it is preferable to combine substitution of the 57th Glu with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met and substitution of the 58th Arg with Asp, Glu, or Thr, more preferably to combine substitution of the 57th Glu with Lys, Leu, Arg, or Val and substitution of the 58th Arg with Asp or Glu, and even more preferably to combine substitution of the 57th Glu with Lys or Arg and substitution of the 58th Arg with Asp or Glu.

[0091] The second chimeric cysteine-rich region may have substitutions of one or more amino acids other than the 57th Glu, 58th Arg, and 60th Arg with other amino acids. Examples of amino acids other than the 57th Glu, 58th Arg, and 60th Arg include the 53rd Trp, 54th Asn, 55th Tyr, 56th Leu, etc. from the N-terminus of the amino acid sequence of (a), (b), (c), or (d) above.

[0092] The other amino acid to be substituted for the 53rd Trp is not particularly limited and can be appropriately selected from 19 amino acids excluding Trp among the 20 types of amino acids, but it is preferably selected from Asp and Asn. Substitution of the 53rd Trp with other amino acids can be combined with, for example, substitution of the 57th Glu with other amino acids.

[0093] The other amino acid to be substituted for the 54th Asn is not particularly limited and can be appropriately selected from 19 amino acids excluding Asn among the 20 types of amino acids, but it is preferably Asp. Substitution of the 54th Asn with other amino acids can be combined with, for example, substitution of the 57th Glu with other amino acids.

[0094] The other amino acid to be substituted for the 55th Tyr is not particularly limited and can be appropriately selected from 19 amino acids excluding Tyr among the 20 types of amino acids, but it is preferably selected from Thr, Asp, Gln, and Glu. Substitution of the 55th Tyr with other amino acids can be combined with, for example, substitution of the 57th Glu with other amino acids.

[0095] The other amino acid to be substituted for the 56th Leu is not particularly limited and can be appropriately selected from 19 amino acids excluding Leu among the 20 types of amino acids, but it is preferably selected from Asp, Gln, Thr, Glu, Gly, Asn, and Pro. Substitution of the 56th Leu with other amino acids can be combined with, for example, substitution of the 57th Glu with other amino acids.

[0096] Specific examples of the amino acid sequence having substitution of one or more amino acids selected from the group consisting of the 57th Glu, 58th Arg, and 60th Arg counted from the N-terminus of the amino acid sequence of (a), (b), (c), or (d) above with other amino acids are as follows: In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Lys (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 42), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Lys (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 66), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Leu (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 44), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Leu (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 68), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 60th Arg from the N-terminus is substituted with Lys (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 46), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 60th Arg from the N-terminus is substituted with Lys (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 70), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Arg, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Arg (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 180), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Val, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Val (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 182), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Ala, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Ala (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 270), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Phe, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Phe (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 272), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with His, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with His (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 274), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Ile, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Ile (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 276), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Met, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Met (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 278), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Lys and the 58th Arg is substituted with Asp, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Lys and the 58th Arg is substituted with Asp (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 184), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, an amino acid sequence in which the 57th Glu from the N-terminus is substituted with Lys and the 58th Arg is substituted with Glu, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Lys and the 58th Arg is replaced by Glu (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 186), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Arg and the 58th Arg is replaced by Asp, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Arg and the 58th Arg is replaced by Asp (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 188), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Lys and the 58th Arg is replaced by Thr, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Lys and the 58th Arg is replaced by Thr (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 280), In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 30, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Leu and the 58th Arg is replaced by Glu, In the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 54, the amino acid sequence in which the 57th Glu from the N-terminus is replaced by Leu and the 58th Arg is replaced by Glu (the amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus among the amino acid sequences described in SEQ ID NO: 282), In the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which Glu at the 57th position from the N-terminus is replaced with Val and Arg at the 58th position is replaced with Thr, In the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which Glu at the 57th position from the N-terminus is replaced with Val and Arg at the 58th position is replaced with Thr (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 284), In the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 30, an amino acid sequence in which Glu at the 57th position from the N-terminus is replaced with Val and Arg at the 58th position is replaced with TGlu, In the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 54, an amino acid sequence in which Glu at the 57th position from the N-terminus is replaced with Val and Arg at the 58th position is replaced with Glu (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus among the amino acid sequences set forth in SEQ ID NO: 286) and the like.

[0097] Note that SEQ ID NO: 42 is the amino acid sequence of chimeric A-E57K-HBD (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A-HBD is replaced with Lys), SEQ ID NO: 44 is the amino acid sequence of chimeric A-E57L-HBD (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A-HBD is replaced with Leu), SEQ ID NO: 46 is the amino acid sequence of chimeric A-R60K-HBD (a modified DcR3 in which Arg at the 60th position from the N-terminus in chimeric A-HBD is replaced with Lys), SEQ ID NO: 66 is the amino acid sequence of chimeric A-E57K (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Lys), SEQ ID NO: 68 is the amino acid sequence of chimeric A-E57L (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Leu), SEQ ID NO: 70 is the amino acid sequence of chimeric A-R60K (a modified DcR3 in which Arg at the 60th position from the N-terminus in chimeric A is replaced with Lys), SEQ ID NO: 180 is the amino acid sequence of chimeric A-E57R (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Arg), SEQ ID NO: 182 is the amino acid sequence of chimeric A-E57V (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Val), SEQ ID NO: 184 is the amino acid sequence of chimeric A-E57K_R58D (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Lys and Arg at the 58th position is replaced with Asp), SEQ ID NO: 186 is the amino acid sequence of chimeric A-E57K_R58E (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Lys and Arg at the 58th position is replaced with Glu), SEQ ID NO: 188 is the amino acid sequence of chimeric A-E57R_R58D (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Arg and Arg at the 58th position is replaced with Asp), SEQ ID NO: 270 is the amino acid sequence of chimeric A-E57A (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Ala), SEQ ID NO: 272 is the amino acid sequence of chimeric A-E57F (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with Phe), SEQ ID NO: 274 is the amino acid sequence of chimeric A-E57H (a modified DcR3 in which Glu at the 57th position from the N-terminus in chimeric A is replaced with His).SEQ ID NO: 276 represents the amino acid sequence of Chimera A-E57I (a modified DcR3 in which Glu at the 57th position from the N-terminus in Chimera A is replaced with Ile), SEQ ID NO: 278 represents the amino acid sequence of Chimera A-E57M (a modified DcR3 in which Glu at the 57th position from the N-terminus in Chimera A is replaced with Met), SEQ ID NO: 280 represents the amino acid sequence of Chimera A-E57K_R58T (a modified DcR3 in which Glu at the 57th position from the N-terminus in Chimera A is replaced with Lys and Arg at the 58th position is replaced with Thr), SEQ ID NO: 282 represents the amino acid sequence of Chimera A-E57K_R58T (a modified DcR3 in which Glu at the 57th position from the N-terminus in Chimera A is replaced with Lys and Arg at the 58th position is replaced with Thr), SEQ ID NO: 284 represents the amino acid sequence of Chimera A-E57V_R58T (a modified DcR3 in which Glu at the 57th position from the N-terminus in Chimera A is replaced with Val and Arg at the 58th position is replaced with Thr), SEQ ID NO: 286 represents the amino acid sequence of Chimera A-E57V_R58E (a modified DcR3 in which Glu at the 57th position from the N-terminus in Chimera A is replaced with Val and Arg at the 58th position is replaced with Glu).

[0098] 2-2. Other regions The modified DcR3 of the present invention may or may not include one or more other regions bound to the C-terminal side of the first or second chimeric cysteine-rich region. Examples of other regions include, for example, part or all of the region existing between CRD4 and HBD in wild-type DcR3, part or all of the region following the C-terminal of the amino acid sequence of CRD4 in TNF receptor superfamily (TNFRSF) molecules other than DcR3, part or all of the HBD of wild-type DcR3, and the like. The expression "other regions bound to the C-terminal side of the first or second chimeric cysteine-rich region" is used in the sense of including the case where the other regions are directly bound to the C-terminal of the first or second chimeric cysteine-rich region and the case where the other regions are bound to the C-terminal of the first or second chimeric cysteine-rich region via yet another region.

[0099] In one form, the DcR3 variant of the present invention includes, as another region bound to the C-terminal side of the first or second chimeric cysteine-rich region, a part or all of the region that exists between CRD4 and HBD in wild-type DcR3. In this form, it is preferable that the other region is directly bound to the C-terminus of the first or second chimeric cysteine-rich region.

[0100] In another form, the DcR3 variant of the present invention includes, as another region bound to the C-terminal side of the first or second chimeric cysteine-rich region, a part or all of the region that follows the C-terminus of the amino acid sequence of CRD4 in a TNF receptor superfamily (TNFRSF) molecule other than DcR3. In this form, it is preferable that the other region is directly bound to the C-terminus of the first or second chimeric cysteine-rich region. The description regarding TNFRSF is the same as above. TNFRSF is preferably OPG.

[0101] In the first or second chimeric cysteine-rich region (for example, the first chimeric cysteine-rich region consisting of the amino acid sequences of (b) to (d) above), when the CRD4 of wild-type DcR3 is replaced with the CRD4 of OPG, the DcR3 variant of the present invention preferably contains, as another region bound to the C-terminus of the chimeric cysteine-rich region, a plurality of amino acid residues following the C-terminus of the amino acid sequence of the CRD4 of OPG. That is, when the CR4 of wild-type DcR3 is replaced with the CRD4 of OPG, it is preferable that a plurality of amino acid residues following the C-terminus of the amino acid sequence of the CRD4 of wild-type DcR3 are also replaced with a plurality of amino acid residues following the C-terminus of the amino acid sequence of the CRD4 of OPG. As the plurality of amino acid residues following the C-terminus of the amino acid sequence of the CRD4 of OPG, for example, the 186th to 194th amino acid residues in the amino acid sequence of OPG (SEQ ID NO: 14) are preferable, but the number of amino acid residues to be replaced with the plurality of amino acid residues following the C-terminus of the CRD4 of wild-type DcR3 can be adjusted as appropriate. The number of amino acid residues following the C-terminus of the amino acid sequence of the CRD4 of OPG is usually 1 to 12, preferably 1 to 10, more preferably 1 to 9, 1 to 6, or 1 to 3. Further, in the first or second chimeric cysteine-rich region (for example, the first chimeric cysteine-rich region consisting of the amino acid sequences of (a) and (d) above), when the CRD4 is derived from wild-type DcR3, it is preferable that an amino acid sequence following the C-terminus of the amino acid sequence of the CRD4 in the first chimeric cysteine-rich region is bound to the C-terminus of the amino acid sequence of the CRD4 of wild-type DcR3. Examples of such an amino acid sequence include the 194th to 195th amino acids of SEQ ID NO: 2, but the number of amino acid residues constituting the amino acid sequence following the C-terminus of the amino acid sequence of the CRD4 of wild-type DcR3 can be adjusted as appropriate. The number of amino acid residues constituting the amino acid sequence following the C-terminus of the CRD4 of wild-type DcR3 is usually 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2.

[0102] In yet another embodiment, the DcR3 variant of the present invention includes a part or all of the HBD of wild-type DcR3 as another region bound to the C-terminal side of the first or second chimeric cysteine-rich region. In this embodiment, the other region may be directly bound to the C-terminal of the first or second chimeric cysteine-rich region, or may be bound to the C-terminal of the first or second chimeric cysteine-rich region via a part or all of the region existing between CRD4 and HBD in wild-type DcR3, or via a part or all of the region following the C-terminal of the amino acid sequence of CRD4 in TNFRSF molecules other than DcR3.

[0103] The DcR3 variant of the present invention preferably does not contain HBD and contains the amino acid residues at positions 186 to 194 of the amino acid sequence of OPG (SEQ ID NO: 14) as another region bound to the C-terminal side of the first or second chimeric cysteine-rich region.

[0104] 3. DcR3 variants containing sugar chains The DcR3 variants of the present invention also include DcR3 variants containing at least one sugar chain. Any DcR3 variant containing a sugar chain is included in the present invention as long as at least one sugar chain is bound to the cysteine-rich region or other amino acid residues contained in the above-described DcR3 variant.

[0105] Glycoproteins have one or more sugar chains. When a glycoprotein has two or more sugar chains, the sugar chains of the glycoprotein may be of one type or two or more types. Examples of the sugar chains of glycoproteins include sugar chains that are N-glycosidically linked to amino acid residues (e.g., asparagine residues, etc.) of peptides or proteins, and sugar chains that are O-glycosidically linked to amino acid residues (e.g., serine residues, threonine residues, etc.) of peptides or proteins. Examples of O-type sugar chains include core 1, core 2, etc., and examples of N-type sugar chains include high-mannose type, hybrid type, and complex type sugar chains, with complex type sugar chains being preferred.

[0106] 4. DcR3 variants containing the Fc region The DcR3 variants of the present invention include proteins in which a homologous or heterologous peptide, polypeptide, or protein is directly bound or fused, either at the N-terminal side or the C-terminal side of a chimeric cysteine-rich region (or a chimeric type containing other regions and being a cysteine-rich region), via a suitable peptide linker if necessary. The number of amino acids constituting the peptide linker is not particularly limited, and examples include 4, 5, 6, or 15.

[0107] Examples of the polypeptide or protein to be bound or fused to the chimeric cysteine-rich region include, for example, the constant region or Fc region of an immunoglobulin, a peptide that binds to FcRn (neonatal Fc receptor), albumin, protein A, protein G, β-galactosidase, glutathione-S-transferase (GST), maltose-binding protein, polyhistidine, FLAG peptide, and other polypeptides or proteins. Preferably, it is the Fc region of an immunoglobulin or its variant (mutant Fc region), and more preferably, the Fc region of a mammalian-derived immunoglobulin or its variant (mutant Fc region).

[0108] As the Fc region of an immunoglobulin (also described as an antibody), the Fc region of a human immunoglobulin is preferred for use in humans. The classes and subclasses of immunoglobulins include, but are not limited to, IgG, IgD, IgE, IgM, IgA, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, or IgA1, etc. If it is to be used in humans, it is preferable to use the classes and subclasses of human immunoglobulins. Also, when using the Fc region of an immunoglobulin as the polypeptide or protein to be bound or fused to the chimeric cysteine-rich region, preferably, the Fc region of the immunoglobulin is bound or fused to the C-terminal side of the chimeric cysteine-rich region.

[0109] Immunoglobulins are composed of heavy and light chain polypeptides. The constant region of the heavy chain of human IgG is composed of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain in order from the N-terminus. The Fc region of IgG in the present invention includes a region combining the CH2 domain and the CH3 domain, and a region combining a part or all of the hinge domain, the CH2 domain, and the CH3 domain. Each domain included in the Fc region of IgG in the present invention can be specified by the EU index number. Specifically, the hinge domain is specified by EU index numbers 216 to 230, the CH2 domain is specified by 231 to 340, and CH3 is specified by EU index numbers 341 to 447, respectively.

[0110] In addition, the polypeptide or protein that binds to or fuses with the chimeric cysteine-rich region includes a modified polypeptide or protein in which one or more amino acids are substituted, deleted, inserted, or added for the purpose of changing the biological activity or its properties of the DcR3 variant, the pharmacokinetics such as the blood half-life, or the physical or chemical properties such as the stability of the protein. The modification of the polypeptide or protein that binds to or fuses with the chimeric cysteine-rich region includes both natural mutations and artificial amino acid substitutions, deletions, insertions, or additions. Examples of the modified polypeptide or protein include a mutant Fc region consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence of the Fc region of an immunoglobulin. Examples of the amino acid sequence of the mutant Fc region include an amino acid sequence in which one or more, preferably 2 to 30, more preferably 2 to 10, particularly preferably 2 to 5 amino acids are substituted, deleted, inserted, or added in the amino acid sequence of the Fc region of an immunoglobulin, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more identity with the amino acid sequence of the Fc region of an immunoglobulin.

[0111] The addition or deletion of the above-described glycosylation sites is also included in the modification of the polypeptide or protein that binds or fuses to the chimeric cysteine-rich region. For example, the glycosylation site can be added by adding or inserting a polypeptide containing an N-type glycosylation site. Specific polypeptide sequences include GGNGT or YGNGT consisting of 5 amino acids [International Publication Patent No. 2014 / 153111].

[0112] Examples of the substitution of human IgG1 that reduces or abolishes complement-dependent cytotoxicity (CDC) activity include substitution of one or more amino acids selected from Leu (L234) at position 234, Leu (L235) at position 235, Asp (D265) at position 265, Asp (D265) at position 270, Lys (K322) at position 322, Pro (P329) at position 329, Pro (P331) at position 331, etc. indicated by the EU index with other amino acids. Specifically, substitution of one or more amino acids selected from L234, L235, D270, K322, P329, P331, etc. indicated by the EU index with Ala, substitution of P331 with Ser or Gly, etc. are included [J. Immunol., 2000, 164: p. 4178-4184, Cell. Immunol., 2000, 200: p. 16-26].

[0113] As substitutions of human IgG1 that reduce or eliminate effector activities such as antibody-dependent cell-mediated cytotoxicity (ADCC) activity and antibody-dependent cell phagocytosis (ADCP), substitutions of one or more amino acids selected from the 297th Asn (N297), 234th Leu (L234), 235th Leu (L235), 237th Gly (G237), 226th Cys (C226), 229th Cys (C229), 238th Pro (P238), 233rd Glu (E233), 267th Ser (S267), 328th Leu (L328), 331st Pro (P331), etc. shown in the EU index to other amino acids can be mentioned. Specifically, substitutions such as substitution of N297 with Ala (N297A), substitution of N297 with Gln (N297Q), substitution of N297 with Gly (N297G), or substitution of L234 with Ala (L234A) / substitution of L235 with Ala (L235A) / substitution of G237 with Ala (G237A) and other amino acid substitutions can be mentioned. Note that " / " means "and" (the same applies hereinafter).

[0114] In addition, as modifications that increase the binding activity to the inhibitory receptor FcγRIIb, substitutions such as substitution of Gly at position 236 with Asp (G236D), substitution of Leu at position 328 with Phe (L328F), substitution of Ser at position 239 with Asp (S239D), substitution of Ser at position 267 with Glu (S267E), etc. shown by the EU index can be mentioned [Curr. Opin. Cell. Biol., 2009, 20: p.685-691]. Also, as modifications that enhance the binding to FcRn (neonatal Fc receptor) in the low pH environment within the endosome, avoid the disappearance of the antibody, and extend the blood half-life, substitutions such as substitution of Thr at position 250 with Gln (T250Q), substitution of Met at position 428 with Leu (M428L), substitution of Met at position 252 with Tyr (M252Y), substitution of Ser at position 254 with Thr (S254T), substitution of Thr at position 256 with Glu (T256E), substitution of Met at position 252 with Tyr (M252Y) / substitution of Ser at position 254 with Thr (S254T) / substitution of Thr at position 256 with Glu (T256E), substitution of Met at position 428 with Leu (M428L) / substitution of Asn at position 434 with Ser (N434S), substitution of Asn at position 434 with Ala (N434A), substitution of Asn at position 434 with His (N434H), etc. shown by the EU index can be mentioned [J. Immunol., 2009, 182: p.7663-7671, MAbs, 2017, 9: p.844-853].

[0115] In a preferred embodiment, the variant Fc region has a substitution of Ser for Cys at position 220 as indicated by the EU index in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass. Examples of the variant Fc region according to this embodiment include, for example, the Fc region (EU indices 217 to 447) excluding Glu at position 216 indicated by the EU index contained in the CH1 domain and the hinge domain from the constant region (SEQ ID NO: 153) of the heavy chain of human IgG1, wherein Cys at position 220 involved in binding to the light chain is substituted with Ser (C220S), and the Fc region of an immunoglobulin having an amino acid sequence (hereinafter referred to as g1S, SEQ ID NO: 72); the Fc region (EU indices 216 to 447) excluding the CH1 domain from the constant region (SEQ ID NO: 153) of the heavy chain of human IgG1, and the Fc region of an immunoglobulin having an amino acid sequence in which Cys at position 220 is substituted with Ser (C220S) (hereinafter referred to as Eg1S, SEQ ID NO: 156), and the like. When the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, as the first chimeric cysteine-rich region contained in the DcR3 variant of the present invention, a first chimeric cysteine-rich region in which CRD1 of wild-type DcR3 is replaced with CRD1 of OPG and CRD4 of wild-type DcR3 is replaced with CRD4 of OPG is preferred. Further, when the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the second chimeric cysteine-rich region contained in the DcR3 variant of the present invention preferably has a substitution of another amino acid for Glu at position 57, or a substitution of another amino acid for Glu at position 57 and a substitution of another amino acid for Arg at position 58. The other amino acid substituted for Glu at position 57 is preferably selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and more preferably selected from Lys, Leu, Arg, and Val. The other amino acid substituted for Arg at position 58 is preferably selected from Asp, Glu, and Thr, and more preferably selected from Asp and Glu.When combining substitution of the 57th Glu with other amino acids and substitution of the 58th Arg with other amino acids, it is preferable to combine substitution of the 57th Glu with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met and substitution of the 58th Arg with Asp, Glu or Thr. It is more preferable to combine substitution of the 57th Glu with Lys, Leu, Arg or Val and substitution of the 58th Arg with Asp or Glu. It is even more preferable to combine substitution of the 57th Glu with Lys or Arg and substitution of the 58th Arg with Asp or Glu.

[0116] In another preferred embodiment, the variant Fc region has substitutions of Ser at position 228 with Pro, Leu at position 235 with Glu, and Arg at position 409 with Lys in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG4 subclass, as indicated by the EU index. Examples of the variant Fc region according to this embodiment include, for example, excluding the CH1 domain from the constant region (SEQ ID NO: 154) of the heavy chain of human IgG4, and having an amino acid sequence in which Ser at position 228, Leu at position 235, and Arg at position 409, as indicated by the EU index in International Publication Patent No. 2006 / 33386, are substituted with Pro, Glu, and Lys, respectively. The Fc of an immunoglobulin (hereinafter referred to as g4PEK, SEQ ID NO: 74) is mentioned. When the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, as the first chimeric cysteine-rich region contained in the DcR3 variant of the present invention, the CRD1 of wild-type DcR3 is preferably substituted with the CRD1 of OPG, and the CRD4 of wild-type DcR3 is substituted with the CRD4 of OPG. Further, when the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the second chimeric cysteine-rich region contained in the DcR3 variant of the present invention preferably has a substitution of Glu at position 57 with another amino acid, or a substitution of Glu at position 57 with another amino acid and a substitution of Arg at position 58 with another amino acid. The other amino acid substituted for Glu at position 57 is preferably selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and more preferably selected from Lys, Leu, Arg, and Val. The other amino acid substituted for Arg at position 58 is preferably selected from Asp, Glu, and Thr, and more preferably selected from Asp and Glu.When combining substitution of the 57th Glu with another amino acid and substitution of the 58th Arg with another amino acid, it is preferable to combine substitution of the 57th Glu with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met and substitution of the 58th Arg with Asp, Glu or Thr. It is more preferable to combine substitution of the 57th Glu with Lys, Leu, Arg or Val and substitution of the 58th Arg with Asp or Glu. It is even more preferable to combine substitution of the 57th Glu with Lys or Arg and substitution of the 58th Arg with Asp or Glu.

[0117] In yet another preferred embodiment, the variant Fc region has substitutions of Leu at position 234 with Ala, Leu at position 235 with Ala, and Gly at position 237 with Ala in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass, as indicated by the EU index. Examples of the variant Fc region according to this embodiment include, for example, the Fc region of immunoglobulin having an amino acid sequence in which Leu at position 234 is substituted with Ala, Leu at position 235 is substituted with Ala, and Gly at position 237 is substituted with Ala in the Fc region of g1S represented by SEQ ID NO: 72, or Eg1S represented by SEQ ID NO: 156 (hereinafter referred to as g1S LALAGA or Eg1S LALAGA, SEQ ID NOs: 162, 164). When the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the first chimeric cysteine-rich region contained in the DcR3 variant of the present invention is preferably a first chimeric cysteine-rich region in which CRD1 of wild-type DcR3 is substituted with CRD1 of OPG and CRD4 of wild-type DcR3 is substituted with CRD4 of OPG. Further, when the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the second chimeric cysteine-rich region contained in the DcR3 variant of the present invention preferably has a substitution of Glu at position 57 with another amino acid, or a substitution of Glu at position 57 with another amino acid and a substitution of Arg at position 58 with another amino acid. The other amino acid substituted for Glu at position 57 is preferably selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and more preferably selected from Lys, Leu, Arg, and Val. The other amino acid substituted for Arg at position 58 is preferably selected from Asp, Glu, and Thr, and more preferably selected from Asp and Glu.When combining substitution of the 57th Glu with other amino acids and substitution of the 58th Arg with other amino acids, it is preferable to combine substitution of the 57th Glu with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met and substitution of the 58th Arg with Asp, Glu or Thr. It is more preferable to combine substitution of the 57th Glu with Lys, Leu, Arg or Val and substitution of the 58th Arg with Asp or Glu. It is even more preferable to combine substitution of the 57th Glu with Lys or Arg and substitution of the 58th Arg with Asp or Glu.

[0118] In yet another preferred embodiment, the variant Fc region has a substitution of Ala for Asn at position 434 indicated by the EU index in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass. Examples of the variant Fc region according to this embodiment include the Fc region of an immunoglobulin having an amino acid sequence in which Asn at position 434 is substituted with Ala in the Fc region of g1S represented by SEQ ID NO: 72 or Eg1S represented by SEQ ID NO: 156 (hereinafter referred to as g1S N434A or Eg1S N434A, SEQ ID NOs: 312, 160), and the Fc region of an immunoglobulin having an amino acid sequence in which Asn at position 434 is substituted with Ala in the Fc region of g1S LALAGA represented by SEQ ID NO: 162 or Eg1S LALAGA represented by SEQ ID NO: 164 (hereinafter referred to as g1S LALAGANA or Eg1S LALAGANA, SEQ ID NOs: 313, 166). When the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the first chimeric cysteine-rich region contained in the DcR3 variant of the present invention is preferably the first chimeric cysteine-rich region in which CRD1 of wild-type DcR3 is replaced with CRD1 of OPG and CRD4 of wild-type DcR3 is replaced with CRD4 of OPG. Further, when the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the second chimeric cysteine-rich region contained in the DcR3 variant of the present invention preferably has a substitution of another amino acid for Glu at position 57, or a substitution of another amino acid for Glu at position 57 and a substitution of another amino acid for Arg at position 58. The other amino acid substituted for Glu at position 57 is preferably selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile and Met, and more preferably selected from Lys, Leu, Arg and Val. The other amino acid substituted for Arg at position 58 is preferably selected from Asp, Glu and Thr, and more preferably selected from Asp and Glu.When combining substitution of the 57th Glu with other amino acids and substitution of the 58th Arg with other amino acids, it is preferable to combine substitution of the 57th Glu with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met and substitution of the 58th Arg with Asp, Glu or Thr. It is more preferable to combine substitution of the 57th Glu with Lys, Leu, Arg or Val and substitution of the 58th Arg with Asp or Glu. It is even more preferable to combine substitution of the 57th Glu with Lys or Arg and substitution of the 58th Arg with Asp or Glu.

[0119] In yet another preferred embodiment, the variant Fc region has substitutions of Met at position 252 with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu, as indicated by the EU index, among the amino acid sequences of the heavy chains of antibodies belonging to the human IgG1 subclass. Examples of the variant Fc region according to this embodiment include the Fc region of immunoglobulin having an amino acid sequence in which Met at position 252 is substituted with Tyr, Ser at position 254 is substituted with Thr, and Thr at position 256 is substituted with Glu, among g1S represented by SEQ ID NO: 72 or Eg1S represented by SEQ ID NO: 156 (hereinafter referred to as g1S YTE or Eg1S YTE, SEQ ID NOs: 311, 158). When the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, as the first chimeric cysteine-rich region contained in the DcR3 variant of the present invention, a first chimeric cysteine-rich region in which CRD1 of wild-type DcR3 is substituted with CRD1 of OPG and CRD4 of wild-type DcR3 is substituted with CRD4 of OPG is preferred. Further, when the DcR3 variant of the present invention contains the variant Fc region according to this embodiment, the second chimeric cysteine-rich region contained in the DcR3 variant of the present invention preferably has a substitution of Glu at position 57 with another amino acid, or a substitution of Glu at position 57 with another amino acid and a substitution of Arg at position 58 with another amino acid. The other amino acid substituted for Glu at position 57 is preferably selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and more preferably selected from Lys, Leu, Arg, and Val. The other amino acid substituted for Arg at position 58 is preferably selected from Asp, Glu, and Thr, and more preferably selected from Asp and Glu.When combining substitution of the 57th Glu with other amino acids and substitution of the 58th Arg with other amino acids, it is preferable to combine substitution of the 57th Glu with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met and substitution of the 58th Arg with Asp, Glu or Thr. It is more preferable to combine substitution of the 57th Glu with Lys, Leu, Arg or Val and substitution of the 58th Arg with Asp or Glu. It is even more preferable to combine substitution of the 57th Glu with Lys or Arg and substitution of the 58th Arg with Asp or Glu.

[0120] Examples of the mutant Fc region include, but are not limited to, mutant Fc regions containing or consisting of the amino acid sequences described in SEQ ID NOs: 72, 74, 156, 158, 160, 162, 164, 166, 311, 312 or 313.

[0121] Examples of the peptide linker added to link the same or different peptides, polypeptides or proteins to the N-terminal or C-terminal side of the chimeric cysteine-rich region include, but are not limited to, peptide linkers such as IEGRMD linker or GS linker, and chemical linkers.

[0122] Most preferably, the DcR3 variant of the present invention is a DcR3 variant containing the first or second chimeric cysteine-rich region and the Fc region or mutant Fc region.

[0123] In one aspect, the amino acid sequence included in the DcR3 variant of the present invention includes, for example, the amino acid sequence set forth in SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence. In this aspect, the DcR3 variant of the present invention may consist of the above amino acid sequence or may include the above amino acid sequence, but preferably consists of the above amino acid sequence. Note that the above amino acid sequence includes HBD.

[0124] In another aspect, the amino acid sequence included in the DcR3 variant of the present invention includes, for example, the amino acid sequence set forth in SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284, or 286, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence. In this aspect, the DcR3 variant of the present invention may consist of the above amino acid sequence or may include the above amino acid sequence, but preferably consists of the above amino acid sequence. Note that the above amino acid sequence does not include HBD.

[0125] In another mode, the amino acid sequence included in the DcR3 variant of the present invention includes, for example, the amino acid sequence set forth in SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284, or 286, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence, and, as a mutant Fc region, an amino acid sequence set forth in SEQ ID NO: 72, 74, 156, 158, 160, 162, 164, 166, 311, 312, or 313.

[0126] In yet another aspect, examples of the amino acid sequence included in the DcR3 variant of the present invention include, for example, the amino acid sequences set forth in SEQ ID NOs: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 150, 168, 170, 172, 174, 176, 178, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 288, 290, 292, 294, 296, 298, 300, 302, 304, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 or 337, or an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the amino acid sequence. In this aspect, the DcR3 variant of the present invention may consist of the above amino acid sequence or may include the above amino acid sequence, but preferably consists of the above amino acid sequence. Note that the above amino acid sequence includes an Fc region or a variant thereof (mutant Fc region).

[0127] Note that SEQ ID NO: 76 is the amino acid sequence of chimeric B-Fc(IEGRMD g1S) (a DcR3 variant obtained by fusing chimeric B, IEGRMD linker, and Fc(g1S)), SEQ ID NO: 78 is the amino acid sequence of chimeric C-Fc(IEGRMD g1S) (a DcR3 variant obtained by fusing chimeric C, IEGRMD linker, and Fc(g1S)), SEQ ID NO: 80 is the amino acid sequence of chimeric A-Fc(IEGRMD g1S) (a DcR3 variant obtained by fusing chimeric A, IEGRMD linker, and Fc(g1S)), SEQ ID NO: 82 is the amino acid sequence of chimeric A-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A and Fc(g4PEK)), SEQ ID NO: 84 is the amino acid sequence of 103-123OPG-Fc(g4PEK) (a DcR3 variant obtained by fusing 103-123OPG and Fc(g4PEK)), SEQ ID NO: 86 is the amino acid sequence of N131S / N144S-Fc(g4PEK) (a DcR3 variant obtained by fusing N131S / N144S and Fc(g4PEK)), SEQ ID NO: 88 is the amino acid sequence of T133A / S146A-Fc(g4PEK) (a DcR3 variant obtained by fusing T133A / S146A and Fc(g4PEK)), SEQ ID NO: 90 is the amino acid sequence of N131S / N144S / N157S-Fc(g4PEK) (a DcR3 variant obtained by fusing N131S / N144S / N157S and Fc(g4PEK)), SEQ ID NO: 92 is the amino acid sequence of T133A / S146A / T159A-Fc(g4PEK) (a DcR3 variant obtained by fusing T133A / S146A / T159A and Fc(g4PEK)), SEQ ID NO: 94 is the amino acid sequence of chimeric A-E57K-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57K and Fc(g4PEK)), SEQ ID NO: 96 is the amino acid sequence of chimeric A-E57L-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57L and Fc(g4PEK)), SEQ ID NO: 98 is the amino acid sequence of chimeric A-R60K-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-R60K and Fc(g4PEK)), SEQ ID NO: 150 is the amino acid sequence of chimeric A-Fc(g1S) (a DcR3 variant obtained by fusing chimeric A and Fc(g1S)), SEQ ID NO: 168 is the amino acid sequence of chimeric A-Fc(Eg1S) (chimeric A andThe amino acid sequence of the DcR3 variant fused with Fc(Eg1S), SEQ ID NO: 170 is the amino acid sequence of the chimeric A-Fc(Eg1S-YTE) (the DcR3 variant fused with chimeric A and Fc(Eg1S YTE)), SEQ ID NO: 172 is the amino acid sequence of the chimeric A-Fc(Eg1S-N434A) (the DcR3 variant fused with chimeric A and Fc(Eg1S-N434A)), SEQ ID NO: 174 is the amino acid sequence of the chimeric A-Fc(g1S-LALAGA) (the DcR3 variant fused with chimeric A and Fc(g1S-LALAGA)), SEQ ID NO: 176 is the amino acid sequence of the chimeric A-Fc(Eg1S-LALAGA) (the DcR3 variant fused with chimeric A and Fc(Eg1S-LALAGA)), SEQ ID NO: 178 is the amino acid sequence of the chimeric A-Fc(Eg1S-LALAGANA) (the DcR3 variant fused with chimeric A and Fc(Eg1S-LALAGANA)), SEQ ID NO: 190 is the amino acid sequence of the chimeric A-E57R-Fc(g4PEK) (the DcR3 variant fused with chimeric A-E57R and Fc(g4PEK)), SEQ ID NO: 192 is the amino acid sequence of the chimeric A-E57V-Fc(g4PEK) (the DcR3 variant fused with chimeric A-E57V and Fc(g4PEK)), SEQ ID NO: 194 is the amino acid sequence of the chimeric A-E57K_R58D-Fc(g4PEK) (the DcR3 variant fused with chimeric A-E57K_R58D and Fc(g4PEK)), SEQ ID NO: 196 is the amino acid sequence of the chimeric A-E57K_R58E-Fc(g4PEK) (the DcR3 variant fused with chimeric A-E57K_R58E and Fc(g4PEK)), SEQ ID NO: 198 is the amino acid sequence of the chimeric A-E57R_R58D-Fc(g4PEK) (the DcR3 variant fused with chimeric A-E57R_R58D and Fc(g4PEK)), SEQ ID NO: 200 is the amino acid sequence of the chimeric A-E57K-Fc(Eg1S) (the DcR3 variant fused with chimeric A-E57K and Fc(Eg1S)), SEQ ID NO: 202 is the amino acid sequence of the chimeric A-E57L-Fc(Eg1S) (the DcR3 variant fused with chimeric A-E57L and Fc(Eg1S)), SEQ ID NO: 204 is the amino acid sequence of the chimeric A-E57R-Fc(Eg1S) (the DcR3 variant fused with chimeric A-E57R and Fc(Eg1S)).SEQ ID NO: 206 is the amino acid sequence of chimeric A-E57V-Fc(Eg1S) (a DcR3 variant in which chimeric A-E57V and Fc(Eg1S) are fused), SEQ ID NO: 208 is the amino acid sequence of chimeric A-E57K_R58D-Fc(Eg1S) (a DcR3 variant in which chimeric A-E57K_R58D and Fc(Eg1S) are fused), SEQ ID NO: 210 is the amino acid sequence of chimeric A-E57K_R58E-Fc(Eg1S) (a DcR3 variant in which chimeric A-E57K_R58E and Fc(Eg1S) are fused), SEQ ID NO: 212 is the amino acid sequence of chimeric A-E57R_R58D-Fc(Eg1S) (a DcR3 variant in which chimeric A-E57R_R58D and Fc(Eg1S) are fused), SEQ ID NO: 214 is the amino acid sequence of chimeric A-E57K-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57K and Fc(Eg1S YTE) are fused), SEQ ID NO: 216 is the amino acid sequence of chimeric A-E57L-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57L and Fc(Eg1S YTE) are fused), SEQ ID NO: 218 is the amino acid sequence of chimeric A-E57R-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57R and Fc(Eg1S YTE) are fused), SEQ ID NO: 220 is the amino acid sequence of chimeric A-E57V-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57V and Fc(Eg1S YTE) are fused), SEQ ID NO: 222 is the amino acid sequence of chimeric A-E57K_R58D-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57K_R58D and Fc(Eg1S YTE) are fused), SEQ ID NO: 224 is the amino acid sequence of chimeric A-E57K_R58E-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57K_R58E and Fc(Eg1S YTE) are fused), SEQ ID NO: 226 is the amino acid sequence of chimeric A-E57R_R58D-Fc(Eg1S YTE) (a DcR3 variant in which chimeric A-E57R_R58D and Fc(Eg1S YTE) are fused), SEQ ID NO: 228 is the amino acid sequence of chimeric A-E57K-Fc(Eg1S N434A) (a DcR3 variant in which chimeric A-E57K and Fc(Eg1S N434A) are fused), SEQ ID NO: 230 isThe amino acid sequence of chimeric A-E57L-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57L and Fc(Eg1S N434A)), SEQ ID NO: 232 is the amino acid sequence of chimeric A-E57R-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57R and Fc(Eg1S N434A)), SEQ ID NO: 234 is the amino acid sequence of chimeric A-E57V-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57V and Fc(Eg1S N434A)), SEQ ID NO: 236 is the amino acid sequence of chimeric A-E57K_R58D-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57K_R58D and Fc(Eg1S N434A)), SEQ ID NO: 238 is the amino acid sequence of chimeric A-E57K_R58E-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57K_R58E and Fc(Eg1S N434A)), SEQ ID NO: 240 is the amino acid sequence of chimeric A-E57R_R58D-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57R_R58D and Fc(Eg1S N434A)), SEQ ID NO: 242 is the amino acid sequence of chimeric A-E57K-Fc(Eg1S LALAGA) (a DcR3 variant obtained by fusing chimeric A-E57K and Fc(Eg1S LALAGA)), SEQ ID NO: 244 is the amino acid sequence of chimeric A-E57L-Fc(Eg1S LALAGA) (a DcR3 variant obtained by fusing chimeric A-E57L and Fc(Eg1S LALAGA)), SEQ ID NO: 246 is the amino acid sequence of chimeric A-E57R-Fc(Eg1S LALAGA) (a DcR3 variant obtained by fusing chimeric A-E57R and Fc(Eg1S LALAGA)), SEQ ID NO: 248 is the amino acid sequence of chimeric A-E57V-Fc(Eg1S LALAGA) (a DcR3 variant obtained by fusing chimeric A-E57V and Fc(Eg1S LALAGA)), SEQ ID NO: 250 is the amino acid sequence of chimeric A-E57K_R58D-Fc(Eg1S LALAGA) (a DcR3 variant obtained by fusing chimeric A-E57K_R58D and Fc(Eg1S LALAGA)), SEQ ID NO: 252 is the amino acid sequence of chimeric A-E57K_R58E-Fc(Eg1S LALAGA) (a DcR3 variant obtained by fusing chimeric A-E57K_R58E andThe amino acid sequence of the DcR3 variant fused with Fc(Eg1S LALAGA), SEQ ID NO: 254, is the amino acid sequence of chimeric A-E57R_R58D-Fc(Eg1S LALAGA) (a DcR3 variant fused with chimeric A-E57R_R58D and Fc(Eg1S LALAGA)), SEQ ID NO: 256 is the amino acid sequence of chimeric A-E57K-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57K and Fc(Eg1S LALAGANA)), SEQ ID NO: 258 is the amino acid sequence of chimeric A-E57L-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57L and Fc(Eg1S LALAGANA)), SEQ ID NO: 260 is the amino acid sequence of chimeric A-E57R-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57R and Fc(Eg1S LALAGANA)), SEQ ID NO: 262 is the amino acid sequence of chimeric A-E57V-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57V and Fc(Eg1S LALAGANA)), SEQ ID NO: 264 is the amino acid sequence of chimeric A-E57K_R58D-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57K_R58D and Fc(Eg1S LALAGANA)), SEQ ID NO: 266 is the amino acid sequence of chimeric A-E57K_R58E-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57K_R58E and Fc(Eg1S LALAGANA)), SEQ ID NO: 268 is the amino acid sequence of chimeric A-E57R_R58D-Fc(Eg1S LALAGANA) (a DcR3 variant fused with chimeric A-E57R_R58D and Fc(Eg1S LALAGANA)), SEQ ID NO: 288 is the amino acid sequence of chimeric A-E57A-Fc(g4PEK) (a DcR3 variant fused with chimeric A-E57A and Fc(g4PEK)), SEQ ID NO: 290 is the amino acid sequence of chimeric A-E57F-Fc(g4PEK) (a DcR3 variant fused with chimeric A-E57F and Fc(g4PEK)), SEQ ID NO: 292 is the amino acid sequence of chimeric A-E57H-Fc(g4PEK) (a DcR3 variant fused with chimeric A-E57H and Fc(g4PEK)).SEQ ID NO: 294 is the amino acid sequence of chimeric A-E57I-Fc(g4PEK) (a DcR3 variant in which chimeric A-E57I is fused with Fc(g4PEK)), SEQ ID NO: 296 is the amino acid sequence of chimeric A-E57M-Fc(g4PEK) (a DcR3 variant in which chimeric A-E57M is fused with Fc(g4PEK)), SEQ ID NO: 298 is chimeric A-E57K_R58, The amino acid sequence of T-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57K_R58T and Fc(g4PEK)), SEQ ID NO: 300 is the amino acid sequence of chimeric A-E57L_R58E-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57L_R58E and Fc(g4PEK)), SEQ ID NO: 302 is the amino acid sequence of chimeric A-E57V_R58T-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57V_R58T and Fc(g4PEK)), SEQ ID NO: 304 is the amino acid sequence of chimeric A-E57V_R58E-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57V_R58E and Fc(g4PEK)), SEQ ID NO: 314 is the amino acid sequence of chimeric A-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A and Fc(g1S YTE)), SEQ ID NO: 315 is the amino acid sequence of chimeric A-Fc(g1S N434A) (a DcR3 variant obtained by fusing chimeric A and Fc(g1S N434A)), SEQ ID NO: 316 is the amino acid sequence of chimeric A-Fc(g1S LALAGANA) (a DcR3 variant obtained by fusing chimeric A and Fc(g1S LALAGANA)), SEQ ID NO: 317 is the amino acid sequence of chimeric A-E57K-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57K and Fc(g1S YTE)), SEQ ID NO: 318 is the amino acid sequence of chimeric A-E57L-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57L and Fc(g1S YTE)), SEQ ID NO: 319 is the amino acid sequence of chimeric A-E57R-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57R and Fc(g1S YTE)), SEQ ID NO: 320 is the amino acid sequence of chimeric A-E57V-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57V and Fc(g1S YTE)), SEQ ID NO: 321 is the amino acid sequence of chimeric A-E57K_R58D-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57K_R58D and Fc(g1S YTE)), SEQ ID NO: 322 is the amino acid sequence of chimeric A-E57K_R58E-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57K_R58E and Fc(g1SThe amino acid sequence of the DcR3 variant fused with (YTE), SEQ ID NO: 323 is the amino acid sequence of the chimeric A-E57R_R58D-Fc(g1S YTE) (the DcR3 variant fused with chimeric A-E57R_R58D and Fc(g1S YTE)), SEQ ID NO: 324 is the amino acid sequence of the chimeric A-E57K-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57K and Fc(g1S N434A)), SEQ ID NO: 325 is the amino acid sequence of the chimeric A-E57L-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57L and Fc(g1S N434A)), SEQ ID NO: 326 is the amino acid sequence of the chimeric A-E57R-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57R and Fc(g1S N434A)), SEQ ID NO: 327 is the amino acid sequence of the chimeric A-E57V-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57V and Fc(g1S N434A)), SEQ ID NO: 328 is the amino acid sequence of the chimeric A-E57K_R58D-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57K_R58D and Fc(g1S N434A)), SEQ ID NO: 329 is the amino acid sequence of the chimeric A-E57K_R58E-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57K_R58E and Fc(g1S N434A)), SEQ ID NO: 330 is the amino acid sequence of the chimeric A-E57R_R58D-Fc(g1S N434A) (the DcR3 variant fused with chimeric A-E57R_R58D and Fc(g1S N434A)), SEQ ID NO: 331 is the amino acid sequence of the chimeric A-E57K-Fc(g1S LALAGANA) (the DcR3 variant fused with chimeric A-E57K and Fc(g1S LALAGANA)), SEQ ID NO: 332 is the amino acid sequence of the chimeric A-E57L-Fc(g1S LALAGANA) (the DcR3 variant fused with chimeric A-E57L and Fc(g1S LALAGANA)), SEQ ID NO: 333 is the amino acid sequence of the chimeric A-E57R-Fc(g1S LALAGANA) (the DcR3 variant fused with chimeric A-E57R and Fc(g1S LALAGANA)), SEQ ID NO: 334 is the amino acid sequence of the chimeric A-E57V-Fc(g1SThe amino acid sequence of the DcR3 variant (chimera A-E57V fused with Fc(g1S LALAGANA)), SEQ ID NO: 335, is the amino acid sequence of the chimera A-E57K_R58D-Fc(g1S LALAGANA) (DcR3 variant in which chimera A-E57K_R58D is fused with Fc(g1S LALAGANA)), SEQ ID NO: 336 is the amino acid sequence of the chimera A-E57K_R58E-Fc(g1S LALAGANA) (DcR3 variant in which chimera A-E57K_R58E is fused with Fc(g1S LALAGANA)), SEQ ID NO: 337 is the amino acid sequence of the chimera A-E57R_R58D-Fc(g1S LALAGANA) (DcR3 variant in which chimera A-E57R_R58D is fused with Fc(g1S LALAGANA)).

[0128] In any of the above aspects, the mutations (modifications) added to the above amino acid sequence include any of natural mutations and artificial amino acid substitutions, deletions, insertions or additions. Also, in any of the above aspects, as the sequence in which 1 to 30 amino acids are deleted, substituted, inserted or added in the above amino acid sequence, there are amino acid sequences in which 1 or 2 or more, preferably 2 to 30, more preferably 2 to 10, particularly preferably 2 to 5 amino acids are substituted, deleted, inserted or added, or amino acid sequences having 80% or more, preferably 85% or more, more preferably 90% or more, for example 93% or more, 95% or more, 97% or more, 98% or more or 99% or more identity with the amino acid sequence.

[0129] The DcR3 variant of the present invention may be further chemically modified for the purpose of changing biological activity or its properties, pharmacokinetics such as blood half-life, or physical or chemical properties such as protein stability.

[0130] Examples of chemical modifications include, for example, polyethylene glycol (PEG)ylation, acetylation, amidation, or phosphorylation, etc., and PEGylation is particularly preferred. PEGylation is, for example, to bind one or more PEG molecules to the amino group at the N-terminus of a protein, or to an amino acid residue having a functional group such as the ε-amino group of Lys, carboxyl group, thiol group, or hydroxyl group in the side chain.

[0131] The average molecular weight of the PEG molecule is not limited hereinafter, but it can be used in the range of about 3000 to about 50000.

[0132] As a method for binding a PEG molecule to a DcR3 variant, an active group such as a carboxyl group, formyl (aldehyde) group, N-hydroxysuccinimide ester group, amino group, thiol group, or maleimide group is introduced into the PEG terminal portion, and it is reacted with a functional group such as an amino group, carboxyl group, thiol group, or hydroxyl group in the side chain of the DcR3 variant.

[0133] The signal peptide is not limited, but when the N-terminal CRD domain contained in the DcR3 variant is derived from human DcR3, the signal peptide of human DcR3 consisting of the amino acid sequence of positions 1 to 29 of SEQ ID NO: 2 can be mentioned. Also, when the N-terminal CRD domain contained in the DcR3 variant is derived from human OPG, the signal peptide derived from human OPG consisting of the amino acid sequence of positions 1 to 21 of SEQ ID NO: 14 can be mentioned. Also, any of an artificial sequence, a sequence derived from an expression vector, and a sequence derived from another protein suitable for the host cell expressing the DcR3 variant is included in the signal peptide of the present invention. The DcR3 variant containing the signal peptide is an immature polypeptide, and in one embodiment, the signal peptide is cleaved during the maturation process. The DcR3 variant of the present invention also includes those having different N-termini cleaved at positions different from the predicted site of the above signal peptide cleavage.

[0134] 5. Method for producing DcR3 variant The DcR3 variant of the present invention can be produced by expressing the DNA encoding the DcR3 variant in a host cell by, for example, the following method using the methods described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press (2001) and the like.

[0135] The signal peptide is not limited, but when the N-terminal CRD domain contained in the DcR3 variant is derived from human DcR3, the signal peptide of human DcR3 consisting of the amino acid sequence of positions 1 to 29 of SEQ ID NO: 2 can be mentioned. Further, when the N-terminal CRD domain contained in the DcR3 variant is derived from human OPG, the signal peptide derived from human OPG consisting of the amino acid sequence of positions 1 to 21 of SEQ ID NO: 14 can be mentioned. In addition, any of an artificial sequence, a sequence derived from an expression vector, and a sequence derived from another protein suitable for the host cell expressing the DcR3 variant can be used for the production of the DcR3 variant of the present invention. The DcR3 variant of the present invention also includes those having different N-termini cleaved at positions different from the predicted site of cleavage of the above signal peptide.

[0136] The DcR3 variant of the present invention can also be obtained by artificially designing it based on the amino acid sequence of the cysteine-rich region of the wild-type DcR3 before amino acid substitution, the amino acid sequence of the cysteine-rich region of the DcR3 variant, or the amino acid sequence of the DcR3 variant, or by analyzing mutants. As the mutagenesis method, site-directed mutagenesis using the PCR method using primers is preferable (Kunkel et al., Proc. Natl. Acad. Sci. USA, 1985, 82: 488-492). In addition, methods for fully synthesizing the mutated gene, performing PCR separately on the part before and after the mutation site using primers containing the mutation, ligating the two fragments by overlap of the region containing the mutation, and inserting them into a vector by the In-fusion cloning method (Clontech) and the like can be mentioned.

[0137] As a method for identifying a mutation having a target binding mode, there is a method of preparing a library into which mutations are randomly introduced, displaying it on phage, yeast, etc., and screening by binding activity to obtain a mutant having the target binding mode. Alternatively, there is a method of producing a mutant having the target binding mode by expressing a vector into which a DNA mutation for substituting a specific amino acid with a different amino acid is introduced in a host cell. The DNA encoding the DcR3 variant of the present invention can be synthesized by a DNA synthesizer by designing a base sequence encoding the DcR3 variant of the present invention from the amino acid sequence of the DcR3 variant of the present invention. It can also be isolated by PCR using cDNA of humans or the like as a template.

[0138] By inserting the DNA encoding the DcR3 variant of the present invention obtained above downstream of the promoter of an appropriate expression vector, a recombinant vector is prepared, and the recombinant vector is introduced into a host cell compatible with the expression vector.

[0139] As the base sequence of the DNA encoding the DcR3 variant, the bases can be substituted so as to be codons optimal for expression in the host, thereby improving the production rate of the target DcR3 variant. When preparing the DNA encoding the above DcR3 variant, a DNA encoding a signal peptide of a secreted protein is added to its 5' end, and using this DNA, a recombinant vector is prepared in the same manner as above and introduced into a host cell, whereby the peptide can be secreted into the medium for production. Examples of the signal peptide include sequences derived from DcR3 or OPG, artificial sequences, sequences derived from expression vectors, or sequences derived from other proteins suitable for host cells.

[0140] In one aspect, examples of the base sequence of the DNA encoding the amino acid sequence of the DcR3 variant include the base sequences set forth in SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45.

[0141] In another aspect, examples of the base sequence of the DNA encoding the amino acid sequence of the DcR3 variant include the base sequences described in SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 179, 181, 183, 185, 187, 269, 271, 273, 275, 277, 279, 281, 283, and 285.

[0142] In yet another aspect, examples of the base sequence of the DNA encoding the amino acid sequence of the DcR3 variant include the base sequences described in SEQ ID NOs: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 149, 167, 169, 171, 173, 175, 177, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 287, 289, 291, 293, 295, 297, 299, 301, and 303.

[0143] As the expression vector, any can be used as long as it can autonomously replicate or be integrated into the chromosome in the host cell to be used and contains an appropriate promoter at a position where the DNA encoding the polypeptide can be transcribed.

[0144] As the host cell, any can be used as long as it can express the gene encoding the DcR3 variant, such as yeast, insect cells, animal cells, etc. Preferably, yeast, animal cells, or insect cells that can glycosylate proteins are mentioned.

[0145] Examples of yeast include Saccharomyces ( Saccharomyces ), Schizosaccharomyces ( Schizosaccharomyces ), Kluyveromyces ( Kluyveromyces ), Trichosporon ( Trichosporon ), Schwanniomyces ( Schwanniomyces ), Pichia (Pichia ) genus, Candida Candida ) microorganisms belonging to, for example, Saccharomyces cerevisiae , Schizosaccharomyces pombe , Kluyveromyces lactis , Trichosporon pullulans , Schwanniomyces alluvius , or Candida utilis etc. can be mentioned.

[0146] Examples of insect cells include Sf9, Sf21 (ovary cells of Spodoptera frugiperda) [Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)], High 5 (manufactured by Invitrogen), which are ovary cells of Trichoplusia ni, or S2 (Schneider 2) cells (Thermo Scientific) derived from late embryos of Drosophila melanogaster, etc.

[0147] Examples of animal cells include, for example, Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cellgenetics, Appendix I, II (pp. 883-900)), CHO / DG44, CHO-K1 (ATCC No.: CCL-61), Freestyle CHO-S cells, CHO cells deficient in the dihydrofolate reductase gene [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO cells deficient in the 6-fucosyltransferase gene (International Publication No. WO 2005 / 035586, International Publication No. WO 02 / 31140), 293 cells, which are human cells (ATCC No.: CRL-1573), Freestyle 293F cells, Expi293 cells (Thermo Scientific), DUKXB11 (ATCC No.: CCL-9096), Pro-5 (ATCC No.: CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3, rat myeloma cell YB2 / 3HL.P2.G11.16Ag.20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14, or Syrian hamster cell BHK, HBT5637 (Japanese Patent Laid-Open No. 63-299), and the like.

[0148] When using yeast as a host cell, examples of expression vectors include YEP13 (ATCC number: 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19, or pHS15, etc.

[0149] As the promoter, any promoter that can be expressed in the yeast strain can be used. For example, promoters of glycolytic genes such as hexokinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, or CUP 1 promoter, etc. can be mentioned.

[0150] As a method for introducing the recombinant vector, any method for introducing DNA into yeast can be used. For example, the electroporation method [Methods Enzymol., 194, 182 (1990)], the spheroplast method [Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)], the lithium acetate method [J. Bacteriology, 153, 163 (1983)], or the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), etc. can be mentioned.

[0151] When using insect cells as a host, for example, according to the methods described in Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992), Bio / Technology, 6, 47 (1988), etc., the peptide can be expressed.

[0152] That is, after co-introducing the recombinant gene-introducing vector and the defective baculovirus genome into insect cells to obtain a recombinant virus in the insect cell culture supernatant, the recombinant virus can be further used to infect insect cells to express the peptide.

[0153] Examples of the gene transfer vector used in this method include pVL1392, pVL1393 (manufactured by Becton Dickinson), pBlueBac4.5 (manufactured by Invitrogen), etc.

[0154] Examples of the baculovirus that can be used include Autographa californica nuclear polyhedrosis virus, which is a virus that infects insects of the family Noctuidae.

[0155] Examples of the co-introduction method of the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

[0156] When using S2 (Schneider 2) cells (Thermo Scientific), which are derived from the late embryos of the insect cell Drosophila melanogaster, as a host, for example, a gene transfer vector such as pMTBiPV5-HisA (Thermo Scientific) can be introduced into the host cell by the calcium phosphate method according to the method described in Mol. Biotechnol., 2015, 10: p. 914-922, etc., to express the peptide.

[0157] When using animal cells as a host, examples of expression vectors include pCI mammalian expression vector (Promega), pcDNA3.1(+) (manufactured by Invitrogen), pcDNA I / Amp, pcDNA I, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3, 133 (1990)], pAS3-3 [Japanese Patent Laid-Open No. 2-227075], pCDM8 [Nature, 329, 840 (1987)], pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], pAGE210, pME18SFL3, or pKANTEX93 (International Publication No. 97 / 10354), etc.

[0158] As the promoter, any promoter that functions in animal cells can be used. For example, the promoter of the IE (immediate early) gene of cytomegalovirus (CMV), the early promoter of SV40, the promoter of retrovirus, the metallothionein promoter, the heat shock promoter, or the SRα promoter, etc. can be mentioned. Also, the enhancer of the IE gene of human CMV may be used together with the promoter.

[0159] As a method for introducing the recombinant vector into animal cells, any method for introducing DNA into animal cells can be used. For example, the electroporation method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], or the method described in Virology, 52, 456 (1973), etc. can be mentioned.

[0160] Transient expression of the DcR3 variant of the present invention obtained by the above-described method, or an expression vector modified therefrom, can be carried out.

[0161] As the host cell into which the expression vector is introduced, any cell can be used as long as it is a host cell capable of expressing the DcR3 variant. For example, COS-7 cells (ATCC number: CRL1651) are used [Methods in Nucleic Acids Res., CRC Press, 283 (1991)]. For introducing the expression vector into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press, (1991)] or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] etc. is used.

[0162] When CHO-S cells or Expi293 cells (Thermo Scientific) are used, for introducing the expression vector, the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] etc. is used.

[0163] Using the expression vector of the DcR3 variant of the present invention obtained by the method described above, or an expression vector modified therefrom, a transformant that stably expresses the DcR3 variant can be obtained. After introducing the expression vector, the transformant that stably expresses the recombinant antibody is selected by culturing in a medium for animal cell culture containing a drug such as G418 sulfate (Japanese Patent Laid-Open No. 2-257891).

[0164] When the transformant is a transformant obtained using a eukaryote such as yeast as the host, as the medium for culturing the transformant, a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, and / or inorganic salts etc. that the transformant can assimilate and can efficiently culture the transformant.

[0165] As the carbon source, any that the transformant can assimilate may be used. For example, carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch or starch hydrolysates, organic acids such as acetic acid and propionic acid, alcohols such as ethanol or propanol etc. can be used.

[0166] As the nitrogen source, for example, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and / or ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented microbial cells and their digests, etc. can be used.

[0167] As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and / or calcium carbonate, etc. can be used.

[0168] The culture is preferably carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 °C, and the culture time is usually preferably 16 hours to 7 days. The pH during the culture is preferably maintained at 3.0 to 9.0. The adjustment of the pH can be carried out using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, or ammonia, etc.

[0169] Also, if necessary during the culture, antibiotics such as ampicillin or tetracycline can be added to the medium.

[0170] As the medium for culturing the transformant obtained using insect cells as the host, generally used TNM-FH medium (manufactured by Becton Dickinson), Sf-900 II SFM medium (manufactured by Invitrogen), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace's insect medium [Nature, 195, 788 (1962)], or Schneider's Medium (Thermo Fisher), etc. can be used.

[0171] The cultivation of the transformant obtained using insect cells as a host is usually preferably carried out at a pH of 6 to 7 and a temperature of 25 to 30°C for preferably 1 to 5 days. Also, if necessary during cultivation, an antibiotic such as gentamicin may be added to the medium.

[0172] For media for animal cell culture, RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRHBiosciences), EX-CELL325 PF CHO serum-Free medium (Sigma Aldrich), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM medium (manufactured by Invitrogen), Eagle's Minimal Essential Medium (MEM) [Science, 122, 501 (1952)], Dulbecco's Modified Eagle Medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], or a medium obtained by adding various additives such as FBS to these media is used.

[0173] The cultivation of animal cells is usually preferably carried out in the presence of 5% CO 2 at a pH of 6 to 8 and a temperature of 30 to 40°C for preferably 1 to 7 days. Also, if necessary during cultivation, an antibiotic such as kanamycin or penicillin may be added to the medium.

[0174] By culturing the obtained transformant strain in a medium, the DcR3 variant or the DcR3 variant is expressed and accumulated in the culture supernatant. Also, the expression level of the DcR3 variant or the DcR3 variant can be increased using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891) or the like.

[0175] By generating and accumulating the DcR3 variant according to the present invention in the culture as described above and collecting it from the culture, the DcR3 variant according to the present invention can be produced.

[0176] In order to isolate and purify the peptide produced by the transformant, ordinary protein isolation and purification methods can be used.

[0177] For example, when the DcR3 variant of the present invention is secreted extracellularly, the DcR3 variant can be recovered from the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a method such as centrifugation, and from the culture supernatant, ordinary protein isolation and purification methods, namely, solvent extraction method, salting-out method with ammonium sulfate or the like, desalting method, precipitation method with an organic solvent, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchange chromatography method using a resin such as S-Sepharose FF (GE Healthcare), hydrophobic chromatography method using a resin such as butyl Sepharose or phenyl Sepharose, gel filtration method using a molecular sieve, affinity chromatography method, chromatofocusing method, or electrophoresis method such as isoelectric focusing electrophoresis, etc. are used alone or in combination to obtain a purified product.

[0178] For example, when the DcR3 variant of the present invention has an Fc of an immunoglobulin capable of binding to protein G or protein A, a protein G chromatography method or a protein A chromatography method in which protein G or protein A is bound to a carrier as an affinity ligand can be used as an affinity chromatography method. [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Also, methods used in protein purification such as gel filtration, ion exchange chromatography, and ultrafiltration can be combined.

[0179] In addition, from the DcR3 variants obtained by the above-described method, DcR3 variants further chemically modified with a peptide, sugar chain, PEG, or the like can also be obtained by using a conventional chemical modification method.

[0180] 6. Methods for Evaluating the Biological Activity, Physical Properties, and Kinetics of DcR3 Variants Preferred DcR3 variants of the present invention include DcR3 variants having neutralizing activity against at least one of LIGHT, TL1A, and FasL, DcR3 variants having neutralizing activity against all of LIGHT, TL1A, and FasL, DcR3 variants having no neutralizing activity against FasL and having neutralizing activity against any one of LIGHT and TL1A, DcR3 variants having no neutralizing activity against FasL and having neutralizing activity against LIGHT and TL1A, and the like.

[0181] The DcR3 variants of the present invention can measure biological activities such as neutralizing activity, physical properties, and in vivo kinetics by using the following methods.

[0182] (1) Preparation of Ligands The LIGHT, TL1A, and FasL used in the present invention are not limited in their origin, and examples include LIGHT, TL1A, and FasL derived from eukaryotes. Examples of LIGHT, TL1A, and FasL derived from eukaryotes include yeast, insects, or mammals. Preferably, LIGHT, TL1A, and FasL derived from primates including humans, or rodents including mice, etc. are mentioned.

[0183] LIGHT, TL1A, or FasL, or cells expressing these ligands, can be obtained by introducing an expression vector containing cDNA encoding the full-length or partial-length of LIGHT, TL1A, or FasL into appropriate host cells such as Escherichia coli, yeast, insect cells, or animal cells. In addition, these ligands can also be obtained by purifying them from various human cultured cells or human tissues that highly express LIGHT, TL1A, or FasL. Moreover, the cultured cells or tissues can be used directly as ligands. Furthermore, synthetic peptides having partial sequences of LIGHT, TL1A, or FasL can be prepared by chemical synthesis methods such as the Fmoc method or the tBoc method.

[0184] In addition, LIGHT, TL1A, and FasL can be produced by introducing and expressing DNA encoding LIGHT, TL1A, or FasL into host cells using methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), etc., for example, by the following methods.

[0185] Soluble LIGHT, TL1A, and FasL are generated by the shedding of the extracellular region by proteases after being expressed on the cell membrane as membrane-bound LIGHT, membrane-bound TL1A, and membrane-bound FasL, respectively. The cleavage sites have been identified for each ligand, but when preparing recombinant soluble ligands, they can contain any sequence near the cleavage site. For example, recombinant soluble LIGHT can be prepared from the region from the 66th to 240th, 74th to 240th, 83rd to 240th positions from the N-terminus in the amino acid sequence of membrane-bound LIGHT. Recombinant soluble TL1A can be prepared from the region from the 72nd to 251st positions from the N-terminus in the amino acid sequence of membrane-bound TL1A. Soluble FasL can be prepared from the region from the 130th to 281st, 134th to 281st positions from the N-terminus in the amino acid sequence of membrane-bound FasL.

[0186] The recombinant soluble ligand can be prepared by adding a His tag, a FLAG tag, etc. to the N-terminus of the above region and can be obtained by affinity purification. Examples of the amino acid sequences of the soluble recombinants of LIGHT, TL1A, and FasL include the amino acid sequences described in SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, 128, 130, and 306. Examples of the nucleotide sequences of the DNA encoding the amino acid sequences of the soluble recombinants of LIGHT, TL1A, and FasL include the nucleotide sequences described in SEQ ID NOs: 113, 115, 117, 119, 121, 123, 125, 127, 129, and 305.

[0187] Functional LIGHT, TL1A, or FasL can form homotrimers. The soluble LIGHT, TL1A, or FasL prepared by any of the above methods can be analyzed by SEC-MALS to determine the molecular weight. SEC-MALS is an analytical method in which the molecular weight is calculated using the maximum value of the scattering intensity detected by a multi-angle light scattering detector (MALS) for each peak detected at a wavelength of 215 nm separated by SEC (Size Exclusion chromatography)-HPLC. Examples of the HPLC system include Prominence manufactured by Shimadzu Corporation, examples of the SEC column include TSKgel manufactured by Tosoh Corporation, and examples of the MALS detector include miniDAWN TREOS manufactured by Wyatt Technology Corporation. Further, as a method for isolating the homotrimer from a crude product forming other than trimers, for example, SEC purification can be mentioned. As a method for SEC purification, for example, using AKTApurifier manufactured by GE Healthcare as the HPLC system and Superdex 200 Increase 10 / 300 GL manufactured by GE Healthcare as the SEC column, the crude product is fractionated by molecular weight size, and only the molecular weight fraction of the homotrimer is recovered.

[0188] (2) Evaluation of the binding activity of the DcR3 variant containing the human Fc region As a method for measuring the binding activity of the DcR3 variant to soluble LIGHT, soluble TL1A, or soluble FasL, for example, a binding assay by enzyme immunoassay (ELISA) and kinetics analysis by Biacore can be mentioned. As the ligand, a gene-introduced cell or recombinant protein obtained by introducing an expression vector containing a DNA sequence encoding the extracellular domain of each ligand of LIGHT, TL1A, or FasL into Escherichia coli, yeast, insect cells, or animal cells, etc., and a serum, plasma, or culture supernatant containing a ligand obtained from human tissue or human cells are used.

[0189] In the case of ELISA, for example, an anti-human IgG antibody is immobilized on a plate with 96 wells or the like, and after reacting with the DcR3 variant, each ligand is dispensed and allowed to bind. After washing, an unlabeled anti-ligand antibody or a receptor-Fc fusion of each ligand is reacted, and then an anti-Fc antibody labeled with biotin, an enzyme, a chemiluminescent substance, or the like is reacted, or a labeled anti-ligand antibody or a receptor-Fc fusion of each ligand is reacted, detection is performed according to the labeling substance, and the binding of the DcR3 variant to the ligand can be measured. In the case of Biacore, for example, Biacore T100 or Biacore T200 is used to measure the kinetics in the binding of each ligand to the DcR3 variant, and the result is analyzed with the analysis software attached to the instrument. Specifically, after immobilizing an anti-human IgG antibody on the sensor chip CM5 by the amine coupling method, the DcR3 variant is flowed to bind an appropriate amount on the sensor chip, and then a plurality of ligands with known concentrations are flowed to measure the binding and dissociation. The obtained data is subjected to kinetics analysis using the software attached to the instrument according to the 1:1 binding model to obtain various parameters. Or, after immobilizing each ligand on the sensor chip, for example, by the amine coupling method, a plurality of DcR3 variants with known concentrations are flowed to measure the binding and dissociation. The obtained data is subjected to kinetics analysis using the software attached to the instrument according to the bivalent binding model to obtain various parameters. Alternatively, on the sensor chip ProteinA with the MabSelectSure ligand, which is a modified protein of the IgG binding domain of ProteinA, immobilized on the sensor chip, the DcR3 variant is flowed to bind an appropriate amount on the sensor chip, and then a plurality of ligands with known concentrations are flowed to measure the binding and dissociation. The obtained data is subjected to kinetics analysis using the software attached to the instrument according to the 1:1 binding model to obtain various parameters.

[0190] The binding activity of the DcR3 variant to membrane-bound LIGHT, membrane-bound TL1A, or membrane-bound FasL can be measured by flow cytometry, for example, using a gene-introduced cell obtained by introducing an expression vector containing a DNA sequence encoding the full length of each ligand of LIGHT, TL1A, and FasL into an animal cell or the like, or a human cell such as PBMC or HUVEC that has been stimulated with a mitogen such as PHA-L, PMA, or ionomycin, or stimulated with anti-CD3 antibody and anti-CD28 antibody, or appropriately stimulated with a cytokine, IgG, or immune complex to induce the expression of the membrane-bound ligand. Specifically, it can be measured by reacting the DcR3 variant with a cell expressing the membrane-bound ligand and then reacting with a fluorescently labeled anti-Fc antibody or the like, or by reacting a DcR3 variant labeled with biotin, a fluorescent dye, or the like, washing, and then detecting the fluorescence intensity corresponding to the labeled substance using a flow cytometer.

[0191] Examples of the DcR3 variant of the present invention measured by the above method include a DcR3 variant having a binding activity to at least one or more of LIGHT, TL1A, and FasL, a DcR3 variant having a binding activity to all of LIGHT, TL1A, and FasL, a DcR3 variant having no binding activity to FasL but having a binding activity to either LIGHT or TL1A, or a DcR3 variant having no binding activity to FasL but having a binding activity to both LIGHT and TL1A.

[0192] (3) Evaluation of the neutralizing activity of the DcR3 variant Examples of the method for measuring the neutralizing activity of the DcR3 variant include a method for measuring the binding between LIGHT, TL1A, or FasL and its corresponding receptor in a solution to which the DcR3 variant has been added, or a method for adding the corresponding ligand to receptor-expressing cells of LIGHT, TL1A, or FasL in a medium to which the DcR3 variant has been added and measuring cell functions such as cytokine production and cell proliferation.

[0193] The inhibitory activity against the binding of human LIGHT to its corresponding receptor can be measured by the following method, for example, using the method described in US Patent US8,974,787 B2. In a reaction solution containing a DcR3 variant, HVEM or LTβR labeled with biotin, a fluorescent dye, etc. is reacted with cells expressing membrane-bound LIGHT. After washing, the fluorescence intensity corresponding to the labeling substance is detected using a flow cytometer to measure the inhibitory activity. Alternatively, in a reaction solution containing a DcR3 variant, LIGHT labeled with biotin, a fluorescent dye, etc. is reacted with cells expressing HVEM or LTβR. After washing, the fluorescence intensity corresponding to the labeling substance is detected using a flow cytometer to measure the inhibitory activity. The inhibitory activity of the DcR3 variant against the binding of LIGHT to its corresponding receptor is confirmed by the decrease in the fluorescence intensity compared to the case where the DcR3 variant is not added.

[0194] Using the same method as described above, the inhibitory activity against the binding of TL1A or FasL to its corresponding receptor can also be measured.

[0195] Preferred DcR3 variants of the present invention include DcR3 variants having inhibitory activity against at least one of the bindings of LIGHT, TL1A, and FasL to their corresponding receptors, DcR3 variants having inhibitory activity against all of the bindings of LIGHT, TL1A, and FasL to their corresponding receptors, DcR3 variants having no inhibitory activity against the binding of FasL to its corresponding receptor but having inhibitory activity against any one of the bindings of LIGHT and TL1A to their corresponding receptors, or DcR3 variants having no inhibitory activity against the binding of FasL to its corresponding receptor but having inhibitory activity against the bindings of LIGHT and TL1A to their corresponding receptors. In the present invention, the expression "having no inhibitory activity against the binding of a certain ligand to its corresponding receptor" is used in the meaning including that the inhibitory activity is significantly lower than that of wild-type DcR3.

[0196] The inhibitory activity against cell functions induced by the addition of LIGHT can be measured as follows, using, for example, the method described in US Patent US8,974,787 B2. In a medium supplemented with a DcR3 variant, cells such as HT-29 (ATCC number: HTB-38) in which the expression of LIGHT receptors HVEM and LTβR has been confirmed are used, and LIGHT-dependent chemokine production such as IL-8, CCL5, or CCL20 is measured by ELISA using the culture supernatant, alphaLISA (Perkin Elmer), or CBA assay (BD Biosciences). Alternatively, chemokine production such as soluble or membrane-bound LIGHT-dependent CXCL-10 is measured in a similar manner using cells such as intestinal myofibroblasts (Lonza) stimulated with IFN-γ. Alternatively, instead of soluble or membrane-bound LIGHT, LIGHT induced by expression from PBMCs or T cells stimulated with anti-CD3 antibody and anti-CD28 antibody, or PMA and ionomycin, can be used to measure in a similar manner. The inhibitory activity of the DcR3 variant against cell functions induced by the addition of soluble or membrane-bound LIGHT is confirmed by a decrease in the production amount of the chemokine, in comparison with the case where the DcR3 variant is not added. Also, in vivo, for example, using the method described in US Patent US8,974,787 B2, in an acute GVHD (Graft versus host disease) model in which human PBMCs are xenografted into immunodeficient mice, improvement in survival, body weight, disease state, pathology, engraftment of human cells, etc. by administration of the DcR3 variant can be evaluated.

[0197] The inhibitory activity on cell functions induced by the addition of TL1A can be measured as follows, using, for example, the methods described in Mucosal Immunology, 2015, 8: p. 545-558. In a medium supplemented with the DcR3 variant, using blood, PBMC, pan T cells, CD4-positive T cells, or memory CD4-positive T cells derived from primates such as humans or rodents such as mice, the cells are stimulated with cytokine cocktails of IL-12, IL-18, and TL1A, or IL-12, IL-18, IL-15, and TL1A, and the production of cytokines such as TL1A-dependent IFN-γ, IL-6, GM-CSF, TNF-α, IL-5, IL-13, IL-17, or IL-22 is measured by ELISA, alphaLISA, or CBA assay using the culture supernatant. Alternatively, for example, using the methods described in Immunity, 2002, 16: p. 479-492, in a medium supplemented with the DcR3 variant, pan T cells, CD4-positive T cells, or memory CD4-positive T cells derived from primates such as humans or rodents such as mice are stimulated with anti-CD3 antibody and anti-CD28 antibody, and the production of TL1A-dependent IFN-γ and IL-2 is measured by the same method as described above. Alternatively, it can also be measured in the same way using a forced expression strain of membrane-type TL1A instead of soluble TL1A. Alternatively, it can also be measured in the same way using TL1A induced by stimulation of PBMC or monocytes with IgG or immune complexes. The inhibitory activity of the DcR3 variant on cell functions induced by the addition of TL1A is confirmed by a decrease in the cytokine or inhibition of cell proliferation in comparison with the case where the DcR3 variant is not added. Furthermore, in vivo, for example, in the TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced colitis model described in Mucosal Immunology, 2011, 4: p. 172-185, or the DSS (dextran sodium sulfate)-induced colitis model described in Mucosal Immunology, 2014, 7: p. 1492-1503, the improvement of survival, body weight, disease state, and pathology by administration of the DcR3 variant can be evaluated.In addition, the effect of improving the pathological condition can be evaluated in a model of a rodent such as a mouse with TL1A-dependent inflammation, allergic disease, or autoimmune disease.

[0198] The inhibitory activity against the cell function induced by the addition of FasL can be measured as follows, for example, using the method described in J. Rheumatol., 2013, 40: p. 1316-1326. In a medium supplemented with the DcR3 variant, using Jurkat cells (DSMZ number: ACC 282), soluble FasL-dependent apoptosis crosslinked with soluble FasL or an antibody is measured by Annexin V / Propium Iodide staining or the BrdU incorporation or ATP amount of living cells. Alternatively, it can also be measured in the same manner using a membrane-type FasL overexpression strain instead of soluble FasL. Or it can also be measured in the same manner using FasL induced from stimulated PBMCs or T cells. The inhibitory activity of the DcR3 variant against the cell function induced by the addition of FasL is confirmed by the decrease in the apoptosis as compared with the case where the DcR3 variant is not added.

[0199] Examples of the DcR3 variant of the present invention measured by the above method include a DcR3 variant having inhibitory activity against at least one or more of the cell functions induced by the addition of LIGHT, TL1A, and FasL, a DcR3 variant having inhibitory activity against all of the cell functions induced by the addition of LIGHT, TL1A, and FasL, a DcR3 variant having no inhibitory activity against the cell function induced by the addition of FasL and having inhibitory activity against any one or more of the cell functions induced by the addition of LIGHT and TL1A, or a DcR3 variant having no inhibitory activity against the cell function induced by the addition of FasL and having inhibitory activity against the cell functions induced by the addition of LIGHT and TL1A. In the present invention, the expression "having no inhibitory activity against the cell function induced by the addition of a certain ligand" is used in a meaning including that the inhibitory activity is significantly lower than that of wild-type DcR3.

[0200] (4) Evaluation of the OPG ligand reactivity of the DcR3 variant One feature of the DcR3 variant of the present invention is that any one or all of a part of CRD1, CRD4, and CRD3, which are not involved in ligand binding, are sequences derived from OPG.

[0201] The fact that the DcR3 variant does not have binding activity to RANKL and TRAIL, which are OPG ligands, can be evaluated by, for example, the same method as the method for measuring the binding activity to LIGHT, TL1A, and FasL described above.

[0202] One feature of the DcR3 variant of the present invention is that it further does not have neutralizing activity against RANKL and TRAIL.

[0203] The neutralizing activity of the DcR3 variant against RANKL can be evaluated by using, for example, the measurement of TRAP (tartrate-resistant acid phosphatase) activity, which is a differentiation assay of osteoclast precursor cells by RANKL stimulation, as described in J. Immunol., 2012, 189: p. 245-252.

[0204] In addition, the neutralizing activity of the DcR3 variant against TRAIL can be evaluated by, for example, inducing apoptosis by soluble TRAIL or cross-linked soluble TRAIL in a human cancer cell line expressing DR4 or DR5, by the same method as the method for measuring the neutralizing activity against FasL described above.

[0205] (5) Pharmacokinetics evaluation of the DcR3 variant As the DcR3 variant of the present invention, a DcR3 variant in which the binding to heparan sulfate contained in heparan sulfate proteoglycan (HSPG) on the cell membrane is reduced or lost is preferred, and a variant having no heparan sulfate binding domain (HBD) is more preferred.

[0206] The presence or absence of binding to heparan sulfate on the cell membrane via HBD can be measured as follows using, for example, the method described in J. Immunol., 2006, 176: p. 173-180. For any cell, such as CHO cells, human cell lines, vascular endothelial cells, hepatocytes, or blood cells, etc., react with wild-type DcR3 or a DcR3 variant, and then react with a fluorescently labeled detection antibody or the like, or react with wild-type DcR3 or a DcR3 variant labeled with biotin, a fluorescent dye, etc. After washing, the fluorescence intensity corresponding to the labeled substance can be detected using a flow cytometer (FCM) for measurement. The decrease or disappearance of binding to the cell membrane due to the deletion of the HBD of DcR3 can be examined by the decrease in fluorescence intensity by FCM.

[0207] As a method for evaluating whether the above binding to the cell membrane is binding via the HBD of DcR3, for example, using the method described in J. Immunol., 2006, 176: p. 173-180, during the reaction of wild-type DcR3 or a DcR3 variant, add GAGs such as heparin and heparan sulfate as inhibitors, or pretreat the cells with enzymes such as heparinase or trypsin, and then react with wild-type DcR3 or a DcR3 variant. This is an example of such a method.

[0208] One feature of the DcR3 variant of the present invention is that its pharmacokinetics is improved compared to wild-type DcR3 because the disappearance via heparan sulfate in vivo is reduced. In the present invention, "the pharmacokinetics is improved" means that the blood half-life is longer than that of wild-type DcR3, or the area under the concentration-time curve (AUC) value up to an infinite time is higher.

[0209] In addition, as a comparison target, instead of wild-type DcR3, a molecule containing the CRD of wild-type DcR3 can also be used. Specifically, DcR3 FL-Fc (amino acid sequence: SEQ ID NO: 100, DNA base sequence: SEQ ID NO: 99), DcR3 FL-FLAG (amino acid sequence: SEQ ID NO: 104, DNA base sequence: SEQ ID NO: 103), S195-Fc (amino acid sequence: SEQ ID NO: 102, DNA base sequence: SEQ ID NO: 101) obtained by fusing the CRD of wild-type DcR3 and Fc, and R128Q-Fc [US Patents US6,835,814 B1, US6,965,01 B1] obtained by fusing Fc to the C-terminus of full-length DcR3 (amino acid sequence: SEQ ID NO: 112, DNA base sequence: SEQ ID NO: 111) having one amino acid mutation in the HBD, etc. can be mentioned. When these molecules containing the CRD of wild-type DcR3 are used as comparison targets, they are also described as wild-type DcR3 controls.

[0210] In rodents such as mice and non-human primates such as cynomolgus monkeys, the change in blood concentration of the DcR3 variant can be measured by administering any dose intravenously or subcutaneously, collecting blood at any time, and using a detection system for DcR3 or human Fc. Pharmacokinetic parameters such as blood half-life and AUC can be calculated from the change in blood concentration using methods described in, for example, Pharmacokinetics, 1999, 14: p. 286-293 and other methods, and using techniques such as moment analysis.

[0211] In addition, since it is known that the pharmacokinetics are improved when the number of sialic acids added to the end of the N-type sugar chain is large (J. Pharm. Sci., 2015, 104: p. 1866-1884), it is also preferable that the DcR3 variant of the present invention having an N-type sugar chain has a large number of added sialic acids. The number of added sialic acids per protein molecule can be calculated, for example, by separating the sialic acid labeled with a sialic acid fluorescent labeling reagent kit (Takara) etc. by reverse phase HPLC and comparing it with the standard curve of sialic acid.

[0212] (6) Physical property evaluation of DcR3 variant One of the characteristics of the DcR3 variant of the present invention is that when expressed, isolated or purified using mammalian cells, the content of aggregates is lower than that of wild-type DcR3.

[0213] Whether aggregates are formed during protein expression and secretion can be determined, for example, by recovering host cells into which a recombinant vector carrying the DcR3 variant has been introduced and / or culture supernatants, and, in the case of an anti-DcR3 antibody or an Fc fusion or an adduct with a tag such as His or FLAG, examining the approximate molecular weight under non-reducing conditions by immunoblotting with an antibody against them. When the protein aggregates during protein expression and / or secretion, one or more bands of a size larger than the predicted molecular weight are detected.

[0214] Examples of methods for calculating the molecular weight of a protein include methods calculated from the amino acid sequence. Further, when a more accurate molecular weight is required, an analysis method by SEC-MALS can be mentioned. For each peak detected at a wavelength of 215 nm separated by SEC (Size Exclusion chromatography)-HPLC, the molecular weight can be calculated using the maximum value of the scattering intensity detected by a multi-angle light scattering detector (Multi Angle Light Scattering; MALS). Examples of the HPLC system include Prominence from Shimadzu Corporation, examples of the SEC column include TSKgel from Tosoh Corporation, and examples of the MALS detector include miniDAWN TREOS from Wyatt Technology Corporation, etc.

[0215] Whether the isolated or purified protein is aggregated can be examined by subjecting the protein to SDS-PAGE under non-reducing conditions and detecting it by CBB staining or the like, or by immunoblotting by the same method as described above. Further, the content of aggregates can be calculated for each peak detected at a wavelength of 215 nm separated by gel filtration chromatography (SEC) using HPLC by calculating the ratio of each peak from its area. Examples of SEC columns include TSKgel G3000SW manufactured by Tosoh Corporation, or ACQUITY UPLC Protein BEH SEC manufactured by Waters Corporation. The preferred content of aggregates when analyzed by the above method is 0 to 60%, more preferably 0 to 40%, still more preferably 0 to 30%, even more preferably 0 to 20%, and most preferably 0 to 10%.

[0216] Further, the DcR3 variant of the present invention is characterized by having lower hydrophobicity than wild-type DcR3. The hydrophobicity of a protein can be measured using a hydrophobic interaction chromatography (HIC) column that interacts with hydrophobic regions present on the surface of the protein. Examples of HIC columns include TSKgel Butyl-NPR manufactured by Tosoh Corporation.

[0217] Further, the DcR3 variant of the present invention is characterized by having improved thermal stability compared to wild-type DcR3. Methods for measuring the thermal stability of a protein include calorimetric methods such as differential scanning calorimetry (DSC), spectroscopic methods for obtaining autofluorescence spectra or circular dichroism (CD) spectra during thermal denaturation or chemical denaturation, differential scanning fluorimetry (DSF) for detecting the exposure of hydrophobic regions present inside the protein with the increase in temperature using a fluorescent dye (such as Sypro Orange) [J Am Chem Soc, 2009, 131: p. 3794-3795].

[0218] DSF for evaluating the thermal stability of proteins can measure the fluorescence intensity at each temperature using, for example, the method described in J.Pharm.Sci.,2013,102:p.2471-2483. Also, software such as CFX manager from BioRad can be used to draw a melting curve and calculate the Tm (thermal unfolding transition midpoints) value. Similarly, DSC for evaluating the thermal stability of proteins can calculate the heat capacity and Tm value at each temperature using, for example, the method described in J.Pharm.Sci.,2012,101:p.955-964.

[0219] 7. DcR3 Variant Composition Examples of the DcR3 variant composition of the present invention include compositions consisting of DcR3 variant molecules. Examples of the DcR3 variant composition of the present invention include compositions containing a plurality of DcR3 variant molecules that have the same primary amino acid sequence constituting the DcR3 variant molecule and can be generated by post-translational modifications such as oxidation / reduction reactions, glycosylation reactions, and sulfation addition reactions in the amino acid sequence. The DcR3 variants of the present invention may include, for example, DcR3 variants having one or more N-glycoside-linked complex sugar chains and DcR3 variants having no N-glycoside-linked complex sugar chains. The proportion of DcR3 variants having one or more N-glycoside-linked complex sugar chains is, for example, preferably 70 to 100%, more preferably 90 to 99%, and particularly preferably 95 to 98% with respect to the total number of DcR3 variants of the present invention.

[0220] 8. Pharmaceutical Composition Containing DcR3 Variant One embodiment of the present invention is a composition comprising an effective amount of the DcR3 variant of the present invention. The composition comprising the DcR3 variant of the present invention can be used as an active ingredient of a prophylactic or therapeutic agent for autoimmune diseases, inflammatory diseases, or allergic diseases, including mucosal diseases. That is, by administering a pharmaceutical composition comprising the DcR3 variant of the present invention to a patient in need of prophylaxis or treatment of an autoimmune disease, inflammatory disease, or allergic disease, the autoimmune disease, inflammatory disease, or allergic disease can be prevented or treated.

[0221] The pathological conditions or diseases for which the composition of the present invention is used include, but are not limited to, for example, inflammatory bowel disease (IBD), systemic lupus erythematosus, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, ulcerative colitis, inflammatory bowel disease, multiple sclerosis, celiac disease, idiopathic thrombocytopenic purpura, myasthenia gravis, Sjogren's syndrome, scleroderma, asthma, uveitis, epidermal hyperplasia, alopecia areata, Behcet's disease, Takayasu arteritis, cartilage inflammation, bone resorption, arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic-onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthritis syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular arthritis, polyarticular arthritis, systemic-onset arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary cirrhosis, sclerosing cholangitis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, chronic obstructive pulmonary disease, Guillain-Barré syndrome, type 1 diabetes, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, or Wiskott-Aldrich syndrome, etc., inflammatory diseases, autoimmune diseases, or allergic diseases.

[0222] The pharmaceutical composition containing the DcR3 variant of the present invention can contain, as an active ingredient, the DcR3 variant or a mixture with an active ingredient for any other treatment. Further, these pharmaceutical preparations are produced by mixing the active ingredient with one or more pharmaceutically acceptable carriers and by any method well known in the technical field of pharmacy.

[0223] The content of the DcR3 variant of the present invention in the pharmaceutical composition varies depending on the dosage form, the pharmaceutically acceptable dosage of the DcR3 variant of the present invention, etc., but is, for example, about 0.01 to 100% by weight. Further, the content of the pharmaceutically acceptable carrier in the pharmaceutical preparation varies depending on the dosage form, the pharmaceutically acceptable dosage of the DcR3 variant of the present invention, etc., but is, for example, 0 to 99.9% by weight.

[0224] As the administration route, it is desirable to use the most effective one for treatment. Examples include oral administration or parenteral administration such as intravenous, subcutaneous, intraoral, intratracheal, rectal, intramuscular, or intraperitoneal administration.

[0225] Examples of the administration form include tablets, powders, granules, syrups, or injections.

[0226] Examples of the preparations suitable for oral administration include, for example, liquid preparations such as syrups can be produced using water, saccharides such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, or soybean oil, preservatives such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor or peppermint. Further, tablets, powders, granules, etc. can be produced using excipients such as lactose, glucose, sucrose, or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, binders such as polyvinyl alcohol, hydroxypropylcellulose, or gelatin, surfactants such as fatty acid esters, plasticizers such as glycerin, etc.

[0227] Formulations suitable for parenteral administration preferably consist of sterile aqueous preparations containing the active compound which is isotonic with the recipient's blood. For example, in the case of an injection, a solution for injection is prepared using a carrier consisting of a salt solution, a glucose solution, or a mixture of a saline solution and a glucose solution, etc.

[0228] Also, in these parenteral preparations, one or more auxiliary components selected from diluents, preservatives, flavorings, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., exemplified for oral preparations, can also be added.

[0229] The medicament containing the DcR3 variant of the present invention can be safely administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, or monkeys, etc.).

[0230] The dosage and frequency of administration of the DcR3 variant of the present invention vary depending on the administration form, the age, weight, disease, nature or severity of the symptoms to be treated of the patient. However, in the case of oral administration, usually 0.01 mg to 1 g per adult per day, preferably 0.05 to 50 mg, is administered once or several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 100 mg per adult per day, preferably 0.01 to 10 mg, is administered once or several times a day. However, these dosages and frequencies of administration vary depending on the various conditions described above.

Example

[0231] Examples of the present invention are shown below. However, the present invention is not limited by these examples.

[0232] [Example 1] Evaluation of the aggregability of wild-type DcR3 in mammalian cells The fusion proteins DcR3 FL-Fc (Figure 3A, SEQ ID NO: 100), which is a fusion of wild-type DcR3 (also referred to as full-length DcR3, SEQ ID NO: 4), the linker sequence IEGRMD (SEQ ID NO: 106), and the human IgG1 Fc region (g1S, SEQ ID NO: 72); S195-Fc (Figure 3B, SEQ ID NO: 102), which is a fusion of human DcR3 lacking the HBD region (SEQ ID NO: 108), the linker sequence IEGRMD (SEQ ID NO: 106), and the human IgG1 Fc region (SEQ ID NO: 72); and DcR3 FL-FLAG (SEQ ID NO: 104), which is a protein with a FLAG tag (SEQ ID NO: 110) added to full-length DcR3 (SEQ ID NO: 4), were prepared as follows.

[0233] For DcR3 FL-Fc, DNA fragments of the signal peptide sequence, the DNA fragment of human DcR3 (SEQ ID NO: 3), the DNA fragment of the linker sequence IEGRMD (SEQ ID NO: 105), and the DNA fragment of Fc (g1S) (SEQ ID NO: 71) were artificially synthesized (by GENEWIZ or Sigma), digested with restriction enzymes NheI and SalI (New England Biolabs), and inserted into the pCIpuro vector (a partial modification of pCI from Promega) using the In-Fusion HD Cloning Kit (Clontech). Escherichia coli DH5α competent cells (TOYOBO) were transformed to obtain transformants into which the DcR3 FL-Fc DNA fragment (SEQ ID NO: 99) was inserted. Similarly, for S195-Fc, DNA fragments of the signal peptide sequence, the DNA fragment of human DcR3 excluding HBD (SEQ ID NO: 107), the DNA fragment of the linker sequence IEGRMD (SEQ ID NO: 105), and the DNA fragment of Fc (g1S) (SEQ ID NO: 71) were artificially synthesized to obtain transformants into which the S195-Fc DNA fragment (SEQ ID NO: 101) was inserted. For DcR3 FL-FLAG, DNA fragments of the signal peptide sequence, the DNA fragment of human DcR3 (SEQ ID NO: 3), and the DNA fragment of the FLAG tag (SEQ ID NO: 109) were artificially synthesized to obtain transformants into which the DcR3 FL-FLAG DNA fragment (SEQ ID NO: 103) was inserted.

[0234] Each plasmid obtained from each transformant was introduced into any one of the host cells of Freestyle CHO-S cells, Freestyle 293F cells, and Expi293 cells (all from Thermo Scientific) to transiently express proteins. For plasmid introduction, any one of Freestyle MAX Reagent, 293Fectin Transfection Reagent, and ExpiFectamine 293 (all from Thermo Scientific) was used.

[0235] After culturing the transfected cells for several days, the culture supernatant was collected. DcR3 FL-Fc and S195-Fc were affinity-purified using MabSelect SuRe (GE Healthcare), and DcR3 FL-FLAG was affinity-purified using Anti-FLAG M2 affinity gel (Sigma). The culture supernatant of DcR3 FL-Fc and S195-Fc was passed through a column filled with resin, washed with PBS (Nacalai Tesque), eluted with elution buffer (20 mM citric acid, 50 mM NaCl, pH 3.4), and immediately neutralized with neutralization buffer (1 M sodium phosphate, pH 7.0). The culture supernatant of DcR3 FL-FLAG was similarly charged onto the resin, washed with PBS, eluted with elution buffer (100 mM glycine-HCl, pH 3.5), and immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 8.0). The absorbance (A) at 280 nm of each elution fraction 280It was measured, and the continuous fractions with high measured values were collected. The buffer of the collected fractions was replaced with PBS using a NAP column (GE Healthcare), and the filtrate passed through a 0.22-μm filter was used as the purified protein. The absorbance coefficients of DcR3 FL-Fc, S195-Fc, and DcR3 FL-FLAG at 280 nm were 1.03, 1.17, and 0.77, respectively, and the concentrations were calculated. After SDS-PAGE under non-reducing and reducing conditions with 100 mM DTT, the gel was stained with Coomassie (Nacalai Tesque), and the molecular weights were confirmed. The estimated molecular weights predicted from the amino acid sequences of DcR3 FL-Fc, S195-Fc, and DcR3 FL-FLAG are approximately 56.4 kDa, 44.7 kDa, and 31.0 kDa, respectively, for the monomer. Under non-reducing conditions, since DcR3 FL-Fc and S195-Fc, which are Fc fusions, exist as dimers, the estimated molecular weights are approximately 112.8 kDa and 89.4 kDa, respectively.

[0236] As a result of SDS-PAGE under non-reducing conditions, most of DcR3 FL-Fc, S195-Fc, and DcR3 FL-FLAG transiently expressed in mammalian cells remained in the sample wells, and those that migrated also showed smears or ladders, indicating that all the recombinants were highly aggregated (Figure 1A).

[0237] Furthermore, for a commercially available full-length DcR3-Fc (Abcam) prepared using HEK293 cells as host cells, immunoblotting was performed under non-reducing conditions using a rabbit anti-human IgG Fc polyclonal antibody as the primary antibody and a goat anti-rabbit IgG antibody (Dako) as the secondary antibody. Similar to the above-described purified product, most of it existed as aggregates (Figure 1B).

[0238] On the other hand, commercially available DcR3 FL-Fc (R&D) expressed in insect cells Sf21 showed almost no aggregation, and differences in aggregation were observed depending on the type of host cell producing wild-type DcR3 (Figure 1C). Also, using the following method with insect cells S2 as the host cell, DcR3 FL-Fc (SEQ ID NO: 100), R218Q-Fc having an R218Q mutation in which the 218th Arg of human DcR3 (SEQ ID NO: 2) was replaced with Gln (US Patents US6835814B1, US6965012B1, SEQ ID NO: 340), and S195-Fc lacking HBD (SEQ ID NO: 102) were prepared and their aggregability was evaluated. Either a DNA fragment of human DcR3 (SEQ ID NO: 3), a DNA fragment in which the R218Q mutation was introduced into human DcR3 (SEQ ID NO: 111), or a DNA fragment of human DcR3 excluding HBD (SEQ ID NO: 107), a DNA fragment of the linker sequence IEGRMD (SEQ ID NO: 105), and a DNA fragment of Fc (g1S) (SEQ ID NO: 71) were artificially synthesized, and S2 cell lines stably expressing DcR3 FL-Fc, R218Q-Fc, and S195-Fc were obtained using pMTBiPV5-HisA (Thermo Scientific) and Drosophila Expression System (Thermo Scientific). Affinity purification with MabSelect SuRe was performed from the culture supernatant of the stable expression strain by the same method as described above.

[0239] As a result, DcR3 FL-Fc, R218Q-Fc, and S195-Fc produced in S2 cells showed almost no aggregation (Figure 1C). Quantitative evaluation of the aggregate content was performed by gel filtration chromatography (SEC) using HPLC (Shimadzu) (TSKgel G3000 SWXL 7.8 mm × 300 mm) (Tosoh). Expi293-derived and S2-derived S195-Fc were analyzed, and the ratios (%) of monomer, aggregate, and degradation product calculated from the peak areas are shown in Table 1.

[0240]

Table 1

[0241] From the above results, it was revealed that when wild-type DcR3 was expressed in mammalian cells, the amount of aggregate formation increased.

[0242] [Example 2] Preparation of a DcR3 variant that does not aggregate in a mammalian cell expression system A human DcR3 variant that does not aggregate in a mammalian cell expression system has not been known so far. Therefore, an attempt was made to prepare a DcR3 variant in which the amount of aggregate formation is reduced while maintaining the activity of DcR3. DcR3 is a soluble molecule consisting of 300 residues, has a signal peptide on the N-terminal side, and then four Cysteine rich domains (CRD1, CRD2, CRD3, CRD4) characteristic of the TNF receptor superfamily (TNFRSF), and the C-terminal side consists of a Heparan Sulfate Binding Domain (HBD) rich in basic amino acids containing a heparan sulfate binding motif (Figs. 2, 3A). LIGHT, TL1A, and FasL, which are DcR3 ligands, all bind via CRD2 and CRD3 of DcR3. Therefore, while retaining the region containing CRD2 and CRD3 of DcR3, CRD1 and / or CRD4 of DcR3 were replaced with the soluble decoy receptor Osteoprotegerin (OPG), which is a related TNFRSF molecule, to prepare a DcR3 variant.

[0243] In human DcR3, chimeric A (SEQ ID NO: 54) in which CRD1 and CRD4 are the amino acid sequences of human OPG, CRD2 and CRD3 are the amino acid sequences of human DcR3, and the amino acid sequence of HBD is removed, and an Fc sequence (IEGRMD g1S (SEQ ID NO: 339), or g4PEK (SEQ ID NO: 74) in which Ser at position 228, Leu at position 235, and Arg at position 409 of the heavy chain of human IgG4 indicated by the EU index are substituted with Pro, Glu, and Lys, respectively) are fused to form chimeric A-Fc (FIG. 3C, SEQ ID NO: 80 or 82); in human DcR3, chimeric B (SEQ ID NO: 50) in which CRD1 is the amino acid sequence of human OPG, CRD2, CRD3, and CRD4 are the amino acid sequences of human DcR3, and the amino acid sequence of HBD is removed, and an Fc sequence (IEGRMD g1S) are fused to form chimeric B-Fc (FIG. 3G, SEQ ID NO: 76); in human DcR3, chimeric C (SEQ ID NO: 52) in which CRD1, CRD2, and CRD3 are the amino acid sequences of human DcR3, CRD4 is the amino acid sequence of human OPG, and the amino acid sequence of HBD is removed, and an Fc sequence (IEGRMD g1S) are fused to form chimeric C-Fc (FIG. 3H, SEQ ID NO: 78); various DcR3 variants having various structures of 103-123OPG-Fc (g4PEK) (FIG. 3D, SEQ ID NO: 84) in which the amino acid sequence containing the 18th to 36th positions and the 2 amino acids on the C-terminal side of CRD3 of chimeric A are substituted with human OPG and an Fc sequence (g4PEK) are prepared by the following method. For DcR3 variants in which CRD4 is derived from DcR3, the TS sequence, which is the amino acid sequence at positions 194 to 195 of DcR3 (SEQ ID NO: 2), is added to the C-terminus of CRD4, and for DcR3 variants in which CRD4 is derived from OPG, the SGNSESTQK sequence, which is the amino acid sequence at positions 186 to 194 of OPG (SEQ ID NO: 14), is added to the C-terminus of CRD4.

[0244] A DNA fragment encoding a signal peptide sequence, chimeric A, chimeric B, chimeric C, or 103-123OPG, and a DNA fragment (chimeric A: SEQ ID NO: 53, chimeric B: SEQ ID NO: 49, chimeric C: SEQ ID NO: 51, 103-123OPG: SEQ ID NO: 55) with the HBD sequence removed was artificially synthesized and ligated with a DNA fragment (SEQ ID NO: 338, 73) encoding an Fc sequence (IEGRMD g1S or g4PEK). It was inserted into a pCIpuro vector in the same manner as in Example 1 to obtain a plasmid into which DNA fragments (chimeric A-Fc: SEQ ID NO: 79 or 81, chimeric B-Fc: SEQ ID NO: 75, chimeric C-Fc: SEQ ID NO: 77, 103-123OPG-Fc: SEQ ID NO: 83) encoding various DcR3 variants were inserted. The obtained plasmid was introduced into any of the host cells of Freestyle CHO-S cells, Freestyle 293F cells, and Expi293 cells in the same manner as in Example 1 to transiently express the protein, and affinity purification using MabSelectSuRe was performed from the culture supernatant.

[0245] The ratios (%) of monomers, aggregates, and degradation products of the various DcR3 variants prepared were calculated by SEC-HPLC in the same manner as in Example 1 or by SEC (Waters) using ACQUITY UPLC Protein BEH SEC 4.6 mm × 150 mm (Table 2).

[0246]

Table 2

[0247] As a result, in all DcR3 variants of chimeric A-Fc, chimeric B-Fc, chimeric C-Fc, and 103-123OPG-Fc, the aggregates were reduced compared to wild-type DcR3. The aggregate reduction effect was highest in chimeric A-Fc, followed by chimeric B-Fc and chimeric C-Fc in that order. Therefore, it was shown that substitution with OPG-derived CRD1 and CRD4 each had an effect of reducing aggregation, and substituting both CRD1 and CRD4 in combination had an additional effect of reducing aggregation.

[0248] When chimeric A-Fc, chimeric B-Fc, and chimeric C-Fc (all IEGRMD g1S) were subjected to SDS-PAGE under non-reducing conditions and reducing conditions with 100 mM DTT, even when produced using mammalian cells as the host, almost no smears or ladders were observed for chimeric A-Fc and chimeric B-Fc (Figure 4).

[0249] To calculate the absolute molecular weight, chimeric A-Fc (IEGRMD g1S or g4PEK) and 103-123OPG-Fc (g4PEK) were analyzed by SEC (TSKgel G3000 SWXL 7.8 mm × 300 mm) (Tosoh Corporation) using HPLC (Prominence, Shimadzu Corporation). As the mobile phase, 50 mmol / L phosphate buffer (pH 7.0, 500 mmol / L NaCl) was used, and after separation at a flow rate of 0.75 mL / min, for each peak detected at a wavelength of 215 nm, the absolute molecular weight (% is uncertainty) was calculated using the scattered light intensity detected by a multi-angle light scattering detector (Multi Angle Light Scattering; MALS) (miniDAWN TREOS, Wyatt Technology Corporation). The results are shown in Table 3.

[0250]

Table 3

[0251] Since the predicted molecular weights of the dimers from the amino acid sequences of chimeric A-Fc (IEGRMD g1S or g4PEK) and 103-123OPG-Fc are 92.0 kDa, 90.1 kDa, and 90.5 kDa, respectively, it was confirmed that all DcR3 variants exist as dimers.

[0252] [Example 3] Analysis of the N-glycosylation rate of chimeric A-Fc (g4PEK) and evaluation of its effect on aggregation In chimeric A-Fc(g4PEK) (SEQ ID NO: 82), the Asn residues predicted to have N-linked glycosylation based on the amino acid sequence are present at three positions (N131, N144, N157) in the CRD4 derived from OPG and at one position (N260) in the Fc region. Therefore, the presence or absence of N-linked glycosylation in chimeric A-Fc(g4PEK) was evaluated by the following method. For sample preparation, the N-linked glycans of reduced and alkylated chimeric A-Fc(g4PEK) were cleaved by PNGaseF treatment, and then the protein was digested with each protease of trypsin, endoprotease Asp-N, and chymotrypsin. The resulting peptide mixture was dissolved in 5% (v / v) acetonitrile / 0.1% formic acid and analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Analysis was performed by gradient elution with 5 - 65% (v / v) acetonitrile / 0.1% formic acid using MAGIC2000 HPLC (Michrom Bioresources) equipped with a C18 reverse-phase column (0.2 mm × 150 mm) (GL Science) and LTQ Orbitrap XL ion trap-Orbitrap hybrid mass spectrometer (Thermo Scientific). The resulting peptide fragments were identified by performing MASCOT analysis (Matrix Science) against the amino acid sequence of chimeric A-Fc(g4PEK). Using the fact that N-linked glycosylation Asn is converted to Asp by PNGaseF treatment and the mass increases by 0.984 Da as an indicator, the peptides with shifted peaks in the MS spectrum and the glycosylation sites contained in the peptides were identified. As a result, it was shown that N-linked glycans were added to all of the Asn residues of N131, N144, N157, and N260. Among these, N157 and N260 had glycans added in almost all peptide fragments, but for N131 and N144, both glycan-added fragments and non-added fragments were detected.

[0253] Next, for the purpose of evaluating the effect on the aggregability of the N - type sugar chains added to three Asn (N131, N144, N157) of the CRD4 derived from OPG, amino acid substitution mutants were prepared to remove the sugar chains at two positions of N131 and N144 or at three positions of N131, N144, and N157. As the two - site sugar chain - removed mutants, N131S / N144S - Fc(g4PEK) (SEQ ID NO: 86) in which N131 and N144 were each substituted with Ser or T133A / S146A - Fc(g4PEK) (SEQ ID NO: 88) in which T133 and S146 were each substituted with Ala were prepared. As the three - site sugar chain - removed mutants, N131S / N144S / N157S - Fc(g4PEK) (SEQ ID NO: 90) in which N131, N144, and N157 were each substituted with Ser or T133A / S146A / T159A - Fc(g4PEK) (SEQ ID NO: 92) in which T133, S146, and T159 were each substituted with Ala were prepared. DNA fragments of the signal peptide sequence and DNA fragments encoding each sugar chain - removed mutant (SEQ ID NOs: 85, 87, 89, 91) were artificially synthesized, inserted into the pCIpuro vector in the same manner as in Example 1, and introduced into Expi293 cells. After transiently expressing each sugar chain - removed mutant, affinity purification using MabSelectSuRe was performed from the culture supernatant. Each prepared sugar chain - removed mutant was analyzed by SEC - UPLC (apparatus: ACQUITY UPLC, column; ACQUITY UPLC Protein BEH SEC 200Å, 1.7μm, 4.6×150mm) (Waters). Table 4 shows the ratios (%) of the monomer, aggregate, and degradation product calculated from the peak areas.

[0254]

Table 4

[0255] The aggregate content of the two-site deglycosylated form was slightly increased in all amino acid substitution variants compared to chimeric A-Fc (Table 2), and further increases in aggregates were observed in all amino acid substitution variants of the three-site deglycosylated form. From this, it was shown that among the three N-linked glycans (N131, N144, N157) added to CRD4 derived from OPG, the glycan added to N157 in particular contributed to reducing the aggregates of chimeric A. On the other hand, even when all three N-linked glycans were removed, the proportion of aggregates was lower compared to DcR3 FL-Fc and S195-Fc (Table 1). Therefore, it was shown that the N-linked glycan and the sequence of OPG each had an effect of reducing the aggregates of wild-type DcR3, and combining the two had an additional effect of reducing the aggregates.

[0256] [Example 4] Physical property evaluation of various DcR3 variants In order to analyze the cause of the aggregation reduction effect of various DcR3 variants prepared in Examples 2 and 3, the following physicochemical analyses were performed. Hydrophobic interaction chromatography (HIC) is an analytical method in which the higher the hydrophobicity of the protein surface, the longer the elution time from the column. Using a hydrophobic chromatography column (TSKgel Butyl-NPR 4.6 mm × 35 mm) (Tosoh Corporation), with a gradient of buffer A (2 mmol / L ammonium sulfate, 20 mmol / L Tris buffer, pH 7.0) and buffer B (20 mmol / L Tris buffer, pH 7.0) as the mobile phase, 8 μg of the sample was separated at a flow rate of 0.5 mL / min, and the elution time (minutes) detected at a wavelength of 215 nm is shown in Table 5.

[0257]

Table 5

[0258] Compared with the single amino acid mutant R218Q-Fc of full-length DcR3 prepared in insect cells S2, S2-derived S195-Fc, mammalian cell-derived chimeric A-Fc (IEGRMD g1S, g4PEK), 103-123OPG-Fc (g4PEK), and the two-site sugar chain-removed form all had shorter elution times, suggesting that their physical properties were improved and the aggregates were reduced due to the decreased hydrophobicity. In addition, for chimeric C and the three-site sugar chain-removed form, in which the aggregate reduction effect was slightly attenuated, their hydrophobicity was equal to or higher than that of R218Q-Fc, indicating a correlation between the proportion of aggregates and the hydrophobicity of the protein.

[0259] Subsequently, the thermal stability of the proteins was evaluated by the Differential Scanning Fluorimetry (DSF) method. In a 96-well white microplate (BioRad), 9.5 μg of various DcR3 variants and 1 μL of SYPRO Orange Protein Gel Stain (invitrogen) diluted 50-fold with water were mixed in a 20 μL system, and the temperature was increased from 20 °C to 95 °C at 0.5 °C intervals for 10 seconds each using a C1000 Thermal Cycler (BioRad). The fluorescence at each temperature was detected in the FRET channel, and the melting curve (Figure 5) and melting temperature (Tm value) (°C) (Table 6) were calculated using CFX Manager software (BioRad).

[0260]

Table 6

[0261] Compared with the single amino acid mutant R218Q-Fc of full-length DcR3 prepared in insect cells S2, the Tm value of S2-derived S195-Fc increased significantly and its thermal stability was improved, suggesting that the HBD region contributed to the thermal instability. In addition, for mammalian cell-derived chimeric A-Fc (IEGRMD g1S), the Tm value was even higher than that of S195-Fc, indicating that the DcR3 variant with a part of the CRD of DcR3 replaced by a part of the CRD of OPG not only had reduced aggregation but also improved thermal stability of the protein (Table 6, Figure 5).

[0262] [Example 5] Evaluation of Reactivity of DcR3 Variant with Heparan Sulfate Binding Domain (HBD) Removed to Human Normal Cells and CHO Cells The pharmacokinetics of wild-type DcR3 and its mutant FLINT (R218Q mutation) in mice and cynomolgus monkeys have been reported to be extremely poor (Drug Metabolism and Disposition, 2003, 31: p. 502-507). One of the reasons for this is thought to be that the heparan sulfate binding domain (HBD) present in wild-type DcR3 directly binds DcR3 to heparan sulfate proteoglycan on the cell membrane (J. Immunol., 2006, 176: 173-180).

[0263] Therefore, the reactivity of the DcR3 variant S195-Fc with the HBD removed, chimeric A-Fc (IEGRMD g1S, g4PEK), and 103-123OPG-Fc (g4PEK) to human primary cells and CHO cells, which are production cells, was analyzed by flow cytometry (FCM). As a positive control, DcR3 FL-Fc containing the HBD region was used, and as a negative control, K194-Fc and an anti-DNP antibody (g4PEK) were used. K194-Fc (amino acid sequence: SEQ ID NO: 152, DNA base sequence: SEQ ID NO: 151) is a protein in which the amino acid sequence of IEGRMD linker (SEQ ID NO: 106) and the amino acid sequence of Fc (g1S) (SEQ ID NO: 72) were fused to the amino acid sequence from the first Met to the 194th Lys of OPG (SEQ ID NO: 14). It was transiently expressed using Expi293 cells by the method described in Example 1 and purified from the culture supernatant using Mabselect SuRe (GE Healthcare). The anti-DNP antibody (g4PEK) was prepared by inserting the variable region of the anti-2,4-dinitrophenol (DNP) antibody described in Clin. Cancer Res., 2005, 11(8), p.3126-3135 into a vector encoding the Fc sequence (g4PEK), introducing it into CHO cells for expression, and using the antibody purified by Protein A from the culture supernatant. As human primary cells, HUVEC (Lonza) and Male Human Hepatocytes (BioreclamationIVT) were used. The human primary cells were cultured using the specified medium for each cell according to the attached document and cultured using a collagen-coated adherent plate (IWAKI). CHO cells were cultured in suspension using EX-CELL 325 PF CHO Serum-Free Medium (Sigma-Aldrich).

[0264] HUVEC and Hepatocyte were detached using a 0.02% EDTA solution and a cell scraper and passed through a cell strainer (40 μm). The detached HUVEC, Hepatocyte, and CHO cells collected from the suspension culture were washed with PBS containing FCM buffer (1% BSA, 1 mmol / L EDTA, 0.05% NaN 3 and then suspended in the FCM buffer.

[0265] Next, seed into a 96-well U-bottom plate (Falcon) at 1×10 5 cells / well, add each prepared Fc fusion protein to a concentration of 10 μg / mL, and react on ice for 1 hour. After washing the cells with FCM buffer, suspend them with the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Molecular Probes) and 0.1 μg / mL of Goat F(ab’) 2 Anti-Human IgG R-phycoerythrin Conjugate (Southern Biotech) and stain on ice for 1 hour. When staining HUVEC and Hepatocytes, add the Human FcR Blocking Reagent (Miltenyi Biotec). After washing the cells with FCM buffer, analyze the fluorescence intensity with a flow cytometer, FACS Fortessa (BD Biosciences).

[0266] As a result of analyzing the PE staining intensity for the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit-negative viable cell fraction, DcR3 FL-Fc (IgG1) (R&D) showed significant binding to all of HUVEC, Hepatocytes, and CHO cells. On the other hand, none of the DcR3 variants with HBD removed showed binding (Figure 6).

[0267] [Example 6] Evaluation of the in vivo kinetics of DcR3 variants

[0268]

Table 7

[0269] The mouse pharmacokinetic tests of S195-Fc and each DcR3 variant shown in Table 7 were carried out. For chimeric A-Fc (g4PEK) and 103-123OPG-Fc (g4PEK), each DcR3 variant was prepared by stable expression in CHO cells by the method shown below. A DNA fragment of the signal peptide sequence and a DNA fragment encoding each DcR3 variant (SEQ ID NOs: 81, 83) were artificially synthesized and inserted into a recombinant vector prepared by the method described in WO 2012 / 081628 in the same manner as in Example 1, and the vector was introduced into CHO cells by electroporation. Culturing and drug selection were performed by general methods, and those in which the proportion of living cells recovered to about 98% were used as stable expression strains. After culturing this stable expression strain in a medium such as EX-CELL 325 PF CHO Serum-Free Medium (Sigma-Aldrich) for a certain period, the culture supernatant was collected, and each DcR3 variant was purified by the method described in Example 1.

[0270] S195-Fc and each DcR3 variant (10 mg / kg) were administered once i.v. to 5- to 6-week-old BALB / c mice (♀) (n = 2 or 3). Blood was collected from the tail vein at any time after administration, and the concentrations of S195-Fc and each DcR3 variant in the blood were measured by the following method. A biotinylated anti-human IgG polyclonal antibody was reacted with streptavidin-conjugated beads, and S195-Fc and each DcR3 variant in the bound serum were detected with an Alexa Fluor 647-labeled anti-human IgG polyclonal antibody. The measurement was carried out using a Gyrolab xP workstation (Gyros AB), and each pharmacokinetic parameter was calculated by the moment analysis method. The standard substance for preparing the calibration curve was the same as the test substance administered to the animals. The changes in blood concentrations of S195-Fc produced in S2 cells and chimeric A-Fc (IEGRMD g1S) produced in CHO-S cells are shown in Fig. 7, and for S195-Fc and each DcR3 variant shown in Table 7, the blood half-life (h) in the elimination phase after single administration and the area under the concentration-time curve up to infinite time, AUC0-∞ (μg*h / mL), are shown in Table 8, respectively.

[0271]

Table 8

[0272] When compared with S195-Fc, which is a wild-type DcR3 control, the blood concentration profile of the DcR3 variant was significantly improved (Figure 7). The half-life was almost the same, but the AUC0-∞ was improved by more than 10-fold (Table 8).

[0273] When wild-type DcR3 and FLINT (R218Q mutation) were intravenously administered to CD-1 mice at a single dose of 0.5 mg / kg, the blood half-lives were 1.2 hours and 3.1 hours, respectively, and the AUC0-∞ (μg*h / mL) was reported to be 0.48 and 0.36, respectively (Drug Metabolism and Disposition, 2003.31: p.502-507.). When comparing the concentration profiles multiplied by the dose ratio assuming linearity, the DcR3 variant showed higher exposure to wild-type DcR3 and FLINT.

[0274] [Example 7] Evaluation of Binding Activity to DcR3 Ligand (1) Preparation of DcR3 Ligand Soluble recombinant forms of DcR3 ligands (LIGHT, TL1A, FasL) of human, cynomolgus monkey, or mouse were prepared (amino acid sequences: SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, 128, 130; DNA base sequences: SEQ ID NOs: 113, 115, 117, 119, 121, 123, 125, 127, 129).

[0275] Soluble recombinant LIGHT of human, cynomolgus monkey, or mouse was added with a His tag (His10) and a GS linker (GGGSGGGSGGGSIEGR) at the N-terminus, and the extracellular region of LIGHT (human LIGHT: Asp74-Val240 (SEQ ID NO: 132), cynomolgus monkey LIGHT: Asp74-Val240 (SEQ ID NO: 134), mouse LIGHT: Asp72-Val239 (SEQ ID NO: 136)) was ligated downstream using the resulting sequence.

[0276] Soluble recombinant TL1A of human, cynomolgus monkey, or mouse was prepared by adding a His tag (His6) and a GS linker (GGGSGGGSGGGS) to the N-terminus, and linking the extracellular region of TL1A (human TL1A: Leu72-Leu251 (SEQ ID NO: 138), cynomolgus monkey TL1A: Leu72-Leu251 (SEQ ID NO: 140), mouse TL1A: Ile94-Leu270 (SEQ ID NO: 142)) downstream thereof.

[0277] Soluble recombinant FasL of human, cynomolgus monkey, or mouse was prepared by adding a His tag (His6) to the N-terminus, and linking the extracellular region of FasL (human FasL: Pro134-Leu281 (SEQ ID NO: 144), cynomolgus monkey FasL: Pro133-Leu280 (SEQ ID NO: 146), mouse FasL: Pro132-Leu279 (SEQ ID NO: 148)) downstream thereof.

[0278] A DNA fragment of the signal peptide sequence and a DNA sequence of the soluble DcR3 ligand with tags were chemically synthesized (GENEWIZ), and inserted downstream of the CMV promoter of the pCI-Hygro2.01 vector (a partial modification of pCI from Promega) using the In-Fusion HD Cloning Kit (Clontech), and Escherichia coli DH5α competent cells (TOYOBO) were transformed.

[0279] The obtained plasmid was introduced into Expi293 cells (Thermo Scientific) to transiently express the protein. The plasmid was introduced using ExpiFectamine 293 (Thermo Scientific), and after culturing for 3 days, the culture supernatant was collected.

[0280] Human FasL and cynomolgus monkey FasL were introduced into host cells of Freestyle CHO-S cells (Thermo Scientific) to transiently express the proteins. The plasmid was introduced using Freestyle MAX Reagent (Thermo Scientific), and after culturing for 3 days, the culture supernatant was collected.

[0281] Protein purification from the culture supernatant of Expi293 cells was performed using Ni Sepharose Fast Flow resin and His Buffer Kit (both from GE Healthcare). The culture supernatant was passed through a column filled with the resin, washed with a washing buffer (60 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4), and then eluted with an elution buffer (250 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4).

[0282] Protein purification from the culture supernatant of Freestyle CHO-S cells was performed using Complete His-Tag Purification Resin (Roche) and His Buffer Kit (GE Healthcare). The culture supernatant was passed through a column filled with the resin, washed with a washing buffer (2 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4), and then eluted with an elution buffer (250 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4).

[0283] Each elution fraction was replaced with PBS using a NAP column (GE Healthcare) and sterilized by passing through a 0.22-μm filter. The purity of the obtained purified protein was confirmed by SDS-PAGE. Analysis of multimer formation by SEC-UPLC (equipment: ACQUITY UPLC, column; ACQUITY UPLC Protein BEH SEC 200 Å, 1.7 μm, 4.6 × 150 mm) (Waters) revealed that almost all recombinant soluble DcR3 ligands had a peak corresponding to the molecular weight of a trimer, but only mouse LIGHT did not show a trimer peak and was monomeric.

[0284] (2) Measurement of binding activity using BIAcore For each of the various wild-type DcR3 controls and DcR3 mutants shown in Table 9, the binding activity to DcR3 ligands (LIGHT, TL1A, FasL) was analyzed by SPR using a BIAcore T-100 (GE Healthcare). HBS-EP+ Buffer was used as the buffer.

[0285] After immobilizing 10,000 RU of anti-human antibody on the Series S Sensor Chip CM5 using the Human Antibody Capture Kit (GE Healthcare), various wild-type DcR3 and DcR3 mutants were flowed at 10 μL / min for 30 seconds for capture. On the other hand, a buffer without protein was flowed through the reference flow cell. Then, as an analyte, human, cynomolgus monkey, or mouse DcR3 ligand diluted to 0.08 - 80 nmol / L was flowed at 10 μL / min for 2 minutes to monitor binding, and then the buffer was flowed for 3 minutes to monitor dissociation. Next, 3 mol / L magnesium chloride was flowed at 20 μL / min for 1 minute to perform a regeneration reaction. Using the BIAcore T-100 evaluation software and the 1:1 Binding model, each DcR3 ligand was considered as a monomer (human LIGHT monomer: 20.8 kDa, human TL1A monomer: 22.1 kDa, human FasL monomer: 17.7 kDa, cynomolgus monkey LIGHT monomer: 20.8 kDa, cynomolgus monkey TL1A monomer: 22.0 kDa, cynomolgus monkey FasL monomer: 17.7 kDa, mouse LIGHT monomer: 20.9 kDa, mouse TL1A monomer: 21.5 kDa, mouse FasL monomer: 17.7 kDa), and each kinetic constant (k a 、k d 、K D ) was calculated (Tables 10 - 12). As a result of the measurement, it was confirmed that various wild-type DcR3 and DcR3 mutants bind to each DcR3 ligand of human, cynomolgus monkey, and mouse, except for mouse LIGHT for which trimers could not be produced.

[0286]

Table 9

[0287]

Table 10

[0288]

Table 11

[0289] [Table 12]

[0290] [Example 8] Evaluation of Binding Activity to OPG Ligands (RANKL, TRAIL) RANKL is known to bind to RANK and be involved in bone resorption, and TRAIL is known to bind to the TRAIL receptor and be involved in cell death. Using the various human DcR3 recombinants prepared, the binding activities to RANKL and TRAIL were analyzed by ELISA. As positive controls for binding to OPG ligands, the C-terminal region deletion mutant K194-Fc of OPG prepared in Example 5, RANK-Fc (Enzo Life Science), and TRAIL R1-Fc (R&D systems) were used. As a negative control, an anti-DNP antibody (IgG1) [the variable region of the anti-2,4-dinitrophenol (DNP) antibody described in Clin. Cancer Res., 2005, 11(8), p. 3126-3135 was inserted into a vector encoding the Fc of IgG1, introduced into CHO cells for expression, and the antibody purified by Protein A was used.

[0291] Anti-human IgG1 (American Qualex) prepared at 10 μg / mL in PBS was dispensed at 50 μL / well into a 96-well plate (MAXISORP NUNC-IMMUNO PLATE, Thermo Scientific) and allowed to adsorb by standing overnight at 4°C. After removing the immobilization solution, 1% Block Ace prepared by dissolving 1 g of Block Ace powder (DS Pharma Biomedical) in 100 mL of water was dispensed at 100 μL / well, allowed to block by standing at room temperature for 1 hour, and washed three times with PBS containing 0.1% Tween (hereinafter referred to as PBST). Next, various human DcR3 recombinants or various DcR3 mutants diluted to 1 μg / mL with 1% BSA-PBS and RANK-Fc were dispensed at 50 μL / well into Plate No. 1, and various wild-type DcR3s or various DcR3 mutants diluted to 1 μg / mL with 1% BSA-PBS and TRAIL R1-Fc were dispensed at 50 μL / well into Plate No. 2, and allowed to stand at room temperature for 1 hour.

[0292] After washing each plate three times with PBST, RANKL (Peprotech) diluted to 0.64 - 50000 pg / mL with 1% BSA-PBS was dispensed at 50 μL / well into Plate No. 1, and TRAIL (Peprotech) diluted to 0.64 - 50000 pg / mL with 1% BSA-PBS was dispensed at 50 μL / well into Plate No. 2, and allowed to stand at room temperature for 1 hour.

[0293] After washing the plate three times with PBST, biotinylated anti-RANKL antibody (Peprotech) diluted to 0.4 μg / mL with 1% BSA-PBS was dispensed at 50 μL / well into Plate No. 1, and biotinylated anti-TRAIL antibody (R&D) diluted to 0.4 μg / mL with 1% BSA-PBS was dispensed at 50 μL / well into Plate No. 2, and allowed to stand at room temperature for 1 hour.

[0294] After washing each plate three times with PBST, streptavidin-HRP (PIERCE) diluted 10,000-fold with 0.1% Block Ace was dispensed at 50 μL / well, and allowed to stand at room temperature for 1 hour.

[0295] After washing each plate three times with PBST, 50 μL / well of TMB + Substrate Chromogen (Dako) was dispensed and left standing at room temperature for 1 minute. 50 μL / well of 0.5 mol / L sulfuric acid solution was dispensed to stop the color reaction, and the absorbance at a sample wavelength of 450 nm and a reference wavelength of 570 nm was measured using a plate reader.

[0296] As a result, K194-Fc bound to RANKL and TRAIL, which are OPG ligands, and RANK-Fc and TRAIL R1-Fc showed binding to RANKL and TRAIL, respectively, but DcR3 FL-Fc (R&D), S195-Fc, chimeric A-Fc (IEGRMD g1S), and chimeric B-Fc (IEGRMD g1S) did not bind to any of the ligands (Figure 8). In combination with the results of Example 7, it was confirmed that the DcR3 variant prepared by replacing a part of the CRD of DcR3 with a part of the CRD of OPG showed binding activity equivalent to that of wild-type DcR3 to the ligands of DcR3 but did not bind to the ligands of OPG.

[0297] [Example 9] Measurement of DcR3 Ligand Neutralizing Activity The neutralizing activities of various wild-type DcR3 controls and DcR3 variants against LIGHT, TL1A, and FasL were measured by the method shown below. The DNP antibody was prepared by the method described in Examples 5 and 8.

[0298] (1) Measurement of LIGHT Neutralizing Activity Using the human colon cancer cell line HT-29 (ATCC number: HTB-38), the neutralizing activities of various wild-type DcR3 and various DcR3 variants were measured using the production of IL-8 by the addition of LIGHT as an index. For cell culture and neutralizing activity evaluation, McCoy's 5A medium (Gibco) supplemented with 10% FBS (Gibco) and penicillin / streptomycin (Nacalai Tesque) was used.

[0299] The HT-29 cell line was seeded at 2x10 in a 96-well adherent culture plate (Sumitomo Bakelite). 4After seeding at cells / well, various wild-type DcR3s or various DcR3 variants were added to a final concentration of 0.1, 1, or 10 μg / mL. Then, human LIGHT (Gly66-Val240) with an N-terminal FLAG tag (DYKDDDDK) (described in JP2013153749) was added to a final concentration of 0.1 μg / mL, and the total volume of the culture medium was adjusted to 200 μL / well. 5% CO 2 After culturing at 37°C for 3 days in an incubator, the culture supernatant was collected, and the IL-8 concentration in the culture supernatant was measured using the AlphaLISA IL-8 Immunoassay Research Kit (Perkin Elmer).

[0300] As a result of the measurement, it was confirmed that in various human DcR3 variants, the amount of IL-8 production decreased in a concentration-dependent manner, indicating LIGHT neutralizing activity (Figure 9).

[0301] (2) Measurement of TL1A neutralizing activity Using human T cells, the neutralizing activities of various wild-type DcR3s and various DcR3 variants were measured with the production of IFN-γ induced by the addition of TL1A as an index. X-VIVO15 medium (Lonza) was used for cell culture and neutralizing activity evaluation.

[0302] Frozen healthy human PBMCs (AllCells) were thawed in a 37°C water bath and suspended in a medium containing DNaseI (STEMCELL) warmed to 37°C. After shaking at low speed for 2 hours at 37°C, T cells were isolated using the EasySep Human T cell Enrichment Kit (STEMCELL). The isolated T cells were seeded at 1x10 in a 96-well suspension culture plate (Sumitomo Bakelite). 5After seeding at cells / well, various wild-type DcR3 controls or various DcR3 mutants were added to achieve final concentrations of 0.1, 1, and 10 μg / mL. Then, recombinant His10 human TL1A prepared in Example 6 was added to achieve a final concentration of 0.1 μg / mL. Human IL-12 (Miltenyi Biotec) at a final concentration of 2 ng / mL and Recombinant Human IL-18 (MBL) at a final concentration of 50 ng / mL were added, and the total volume of the culture medium was adjusted to 200 μL / well. 5% CO 2 After culturing at 37°C for 3 days in an incubator, the culture supernatant was collected, and the IFN-γ concentration in the culture supernatant was measured using the AlphaLISA IFN-γ Immunoassay Research Kit (Perkin Elmer).

[0303] As a result of the measurement, it was confirmed that the production amount of IFN-γ decreased in a concentration-dependent manner with various DcR3 mutants, indicating TL1A neutralizing activity (Figure 10).

[0304] (3) Measurement of FasL neutralizing activity Using the T cell leukemia Jurkat cell line (DSMZ number: ACC 282), the neutralizing activities of various wild-type DcR3 controls and various DcR3 mutants were measured using apoptosis induced by FasL addition as an index. For cell culture and neutralizing activity evaluation, RPMI1640 medium (Nacalai Tesque) supplemented with 10% FBS and penicillin / streptomycin was used.

[0305] The Jurkat cell line was seeded at 5x10 4 cells / well in a 96-well suspension culture plate, and then various wild-type DcR3 or DcR3 mutants were added to achieve final concentrations of 0.01, 0.1, and 1 μg / mL. Then, recombinant human His6 Fas Ligand (CST Japan) was added to achieve a final concentration of 0.1 μg / mL, and the total volume of the culture medium was adjusted to 100 μL / well. 5% CO 2After culturing overnight at 37°C in an incubator, CellTiter-Glo (Promega) was added to the Jurkat culture plate at 100 μL / well, and the number of viable cells was measured using a luminometer (Veritas, Promega) with the amount of ATP production as an indicator.

[0306] As a result of the measurement, it was confirmed that in various DcR3 variants, the amount of ATP production from viable cells increased in a concentration-dependent manner and exhibited FasL neutralizing activity (Figure 11).

[0307] [Example 10] Ligand neutralizing activity and binding activity of FasL-binding decreased variants Based on chimeric A-Fc (g4PEK), among the DcR3 ligands, the production and evaluation of single amino acid substitution variants having binding and neutralizing activities against TL1A and LIGHT but decreased binding and neutralizing activities against FasL were performed. Variants in which any amino acid of CRD2 and CRD3 shown in Table 13 was substituted with Ala or an amino acid other than Ala were prepared, and the neutralizing activity against the DcR3 ligand was measured by the same method as in Example 9. As a result, in the single amino acid substitution variants of chimeric A-E57K-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 94, DNA base sequence: SEQ ID NO: 93) in which Glu at the 57th position from the N-terminus of chimeric A-Fc (g4PEK) was substituted with Lys or Leu, chimeric A-E57L-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 96, DNA base sequence: SEQ ID NO: 95), and chimeric A-R60K-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 98, DNA base sequence: SEQ ID NO: 97) in which Arg at the 60th position was substituted with Lys, the neutralizing activity was selectively decreased against FasL (Table 13).

[0308] For the variants of chimeric A-E57K-Fc(g4PEK) (SEQ ID NO: 94), chimeric A-E57L-Fc(g4PEK) (SEQ ID NO: 96), and chimeric A-R60K-Fc(g4PEK) (SEQ ID NO: 98) with selectively reduced neutralizing activity of FasL, the binding activity to the DcR3 ligand was measured by the same method as in Example 7. As a result, in the variants of chimeric A-E57K-Fc(g4PEK), chimeric A-E57L-Fc(g4PEK), and chimeric A-R60K-Fc(g4PEK), the binding to FasL alone was significantly reduced, while the binding to TL1A and LIGHT was maintained (FIGS. 12A, B, C).

[0309]

Table 13

[0310] [Example 11] Evaluation of the aggregability of commercially available wild-type DcR3 in mammalian cells In the same manner as in Example 1, commercially available full-length DcR3-Fc (Abcam) prepared using HEK293 cells as host cells, commercially available full-length DcR3-Fc (AdipoGen) prepared using CHO cells as host cells, and an Fc fusion of a DcR3 molecule with approximately the C-terminal half of the HBD deleted (Enzo) prepared using HEK293 cells as host cells were electrophoresed under reducing and non-reducing conditions. The molecular weight of the monomer predicted from the electrophoretic mobility under reducing conditions was approximately 50 kDa to 60 kDa, but the electrophoretic mobility under non-reducing conditions was significantly larger than the predicted molecular weight of the dimer, and most of each commercially available product existed as aggregates (FIG. 13).

[0311] [Example 12] Preparation of a variant with reduced FasL binding activity To select a variant of DcR3 ligands that binds to TL1A and LIGHT and has a selectively reduced binding affinity for FasL, a single amino acid substitution variant (E57X; X is any amino acid other than Glu) was prepared by substituting the 57th Glu (E57) from the N-terminus of chimeric A-Fc(g4PEK) (SEQ ID NO: 82) with an amino acid other than Glu. Further, for E57K, E57L, E57R, and E57V in which E57 was substituted with Lys, Leu, Arg, or Val, any one of the neighboring amino acids, Trp (W53), Asn (N54), Tyr (Y55), Leu (L56), and Arg (R58) from the N-terminus of chimeric A-Fc(g4PEK) (SEQ ID NO: 82), was further substituted with a specific amino acid Z (Z is any one of Asp, Glu, Asn, Gln, Pro, Thr, Gly) to prepare a two-amino acid substitution variant (E57X_W53Z, E57X_N54Z, E57X_Y55Z, E57X_L56Z, E57X_R58Z; each two-amino acid substitution variant was assigned a variant number shown in Fig. 14A), and a fusion with Fc(g4PEK) was prepared.

[0312] Among these, for chimeric A-E57K (amino acid sequence: SEQ ID NO: 66, DNA base sequence: SEQ ID NO: 65), chimeric A-E57R (amino acid sequence: SEQ ID NO: 180, DNA base sequence: SEQ ID NO: 179), chimeric A-E57V (amino acid sequence: SEQ ID NO: 182, DNA base sequence: SEQ ID NO: 181), chimeric A-E57K_R58D (variant number: 45-10, amino acid sequence: SEQ ID NO: 184, DNA base sequence: SEQ ID NO: 183), chimeric A-E57K_R58E (variant number: 45-18, amino acid sequence: SEQ ID NO: 186, DNA base sequence: SEQ ID NO: 185), and chimeric A, fusions with a part of various mutagenized Fc sequences shown in Table 14 were prepared (base sequences: SEQ ID NO: 213, 217, 219, 221, 223, 227, 231, 233, 235, 237, 255, 259, 261, 263, 265, 149, 167, 169, 171, 173, 175, 177, amino acid sequences: SEQ ID NO: 214, 218, 220, 222, 224, 228, 232, 234, 236, 238, 256, 260, 262, 264, 266, 317, 319, 320, 321, 322, 324, 326, 327, 328, 329, 331, 333, 334, 335, 336, 150, 168, 170, 172, 174, 176, 178). In Table 14, E216 indicates the 216th Glu of the human IgG1 heavy chain shown by the EU index. C220S, M252Y, S254T, T256E, N434A, L234A, L235A, G237A at the mutagenized sites respectively indicate that the 220th Cys of the human IgG1 heavy chain shown by the EU index was replaced with Ser, the 252nd Met with Tyr, the 254th Ser with Thr, the 256th Thr with Glu, the 434th Asn with Ala, the 234th Leu with Ala, the 235th Leu with Ala, and the 237th Gly with Ala.

[0313]

Table 14

[0314] Chimeric A-Fc(g1S) (nucleotide sequence: SEQ ID NO: 149, amino acid sequence: SEQ ID NO: 150) was prepared by deleting the IEGRMD sequence from chimeric A-Fc(IEGRMD g1S) (SEQ ID NO: 80) containing the linker sequence IEGRMD (SEQ ID NO: 106), which was prepared in Example 2, by stable expression in CHO cells by the method described in Example 6. Chimeric A-Fc(Eg1S) (nucleotide sequence: SEQ ID NO: 167, amino acid sequence: SEQ ID NO: 168) was prepared by stable expression in CHO cells by the method described in Example 6 of a sequence in which chimeric A (SEQ ID NO: 54) and Eg1S were ligated together.

[0315] Chimeric A-Fc(Eg1S YTE, Eg1S N434A, Eg1S LALAGA, Eg1S LALAGANA) (nucleotide sequences: SEQ ID NO: 169, 171, 175, 177, amino acid sequences: SEQ ID NO: 170, 172, 176, 178) were obtained by transient expression in CHO-S cells of sequences in which each Fc was fused to the C-terminus of chimeric A (SEQ ID NO: 54).

[0316] Chimeric A-Fc(g1S YTE, g1S N434A, g1S LALAGA, g1S LALAGANA) (amino acid sequences: SEQ ID NO: 314, 315, 174, 316) were obtained by transient expression in Expi293 cells of sequences in which each Fc was fused to the C-terminus of chimeric A (SEQ ID NO: 54).

[0317] Plasmids expressing the fusion of each amino acid substitution mutant with Fc(g4PEK) were prepared by using the DNA sequence of either chimeric A-Fc(g4PEK) or E57X-Fc(g4PEK; X is K, R or V) as a template, and using PCR primers designed to contain the mutation sites, to PCR amplify two regions, from the NheI site to the site where the amino acid substitution was introduced and from the site where the amino acid substitution was introduced to the SalI site, and inserting them under the CMV promoter of the pCIpuro vector by the same method as in Example 1.

[0318] The fusion of each amino acid substitution variant with the Fc of Eg1S YTE, Eg1S N434A, or Eg1S LALAGANA was prepared by using, as a template, any of the DNA sequences of chimeric A-Fc having the respective mutant Fc sequences, and PCR primers designed to include the mutation sites. Two regions, from the EcoRI site to the site where the amino acid substitution was introduced and from the site where the amino acid substitution was introduced to the Bsu36I site, were each PCR-amplified and inserted into the EcoRI and Bsu36I sites of the chimeric A-various mutant Fc vectors used as templates to prepare plasmids.

[0319] The fusion of each amino acid substitution variant with the Fc of g1S YTE, g1S N434A, or g1S LALAGANA was prepared by using, as a template, any of the DNA sequences of each of the above-mentioned amino acid substitution variant-Fc, and PCR primers designed to exclude E216. Two regions, from the EcoRI site to the site of Glu to be deleted and from the site of Glu to be deleted to the Bsu36I site, were each PCR-amplified and inserted into the EcoRI and Bsu36I sites of the respective amino acid substitution variant-Fc vectors used as templates to prepare plasmids.

[0320] Each plasmid was introduced into Expi293 cells for transient expression, and affinity purification was performed using MabSelect SuRe from the culture supernatant. The content ratios of the monomer, aggregate, and degradation product of the prepared chimeric A-Fc, 1 or 2 amino acid substitution variant-Fc were calculated from the peak areas analyzed by SEC-UPLC (ACQUITY UPLC Protein BEH SEC 4.6 mm×150 mm) (Waters) or SEC-HPLC (TSKgel SuperSW3000 4.0 μm, 4.6 mm x 300 mm) (Tosoh) in the same manner as in Example 2.

[0321] As a result, many of the chimeric A-Fc, 1 and 2 amino acid substitution variant-Fc having various mutant Fc prepared maintained a lower aggregate content ratio than S195-Fc (FIGS. 14A, B, C).

[0322] [Example 13] Evaluation of binding activity to DcR3 soluble trimeric ligand Regarding various chimeric A-Fcs with different DcR3-Fc, S195-Fc, and Fc sequences and various FasL-binding activity-reduced mutants prepared in Example 12, the binding activity evaluations for soluble human LIGHT trimer, soluble human TL1A trimer, and soluble human FasL trimer were carried out by slightly modifying the method described in Example 7.

[0323] (1) Preparation of DcR3 soluble trimer ligand For soluble recombinant human LIGHT, a sequence (FLAG-LIGHT) in which a FLAG tag (DYKDDDDK) was added to the N-terminus and the extracellular region (Asp74-Val240) (SEQ ID NO: 132) of LIGHT was ligated downstream thereof was used (base sequence: SEQ ID NO: 305, amino acid sequence: SEQ ID NO: 306). For soluble recombinant human TL1A, a sequence (His6-TL1A) in which a His tag (His6) and a GS linker (GGGSGGGSGGGS) were added to the N-terminus and the extracellular region (Leu72-Leu251) (SEQ ID NO: 138) of TL1A was ligated downstream thereof was used (base sequence: SEQ ID NO: 115, amino acid sequence: SEQ ID NO: 116). Each plasmid was prepared in the same manner as in Example 7 and transiently expressed using Expi293 cells.

[0324] FLAG-LIGHT was purified using ANTI-FLAG M2 Affinity Gel (Sigma). The culture supernatant was passed through a column filled with the resin, washed with a washing buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.4), and then eluted with an eluent (0.1 M glycine hydrochloride, pH 3.5).

[0325] His6-TL1A was purified by the following method. The culture supernatant was passed through a column packed with Complete His-Tag Purification Resin (Roche), washed with a washing buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl), and then eluted with an elution solution (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM Imidazole). The elution solution replaced with PBS using a NAP column (GE Healthcare) was passed through a column packed with Ni Sepharose Fast Flow resin (GE Healthcare), washed with a washing buffer (60 mM imidazole, 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) prepared using a His Buffer Kit (GE Healthcare), and then eluted with an elution buffer (250 mM imidazole, 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4).

[0326] The elution fractions of FLAG-LIGHT and His6-TL1A were each replaced with PBS using a NAP column (GE Healthcare) and sterilized by passing through a 0.22-μm filter. The obtained purified proteins were fractionated into trimer fractions by gel filtration chromatography (SEC) using HPLC (Shimadzu) (TSKgel G3000 SWXL 7.8 mm x 300 mm) (Tosoh).

[0327] Human soluble recombinant FasL with a His tag (His6) added to the N-terminus and the extracellular region of FasL (Pro134-Leu281) (SEQ ID NO: 144) linked downstream thereof, Human His6 Fas Ligand / TNFSF6 (Cell Signaling TECHNOLOGY), was used. SEC-MALS was performed in the same manner as in Example 2, and it was confirmed that it was a trimer.

[0328] (2) Measurement of binding activity using BIAcore Both DcR3-Fc (from R&D) and S195-Fc were evaluated as those described in Example 7. Chimeric A-g4PEK (SEQ ID NO: 82) was prepared in Example 6. Chimeric A-Fc (g1S, Eg1S) was prepared in Example 12. As various FasL-binding decreased mutants, the Fc (g4PEK) fusions prepared in Example 9 or Example 12 were evaluated.

[0329] The binding activity to human DcR3 trimeric ligand was analyzed by the SPR method. BIAcore T-100 (GE Healthcare) was used for measuring the binding activity to human LIGHT and human TL1A, and BIAcore T-100 (GE Healthcare) or BIAcore T-200 (GE Healthcare) was used for measuring the binding activity to human FasL, respectively. HBS-EP+ Buffer was used as the buffer.

[0330] After immobilizing 10000 RU of anti-human antibody on Series S Sensor Chip CM5 using Human Antibody Capture Kit (both from GE Healthcare), various wild-type DcR3 and DcR3 mutants were flowed at 10 μL / min for 30 seconds and captured. On the other hand, a buffer without protein was flowed through the reference flow cell. Then, as the analyte, each human DcR3 trimeric ligand diluted to 0.02 - 80 nmol / L was flowed at 30 μL / min for 2 minutes to monitor the binding, and then the buffer was flowed for 3 minutes to monitor the dissociation. Next, 3 mol / L magnesium chloride was flowed at 30 μL / min for 1 minute to perform the regeneration reaction. For measuring the binding activity to human LIGHT and human TL1A, BIAcore T-100 evaluation software and the 1:1 Binding model were used. The ligand was used as a trimer (human LIGHT trimer: 62.4 kDa, human TL1A trimer: 66.2 kDa), and each kinetic constant (k a 、k d 、K D) was calculated. For the measurement of the binding activity to human FasL, BIAcore T-100 evaluation software, 1:1 Binding model or BIAcore T-200 evaluation software, 1:1 Binding model was used. The ligand was used as a monomer (human FasL monomer: 19.8 kDa), and each kinetic constant (k a , k d , K D ) was calculated.

[0331] As a result, it was confirmed that all of chimeric A-Fc (g1S), chimeric A-Fc (Eg1S), and chimeric A-Fc (g4PEK) bind to each DcR3 trimer ligand (Figure 15A).

[0332] Among the FasL-binding ability-reduced mutants (g4PEK) prepared in Example 12, the single amino acid substitution chimera A-E57K-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 94, DNA base sequence: SEQ ID NO: 93) (hereinafter sometimes referred to as "E57K-Fc"), chimera A-E57L-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 96, DNA base sequence: SEQ ID NO: 95) (hereinafter sometimes referred to as "E57L-Fc"), chimera A-E57R-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 190, DNA base sequence: SEQ ID NO: 189) (hereinafter sometimes referred to as "E57R-Fc"), chimera A-E57V-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 192, DNA base sequence: SEQ ID NO: 191) (hereinafter sometimes referred to as "E57V-Fc"), chimera A-E57A-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 288, DNA base sequence: SEQ ID NO: 287) (hereinafter sometimes referred to as "E57A-Fc"), chimera A-E57F-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 290, DNA base sequence: SEQ ID NO: 289) (hereinafter sometimes referred to as "E57F-Fc"), chimera A-E57H-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 292, DNA base sequence: SEQ ID NO: 291) (hereinafter sometimes referred to as "E57H-Fc"), chimera A-E57I-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 294, DNA base sequence: SEQ ID NO: 293) (hereinafter sometimes referred to as "E57I-Fc"), chimera A-E57M-Fc (g4PEK) (amino acid sequence: SEQ ID NO: 296, DNA base sequence: SEQ ID NO: 295) (hereinafter sometimes referred to as "E57M-Fc"), and the double amino acid substitution chimera A-E57K_R58D-Fc (g4PEK) (variant number: 45-10, amino acid sequence: SEQ ID NO: 194, DNA base sequence: SEQ ID NO: 193) (hereinafter sometimes referred to as "45-10-Fc"), chimera A-E57K_R58T-Fc (g4PEK) (variant number: 45-11, amino acid sequence: SEQ ID NO: 298, DNA base sequence: SEQ ID NO: 297) (hereinafter sometimes referred to as "45-11-Fc"), chimera A-E57K_R58E-Fc (g4PEK) (variant number: 45-18, amino acid sequence: SEQ ID NO: 196, DNA base sequence: SEQ ID NO: 195) (hereinafter sometimes referred to as "45-18-Fc"),Chimeric A-E57L_R58E-Fc(g4PEK) (variant number: 46-4, amino acid sequence: SEQ ID NO: 300, DNA base sequence: SEQ ID NO: 299) (hereinafter sometimes referred to as "46-4-Fc"), chimeric A-E57R_R58D-Fc(g4PEK) (variant number: 82-5, amino acid sequence: SEQ ID NO: 198, DNA base sequence: SEQ ID NO: 197) (hereinafter sometimes referred to as "82-5-Fc"), chimeric A-E57V_R58T-Fc(g4PEK) (variant number: 85-6, amino acid sequence: SEQ ID NO: 302, DNA base sequence: SEQ ID NO: 301) (hereinafter sometimes referred to as "85-6-Fc"), and chimeric A-E57V_R58E-Fc(g4PEK) (variant number: 85-8, amino acid sequence: SEQ ID NO: 304, DNA base sequence: SEQ ID NO: 303) (hereinafter sometimes referred to as "85-8-Fc") all have a K for LIGHT trimer and TL1A trimer that is less than 3 times that of chimeric A-Fc(g4PEK), D and the K for FasL trimer D is greater than 3 times or the Rmax value is reduced to less than 5, and it was confirmed that the binding selectivity for FasL was reduced (Figures 16A, B).

[0333] Among these, E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, 82-5-Fc (all with Fc being g4PEK) were purified to 95% or more of the monomer by SEC-HPLC (column; TSKgel G3000 SWXL 7.8mm x 300mm, Tosoh Corporation, HPLC; Shimadzu Corporation), and the kinetic constants for each when subjected to BIAcore measurement are shown in Figure 15B.

[0334] [Example 14] Evaluation of Neutralizing Activity of Soluble DcR3 Ligand The evaluation of the neutralizing activity of various DcR3 variants against soluble human LIGHT, soluble human TL1A, and soluble human FasL was carried out with a partial modification of the method described in Example 9.

[0335] (1) Preparation of Various Chimeric A-Fc with Different Fc Sequences Chimeric A-Fc(g1S, Eg1S) prepared in Example 12 was used. Each chimeric A-Fc in which each Fc of Eg1S YTE, Eg1S N434A, Eg1S LALAGA, or Eg1S LALAGANA shown in Table 14 was fused to the C-terminal side of chimeric A prepared in Example 12 was prepared by transient expression in CHO-S cells and purified to 95% or more monomer using SEC (Superdex 200 Increase 10 / 300 GL) (GE Healthcare) with an AKTA purifier (GE Healthcare).

[0336] (2) Soluble human LIGHT neutralizing activity For chimeric A-Fc (g1S, Eg1S, Eg1S YTE, Eg1S N434A, Eg1S LALAGA, Eg1S LALAGANA, g4PEK), the inhibitory activity against LIGHT-dependent CXCL10 production from IFN-γ-stimulated intestinal myofibroblasts (Lonza) was evaluated. Intestinal myofibroblasts were cultured in a collagen I-coated flask (BD) using SmGM-2 Bullet Kit (Lonza) medium. After seeding the cells at 1x10 4 cells / well in a collagen I-coated 96-well plate (BD), IFN-γ was added at a final concentration of 10 ng / mL, trimeric FLAG-LIGHT prepared in Example 13 was added at a final concentration of 20 ng / mL, and each final concentration of various DcR3 variants was 19.5, 78.1, 313, 1250, 5000, 20000 ng / mL, or 4.88, 19.5, 78.1, 313, 1250, 5000 ng / mL, and cultured for 3 days. The culture supernatant was collected, and the CXCL10 concentration in the culture supernatant was measured using a CXCL10 / IP-10 (human) AlphaLisa Detection Kit (Perkin Elmer). As a result, it was confirmed that chimeric A-Fc having any Fc sequence inhibited CXCL10 production in a concentration-dependent manner and had neutralizing activity against soluble LIGHT (Figs. 17A, B).

[0337] For the FasL-binding decreased mutants E57K-Fc, E57L-Fc, E57R-Fc, and E57V-Fc (in all cases, Fc is g4PEK), the inhibitory activity against LIGHT-dependent IL-8 production from HT-29 cells was evaluated in the same manner as in Example 9. As a result, it was confirmed that for each of the mutants, IL-8 production was inhibited in a concentration-dependent manner and that they had neutralizing activity against soluble LIGHT (Figure 17C).

[0338] For the FasL-binding decreased mutants 45-10-Fc, 45-18-Fc, and 82-5-Fc (in all cases, Fc is g4PEK), similar to chimeric A-Fc, the inhibitory activity against LIGHT-dependent CXCL10 production from IFN-γ-stimulated intestinal myofibroblasts was evaluated under the condition that the number of intestinal myofibroblasts per well was 2x10 4 , and each final concentration of the test substance was 19.5, 78.1, 313, 1250, 5000, 20000 ng / mL. As a result, it was confirmed that for each of the mutants, CXCL10 production was inhibited in a concentration-dependent manner and that they had neutralizing activity against soluble LIGHT (Figure 17D).

[0339] (3) Soluble human TL1A neutralizing activity In the same manner as in Example 9, the inhibitory activity of various DcR3 mutants against TL1A-dependent IFN-γ production from IL-12- and IL-18-stimulated human T cells was evaluated. As a result, it was confirmed that chimeric A-Fc having any Fc sequence inhibited the IFN-γ production amount in a concentration-dependent manner and had neutralizing activity against soluble TL1A (Figures 18A, B). Furthermore, it was also confirmed that the FasL-binding decreased mutants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, and 82-5-Fc (in all cases, Fc is g4PEK) similarly had neutralizing activity against soluble TL1A (Figures 18C, D).

[0340] (4) Soluble human FasL neutralizing activity In the same manner as in Example 9, the inhibitory activities of various DcR3 variants against FasL-dependent cell death of Jurkat cells or A3 cells, which are Jurkat subclones, were evaluated. As a result, it was confirmed that chimeric A-Fc having any Fc sequence inhibited cell death of A3 cells in a concentration-dependent manner and had neutralizing activity against soluble FasL (Figs. 19A and B). On the other hand, for FasL-binding activity-reducing variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, 82-5-Fc (in all cases, Fc is g4PEK), it was confirmed that the inhibitory activity against Jurkat cell death was significantly reduced and the neutralizing activity against soluble FasL was selectively reduced (Figs. 19C and D).

[0341] [Example 15] Evaluation of Binding Activity of DcR3 to Membrane Ligand The binding activities of various DcR3 variants to membrane human LIGHT, membrane human TL1A, and membrane human FasL were evaluated by flow cytometry using a strain with forced expression of the membrane ligand. Membrane human LIGHT-overexpressing HEK293 cells described in US8974787 were used. Membrane TL1A and membrane FasL were PCR-amplified with addition of a Met residue and a FLAG tag (DYKDDDDK) to the N-terminus using respective commercially available ORF clones (Origene) as templates (base sequences: SEQ ID NOs: 307, 309, amino acid sequences: SEQ ID NOs: 308, 310), inserted downstream of the CMV promoter of the pCIpuro vector using the In-Fusion HD Cloning Kit (Clontech), and Escherichia coli DH5α competent cells (TOYOBO) were transformed.

[0342] The obtained plasmid was introduced into CHO-K1 cells (ECACC) using a Nucleofector and Cell Line Nucleofector Kit T (both from Lonza), and subjected to drug selection with 10 μg / mL Puromycin (Thermo Fisher Scientific). The obtained drug-resistant cells were stained with a DyLight488-labeled anti-human TL1A antibody (Novus Biologics) or an APC-labeled anti-human FasL antibody (BD Pharmingen), and the high-expression fraction was sorted using a cell sorter (Sony). After expansion culture, the cells were stained with a PE-labeled anti-human TL1A antibody (Novus Biologics) or a PE-labeled anti-human FasL antibody (BioLegend), and sorted again to obtain cell lines with high expression of membrane-type human TL1A or membrane-type human FasL.

[0343] The binding activities of various DcR3 variants to the membrane-type ligand overexpression strains and host cells were evaluated in the same manner as in Example 5, with the following conditions changed. HEK293 and the membrane-type LIGHT overexpression strain were reacted with 1 μg / mL of each protein and 10 ng / mL of the secondary antibody Goat F(ab’) 2 Anti-Human IgG R-phycoerythrin Conjugate (Southern Biotech). For the membrane-type TL1A and membrane-type FasL overexpression strains, the cells were reacted with 1 or 10 μg / mL of each protein and 0.1 or 1 μg / mL of the secondary antibody, respectively.

[0344] As a result, none of the chimeric A-Fc (g1S, Eg1S, g4PEK) reacted with the host cells, 293 or CHO-K1 cells, and specifically reacted with each membrane-type ligand overexpressing strain. Therefore, it was confirmed that all types of chimeric A with different Fc sequences had binding activity to the membrane-type DcR3 ligand (Figs. 20A and B). Furthermore, the binding activities of FasL-binding decreased mutants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, 82-5-Fc (all Fc is g4PEK) to the ligand were similarly evaluated. As a result, it was confirmed that while the binding activities to membrane-type human LIGHT and membrane-type human TL1A were retained, the binding activities to membrane-type human FasL were significantly reduced except for E57L (Figs. 21A, B, and C).

[0345] [Example 16] Evaluation of Binding Activity to DcR3 Primary Ligand The binding activities of various DcR3 mutants to each DcR3 ligand derived from primary cells were evaluated by the following method.

[0346] (1) Primary LIGHT Binding Activity It is known that the expression of membrane-bound LIGHT is induced in activated human T cells (The Journal of Immunology, 2004, 173: p. 502-507.). Cryopreserved healthy human PBMCs (AllCells) were thawed and stimulated overnight with PMA (Sigma) at a final concentration of 50 ng / mL and ionomycin (Sigma) at a final concentration of 1 μg / mL. Subsequently, the binding of chimeric A-Fc (Eg1S) to membrane-bound LIGHT induced in CD3-positive T cells was evaluated by the following method. The anti-DNP antibody (IgG1) described in Example 8 was used as a negative control. The stimulated PBMCs were collected, reacted with Human FcR Blocking Reagent (Miltenyi Biotech), and then chimeric A-Fc or anti-DNP antibody at a final concentration of 0.4 μg / mL labeled with BV421-labeled CD3 antibody (BD Pharmingen), 7-AAD Staining Solution (BD Pharmingen), and Alexa Fluor488 Antibody Labeling Kit (Thermo Scienific) was added respectively. After the reaction, the cells were washed, and the fluorescence intensity of Alexa Fluor488 in CD3-positive T cells in the viable cell fraction was analyzed using a flow cytometer. To confirm the expression of membrane-bound LIGHT in the induced and stimulated PBMCs, the induced and stimulated PBMCs were reacted with Human FcR Blocking Reagent (Miltenyi Biotech), and then BV510-labeled CD3 antibody (BioLegend), 7-AAD Staining Solution (BD Pharmingen), and PE-labeled LIGHT antibody (LSBio) or PE-labeled mouse IgG1κ isotype control antibody (BioLegend) was added respectively. After the reaction, the cells were washed, and the fluorescence intensity of PE in CD3-positive T cells in the viable cell fraction was analyzed using a flow cytometer.

[0347] As a result, it was confirmed that the expression of LIGHT was induced on CD3-positive human T cells of the induced and stimulated PBMCs, and it was confirmed that Alexa Fluor488-labeled chimeric A-Fc bound to membrane-bound LIGHT on activated CD3-positive human T cells (Figs. 22A, B).

[0348] (2) Primary TL1A binding activity HUVEC cells (Lonza) cultured in the same manner as in Example 5 were treated with Recombinant Human IL-1 alpha (R&D) at a final concentration of 10 ng / mL and the TACE inhibitor TAPI-1 (Calbiochem) at a final concentration of 20 μM for 24 hours. The binding of chimeric A-Fc (IEGRMD g1S) to membrane-bound TL1A whose expression was induced was evaluated by a competition experiment with the TL1A antibody 1D1 1.31 (US2015 / 0132311). The TL1A antibody 1D1 1.31 was prepared by ligating the amino acid sequences of VL and VH described in US2015 / 0132311 to the constant region of human IgG1, transiently expressing it using Expi293 cells by the method described in Example 1, and purifying it from the culture supernatant using Mabselect SuRe (GE Healthcare). The anti-DNP antibody (IgG1) described in Example 8 was used as a negative control. The recovered cells were reacted with Human FcR Blocking Reagent (Miltenyi Biotech), and then either the anti-DNP antibody, chimeric A-Fc, or TL1A antibody labeled with the Zenon Alexa Fluor 647 Human IgG Labeling Kit (Molecular Probes) was added to a final concentration of 8.3 μg / mL. After the reaction, the cells were washed, and the fluorescence intensity of Alexa Fluor 647 was analyzed using a flow cytometer. Under competition conditions, either unlabeled DNP antibody, TL1A antibody, or chimeric A-Fc at a final concentration of 50 μg / mL was reacted in advance.

[0349] As a result, since the binding of labeled chimeric A-Fc competed with unlabeled TL1A antibody and the binding of labeled TL1A antibody competed with unlabeled chimeric A-Fc, it was confirmed that chimeric A binds to membrane-bound TL1A on stimulated HUVEC cells (Figure 23).

[0350] (3) Primary FasL binding activity The binding of chimeric A-Fc (IEGRMD g1S) to primary soluble FasL produced from human T cells induced to undergo activation-induced cell death (AICD) was evaluated by the following method. The primary soluble FasL was prepared by the following method. That is, human T cells isolated from cryopreserved healthy human PBMCs by the same method as in Example 9 were seeded in a 96-well U-bottom plate (BD) at 2x10 4 cells / well, cultured for 24 hours with PHA-L (eBioscience) at a final concentration of 1 μg / mL, and then cultured for 5 days after adding IL-2 (Peptrotech) at a final concentration of 1 μg / mL. After 5 days, the cells were collected, seeded in a 96-well U-bottom plate coated with 5 μg / mL of anti-CD3 antibody OKT3 (BioLegend), induced to undergo AICD, cultured overnight, the culture supernatant was collected, and concentrated to 1 / 10 of the solution volume using an Amicon Ultra-15 (Millipore) with a molecular weight cut-off of 10 kDa pre-rinsed with sterile water.

[0351] A 96-well immunoplate (Thermo Scientific) coated with 10 μg / mL of anti-human IgG antibody (American Qualex) was blocked with 1% Block Ace (DS Pharma Biomedical), and then 20 μg / mL of chimeric A-Fc (IEGRMD g1S) or Fas-Fc (R&D) was captured. After washing, recombinant FasL (abcam) used as a standard or 10-fold concentrated AICD culture supernatant was reacted, and after washing, a biotinylated anti-FasL antibody (abcam) was reacted. After washing, streptavidin-HRP (PIERCE) was reacted, washed again, TMB solution (abcam) was added to develop color, the color reaction was stopped with 2N sulfuric acid solution, and the absorbance at 450 nm was measured.

[0352] The results of the plate with immobilized chimeric A-Fc (IEGRMD g1S) are shown in Fig. 24A, and the results of the plate with immobilized Fas-Fc are shown in Fig. 24B. 10×AICDsup. CD3 indicates that the culture supernatant of T cells that induced AICD under anti-CD3 antibody OKT3 stimulation conditions was used, and none indicates that the culture supernatant of T cells that did not induce AICD was used. FasL was detected only in the culture supernatant of T cells that induced AICD for both plates with immobilized chimeric A-Fc or Fas-Fc. From this, it was confirmed that chimeric A-Fc binds to soluble FasL produced from AICD-induced T cells (Figs. 24A, B).

[0353] [Example 17] Physical property evaluation of DcR3 variants For various chimeric A-Fc (g1S, Eg1S, Eg1S YTE, Eg1S N434A, Eg1S LALAGANA, Eg1S LALAGA, g4PEK) and FasL-binding reduced variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, 82-5-Fc (all g4PEK), the elution time (minutes) by hydrophobic interaction chromatography (HIC) and the Tm value (°C) by Differential Scanning Fluorimetry (DSF) method were calculated in the same manner as in Example 4.

[0354] As a result, it was confirmed that there was no significant effect on hydrophobicity by introducing multiple amino acid mutations into the IgG1 Fc sequence. Since the elution times of the FasL-binding reduced variants were not significantly different from those of chimeric A-g4PEK, it was confirmed that there was no effect on hydrophobicity due to one or two amino acid substitutions (Fig. 25).

[0355] Regarding the Tm value determined by DSF, among the amino acid substitutions or insertions in the IgG1 Fc sequence, an effect on thermal stability was observed due to the introduction of the YTE mutation. Since there was no significant difference in the Tm values of the FasL-binding decreased variants compared to chimeric A-g4PEK, it was confirmed that introducing one or two amino acid substitutions into the CRD portion of DcR3 had no effect on thermal stability (Figure 26).

[0356] [Example 18] Evaluation of the in vivo kinetics of DcR3 variants For chimeric A-Fc(Eg1S) and FasL-binding decreased variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, 82-5-Fc (in all cases, Fc is g4PEK), the in vivo kinetics in mice were evaluated by the same method as described in Example 6. Chimeric A-Fc(Eg1S) was prepared by stable expression in CHO-K1 cells, and each FasL-binding decreased variant was prepared by transient expression in CHO-S cells. The purified products with more than 95% monomers were used, which were purified by SEC (Superdex 200 Increase 10 / 300 GL) (GE Healthcare) using an AKTApurifier (GE Healthcare).

[0357] When 10 mg / kg of each DcR3 variant was administered as a single i.v. dose to 5- to 6-week-old BALB / c mice (♀) (n = 2 or 3), the plasma half-life (h) during the elimination phase after a single dose and the area under the concentration-time curve up to infinite time, AUC0-∞ (μg*h / mL), were calculated (Figure 27). When measuring the concentration of the DcR3 variant in the serum of mice administered with E57K-Fc, E57R-Fc, or E57V-Fc, each DcR3 variant prepared by transient expression in Expi293 cells was used as the standard substance.

[0358] As a result, compared to S195-Fc(IEGRMD g1S), the wild-type DcR3 described in Example 6, the AUC of all DcR3 variants was significantly improved.

[0359] [Example 19] In Vivo Efficacy Evaluation of DcR3 Variant The in vivo efficacy evaluation of chimeric A-Fc (g4PEK) was carried out using a mouse acute xenogeneic graft-versus-host disease (GVHD) model.

[0360] (1) Preparation of Mouse Acute Xenogeneic GVHD Model The mouse acute xenogeneic GVHD model was prepared in the same manner as described in JP5209625. Using 6-week-old severe combined immunodeficiency (SCID) female mice, 100 μg of rat anti-mouse IL2 receptor-β (IL2Rβ) chain antibody TMβl (Bio X Cell) was intraperitoneally injected on days -2 and 5 to deplete endogenous mouse natural killer cells. On day -1, the mice were irradiated with a lethal dose of 1.7 Gy or less using a CellRad type X-ray irradiator (Faxitron). On day 0, 3x10 6 human PBMCs (AllCells) were transferred intraperitoneally, and subsequently, 300 μg of chimeric A-Fc or DNP antibody (both g4PEK) prepared in 100 μL of PBS was intraperitoneally injected. For the chimeric A-Fc administration group, 300 μg of chimeric A-Fc was additionally administered on days 4 and 8. After 12 days, the macroscopic disease score due to the GVHD reaction was determined, and the spleen was collected from the sacrificed mice to evaluate the number of human cells in the spleen.

[0361] (2) Evaluation of GVHD Disease Score The macroscopic disease observed on day 12 was scored based on four indicators: coat condition, intestinal redness, activity level, and weight loss (Figure 28A). Each indicator was scored 0, 1, or 2 for none, mild, and severe, respectively, and the total score of all indicators was used to determine the GVHD disease score.

[0362] As a result, an increase in the GVHD disease score due to the transfer of human PBMCs and a decrease in the disease score due to chimeric A-Fc administration compared to the DNP antibody administration group were confirmed, respectively, and the efficacy of chimeric A-Fc was recognized (Figure 28B).

[0363] (3) Measurement of the number of human cells in the spleen Mouse spleens were collected, and the spleens were disrupted using gentleMACS Dissociators (Miltenyi) to prepare splenocyte suspensions. After hemolysis treatment with Lysing buffer (BD Biosciences), cells were stained with PE / Cy7-labeled anti-human CD45 antibody, FITC-labeled anti-human CD3 antibody, APC-labeled anti-human CD4 antibody, and PE-labeled anti-human CD8 antibody (all from BioLegend), and each human cell subset was analyzed by flow cytometry. CountBright Absolute Counting Beads, for flow cytometry (Thermo Fisher Scientific) was used for cell number measurement. The fluorescence bead count detected by flow cytometry and the abundance ratio of each cell subset number were corrected by the known added bead amount to calculate the total cell number in the spleen.

[0364] As a result, human cells were detected in the mouse spleen by transplantation of human PBMC, and a decrease in the number of human cells in the mouse spleen by administration of chimeric A-Fc was confirmed as compared with the DNP antibody administration group (Figure 29).

[0365] [Example 20] Evaluation of binding activity to soluble DcR3 ligand trimer For chimeric A-Fc having various mutant Fcs prepared in Example 12 and FasL-binding activity-reduced mutants, evaluation of binding activity to soluble human LIGHT trimer, soluble cynomolgus monkey LIGHT trimer, soluble human TL1A trimer, soluble cynomolgus monkey TL1A trimer, soluble human FasL trimer, or soluble cynomolgus monkey FasL trimer was performed as follows with a partial modification of the methods described in Example 7 and Example 13.

[0366] (1) Preparation of soluble DcR3 ligand trimer The human soluble DcR3 ligand trimer used was the one prepared in Example 13. The cynomolgus soluble recombinant LIGHT with an N-terminal FLAG tag (DYKDDDDK) and the extrace...

Claims

1. A DcR3 variant which is a variant of a wild-type mammalian Decoy Receptor 3 (hereinafter abbreviated as DcR3), comprising a first chimeric cysteine-rich region or a second chimeric cysteine-rich region; The first chimeric cysteine-rich region is selected from the group consisting of the following (a), (b), (c), and (d): (a) an amino acid sequence in which, in the amino acid sequence of the cysteine-rich region of wild-type DcR3, cysteine-rich domain 1 (hereinafter abbreviated as CRD1) of wild-type DcR3 is substituted with CRD1 of mammalian osteoprotegerin (hereinafter abbreviated as OPG); (b) an amino acid sequence in the cysteine-rich region of wild-type DcR3 in which the CRD4 of wild-type DcR3 is replaced with the CRD4 of OPG; (c) an amino acid sequence of the cysteine-rich region of wild-type DcR3 in which the CRD1 of wild-type DcR3 is replaced with the CRD1 of OPG and the CRD4 of wild-type DcR3 is replaced with the CRD4 of OPG; or (d) an amino acid sequence in which the portion from the 103rd to 123rd positions from the N-terminus in the amino acid sequence of (a), (b) or (c) is replaced with the corresponding portion in the amino acid sequence of the cysteine-rich domain of OPG; Any one of the following: The CRD2 and CRD3 of wild-type DcR3 are retained in the first chimeric cysteine-rich region; and The second chimeric cysteine-rich region has the following structure (e): (e) an amino acid sequence in which 1 to 5 amino acids have been deleted, substituted, inserted or added in the amino acid sequence of the first chimeric cysteine-rich region; Including, The amino acid sequence of (e) is as follows: - Substitution of Glu at the 57th position from the N-terminus of the amino acid sequence of (a), (b), (c) or (d) above with another amino acid; - Substitution of Arg at the 58th position from the N-terminus of the amino acid sequence of (a), (b), (c) or (d) with another amino acid; and - Substitution of Arg at the 60th position from the N-terminus of the amino acid sequence of (a), (b), (c) or (d) above with another amino acid; or having one or more substitutions selected from the group consisting of The amino acid sequence of (e) is the following (f) to (i): (f) substitution of Asn at the 131st and 144th positions from the N-terminus of the amino acid sequence of (b), (c), or (d) with other amino acids; (g) substitution of Asn at the 131st, 144th, and 157th positions from the N-terminus of the amino acid sequence of (b), (c), or (d) above with other amino acids; (h) substitution of Thr at the 133rd position and Ser at the 146th position from the N-terminus of the amino acid sequence of (b), (c) or (d) with another amino acid; or (i) substitution of Thr at the 133rd position, Ser at the 146th position, and Thr at the 159th position from the N-terminus of the amino acid sequence of (b), (c), or (d) with other amino acids; having substitutions selected from DcR3 variants.

2. The DcR3 variant of claim 1, which has one or more complex-type N-glycoside-linked sugar chains.

3. having neutralizing activity against at least one of LIGHT, TL1A and FasL; It has neutralizing activity against all of LIGHT, TL1A and FasL. has no neutralizing activity against FasL and has neutralizing activity against any one or more of LIGHT and TL1A, or 3. The DcR3 variant according to claim 1, which has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A.

4. The DcR3 variant according to any one of claims 1 to 3, wherein the first chimeric cysteine-rich region is composed of an amino acid sequence in which the CRD1 of wild-type DcR3 is replaced with the CRD1 of OPG, and the CRD4 of wild-type DcR3 is replaced with the CRD4 of OPG, in the amino acid sequence of the cysteine-rich region of wild-type DcR3.

5. the amino acid sequence of (a) is an amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 26 or 50, the amino acid sequence (b) is an amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 28 or 52, the amino acid sequence of (c) is an amino acid sequence consisting of the 1st to 164th amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 30 or 54, The DcR3 variant described in any one of claims 1 to 4, wherein the amino acid sequence (d) is an amino acid sequence consisting of amino acids 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 32 or 56.

6. A DcR3 variant described in any one of claims 1 to 5, wherein the amino acid sequence (e) has a substitution of Glu at the 57th position and Arg at the 58th position from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with other amino acids.

7. The amino acid sequence of (e) is as follows: - Substitution of Glu at the 57th position from the N-terminus of the amino acid sequence of (a), (b), (c) or (d) above with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met; - Substitution of Arg at the 58th position from the N-terminus of the amino acid sequence of (a), (b), (c) or (d) above with Asp, Glu or Thr; and - substitution of Arg at the 60th position from the N-terminus of the amino acid sequence of (a), (b), (c) or (d) with Lys; The DcR3 variant of any one of claims 1 to 6, having one or more substitutions selected from the group consisting of:

8. The DcR3 variant of claim 6, wherein the amino acid sequence (e) has a substitution of Glu at the 57th position from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met, and a substitution of Arg at the 58th position from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Asp, Glu or Thr.

9. The amino acid sequence of the above (e) is selected from the following (f') to (i'): (f') substitution of Asn at the 131st and 144th positions from the N-terminus of the amino acid sequence of (b), (c) or (d) with Ser; (g') substitution of Asn at the 131st, 144th, and 157th positions from the N-terminus of the amino acid sequence of (b), (c), or (d) with Ser; (h') substitution of Thr at the 133rd position and Ser at the 146th position from the N-terminus of the amino acid sequence of (b), (c) or (d) with Ala; or (i') substitution of Thr at the 133rd position, Ser at the 146th position, and Thr at the 159th position from the N-terminus of the amino acid sequence of (b), (c), or (d) with Ala; The DcR3 variant of any one of claims 1 to 8, having a substitution selected from:

10. The DcR3 variant described in any one of claims 1 to 9, wherein the amino acid sequence (e) is an amino acid sequence consisting of amino acids 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284 or 286.

11. The DcR3 variant according to any one of claims 1 to 10, wherein the DcR3 variant comprises the first or second chimeric cysteine-rich region and the heparan sulfate binding domain of the wild-type DcR3 bound to the C-terminal side of the first or second chimeric cysteine-rich region, or the DcR3 variant comprises the first or second chimeric cysteine-rich region and does not comprise the heparan sulfate binding domain of the wild-type DcR3.

12. The DcR3 variant of any one of claims 1 to 11, wherein the DcR3 variant comprises an amino acid sequence set forth in SEQ ID NO:50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284 or 286.

13. The DcR3 variant of claim 12 , wherein the DcR3 variant comprises the amino acid sequence set forth in SEQ ID NO: 66 or 186.

14. The DcR3 variant described in any one of claims 1 to 13, comprising an Fc region of a human IgG1 antibody, a human IgG2 antibody, or a human IgG4 antibody, or a mutant Fc region consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of the Fc region.

15. The DcR3 variant of claim 14, wherein the mutant Fc region comprises a substitution of Cys at position 220 of the amino acid sequence of the heavy chain of human IgG1 as shown in the EU index with Ser.

16. The DcR3 variant of claim 15, wherein the mutant Fc region comprises a substitution of Leu at position 234 with Ala, Leu at position 235 with Ala, and Gly at position 237 with Ala in the amino acid sequence of the heavy chain of human IgG1 as shown in the EU index.

17. 17. The DcR3 variant of claim 15 or 16, wherein the mutant Fc region comprises a substitution of Asn at position 434 (EU index) of the amino acid sequence of the heavy chain of human IgG1 with Ala.

18. The DcR3 variant of claim 15, wherein the mutant Fc region comprises a substitution of Met at position 252 with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu in the amino acid sequence of the heavy chain of human IgG1 as shown in the EU index.

19. The DcR3 variant of claim 14, wherein the mutant Fc region comprises a substitution of Ser at position 228 with Pro, Leu at position 235 with Glu, and Arg at position 409 with Lys, as shown in the EU index of the amino acid sequence of the heavy chain of human IgG4.

20. The DcR3 variant of any one of claims 14 to 19, comprising a mutant Fc region consisting of an amino acid sequence set forth in SEQ ID NO: 72, 74, 156, 158, 160, 162, 164, 166, 311, 312 or 313, or an amino acid sequence set forth in SEQ ID NO: 72, 74, 156, 158, 160, 162, 164, 166, 311, 312 or 313 in which 1 to 5 amino acids have been deleted, substituted, inserted or added, and the amino acid sequence in which 1 to 5 amino acids have been deleted, substituted, inserted or added is the amino acid sequence of an Fc region.

21. The DcR3 variant, - a variant Fc region according to SEQ ID NO: 164 or the amino acid sequence of SEQ ID NO: 164 with one amino acid deleted; and a first chimeric cysteine-rich region or a second chimeric cysteine-rich region, (i) a first chimeric cysteine-rich region having a substitution of CRD1 of wild-type DcR3 for CRD1 of OPG, and a substitution of CRD4 of wild-type DcR3 for CRD4 of OPG; and (ii) the second chimeric cysteine-rich region has a substitution of Glu at position 57 with another amino acid selected from Lys, Leu, Arg, and Val, or a substitution of Glu at position 57 with another amino acid selected from Lys, Leu, Arg, and Val and a substitution of Arg at position 58 with another amino acid selected from Asp and Glu; Chimeric cysteine-rich region The DcR3 variant of any one of claims 14 to 19, comprising:

22. The DcR3 variant, - a variant Fc region according to SEQ ID NO: 160 or 166, or the amino acid sequence of SEQ ID NO: 160 or 166 in which one amino acid has been deleted; and a first chimeric cysteine-rich region or a second chimeric cysteine-rich region, (i) a first chimeric cysteine-rich region having a substitution of CRD1 of wild-type DcR3 for CRD1 of OPG, and a substitution of CRD4 of wild-type DcR3 for CRD4 of OPG; and (ii) the second chimeric cysteine-rich region has a substitution of Glu at position 57 with another amino acid selected from Lys, Leu, Arg, and Val, or a substitution of Glu at position 57 with another amino acid selected from Lys, Leu, Arg, and Val and a substitution of Arg at position 58 with another amino acid selected from Asp and Glu; Chimeric cysteine-rich region The DcR3 variant of any one of claims 14 to 19, comprising:

23. The DcR3 variant, - a variant Fc region according to SEQ ID NO: 158 or the amino acid sequence of SEQ ID NO: 158 with one amino acid deleted; and a first chimeric cysteine-rich region or a second chimeric cysteine-rich region, (i) a first chimeric cysteine-rich region having a substitution of CRD1 of wild-type DcR3 for CRD1 of OPG, and a substitution of CRD4 of wild-type DcR3 for CRD4 of OPG; and (ii) the second chimeric cysteine-rich region has a substitution of Glu at position 57 with another amino acid selected from Lys, Leu, Arg, and Val, or a substitution of Glu at position 57 with another amino acid selected from Lys, Leu, Arg, and Val and a substitution of Arg at position 58 with another amino acid selected from Asp and Glu; Chimeric cysteine-rich region The DcR3 variant of any one of claims 14 to 19, comprising:

24. The DcR3 variant of any one of claims 21 to 23, wherein the second chimeric cysteine-rich region has a substitution of Glu at position 57 with Lys or Arg and a substitution of Arg at position 58 with Asp or Glu.

25. The DcR3 variants are selected from SEQ ID NOs: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 150, 168, 170, 172, 174, 176, 178, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 3 62, 264, 266, 268, 288, 290, 292, 294, 296, 298, 300, 302, 304, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 or 337, or an amino acid sequence having 99% or more identity to the amino acid sequence.

26. 26. The DcR3 variant of claim 25, wherein the DcR3 variant comprises an amino acid sequence set forth in SEQ ID NO:214, or an amino acid sequence having 99% or greater identity to SEQ ID NO:

214.

27. ​​The DcR3 variant described in claim 25, which comprises an amino acid sequence set forth in SEQ ID NO:224, or an amino acid sequence having 99% or greater identity to SEQ ID NO:

224.

28. The DcR3 variant described in claim 25, which comprises an amino acid sequence set forth in SEQ ID NO:256, or an amino acid sequence having 99% or greater identity to SEQ ID NO:

256.

29. The DcR3 variant described in claim 25, wherein the DcR3 variant comprises an amino acid sequence set forth in SEQ ID NO:266, or an amino acid sequence having 99% or greater identity to SEQ ID NO:

266.

30. A DNA encoding the DcR3 variant according to any one of claims 1 to 29.

31. A recombinant vector comprising the DNA of claim 30.

32. A transformant obtained by introducing the recombinant vector according to claim 31 into a host cell.

33. The transformant according to claim 32, wherein the host cell is a cell derived from a mammal.

34. The transformant according to claim 33, wherein the mammalian cell is a CHO cell.

35. A method for producing a DcR3 variant or a DcR3 variant composition, comprising culturing a transformant described in any one of claims 32 to 34 in a medium to produce and accumulate a DcR3 variant, and purifying the DcR3 variant from the resulting culture medium.

36. A pharmaceutical composition comprising the DcR3 variant according to any one of claims 1 to 29 as an active ingredient.

37. The pharmaceutical composition according to claim 36, which is an agent for preventing or treating an autoimmune disease, an inflammatory disease, or an allergic disease.

38. Use of the DcR3 variant according to any one of claims 1 to 29 for producing an agent for the prevention or treatment of an autoimmune disease, an inflammatory disease, or an allergic disease.

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